tch346 prevents motor symptoms and loss of striatal fdopa uptake in bilaterally mptp-treated...

Radiotracer imaging in PD

Value of in vivo presynaptic dopaminergic measures in animal models and human disease

ISBN: 978-90-367-3958-0 (boek)ISBN: 978-90-367-3927-6 (digitaal)

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Cover picture: Michelangelo: “The creation of Adam”, circa 1511Sistine Chapel, Rome

The printing of this thesis was financially supported by:Boehringer Ingelheim bvGE HealthcareGlaxoSmithKlineNovartis Pharma B.V.Parkinson Patiënten VerenigingSiemens Nederland N.V.Veenstra Instruments Joure

RIJKSUNIVERSITEIT GRONINGEN

Radiotracer imaging in PD

Value of in vivo presynaptic dopaminergic measures in animal models and human disease

Proefschrift

ter verkrijging van het doctoraat in deMedische Wetenschappen

aan de Rijksuniversiteit Groningenop gezag van de

Rector Magnificus, dr. F. Zwarts,in het openbaar te verdedigen op

maandag 5 oktober 2009om 13.15 uur

door

Sietske Aleida Eshuisgeboren op 8 maart 1971

te Drachten

Promotores:Prof. Dr. K.L. LeendersProf. Dr. R.A.J.O. Dierckx

Copromotores:Dr. P.L. Jager

Beoordelingscommissie:Prof. Dr. B.R. BloemProf. Dr. J.H.A. De KeyserProf. Dr. A.A. Lammertsma

Paranimfen:E.M.P.E. ZeinstraE. Zorgdrager

Opgedragen aan Tjitske Eshuis - de Boer (1931 – 1987), Gerhard Hendrik Eshuis (1934 – 1991) en Aukje Zorgdrager – Bouma (1936 - 2004).

Contents

Chapter 1: Introduction 9

Chapter 2: Parkinson’s disease: symptoms and age dependency. 29 S.A. Eshuis, K.L. Leenders. Functional Neurobiology of Aging; Chapter 68. Edited by Hof and Mobbs.

Chapter 3: Motor behavior correlates with striatal [18F]-DOPA uptake 55 in MPTP-lesioned primates. S.A. Eshuis, R. Kortekaas, G. Andringa, A. Cools, K.L. Leenders, R.P. Maguire. Submitted.

Chapter 4: TCH346 prevents motor symptoms and loss of striatal F-DOPA 69 uptake in bilaterally MPTP-treated primates. G. Andringa, S.A. Eshuis, E. Perentes, R.P. Maguire, D. Roth, M. Ibrahim, K.L. Leenders, A. Cools. Neurobiol Dis 2003;14:205-17

Chapter 5: Comparison of FP-CIT SPECT and F-DOPA PET in patients 93 with de novo and advanced Parkinson’s disease. S.A. Eshuis, R.P. Maguire, K.L. Leenders, S. Jonkman, P.L. Jager. Eur J Nucl Med Mol Imaging 2006;33:200-9

Chapter 6: Direct comparison of FP-CIT SPECT and F-DOPA PET in 115 patients with Parkinson’s disease and healthy controls. S.A. Eshuis, P.L. Jager, R.P. Maguire, S. Jonkman, R.A. Dierckx, K.L. Leenders, Eur J Nucl Med Mol Imaging 2009;36:454-62.

Chapter 7: Summary 135

Chapter 8: Discussion and future perspectives 141

Chapter 9: Nederlandse samenvatting 155

Chapter 10: List of abbreviations 161

Chapter 11: Dankwoord 165

1Introduction

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Aim of this thesis

The main aim of the thesis is to study radiotracer uptake in the “presynaptic” dopaminergic system of the basal ganglia in order to assess its use in Parkinson’s disease. The topics addressed here are:

a) the association between the cerebral dopaminergic system and clinical motor signs

b) the possibility to monitor neuroprotectionc) early clinical diagnosis.

The uptake of two well-known tracers namely FP-CIT (SPECT) and F-DOPA (PET) in patients with several stages of Parkinson’s disease and in healthy volunteers were compared. Both primate MPTP and human PET or SPECT scans in addition to clinical motor assessments constitute the basis of this thesis. We also studied the effects of a presumed neuroprotective agent on radiotracer uptake in the animal model and correlated this with motor behaviour and post-mortem histopathological findings.

1. Parkinson’s disease Parkinson’s disease is a common neurodegenerative disorders, mainly affecting the elderly. It is a slowly progressive disorder in which progression is believed to be fastest in the beginning of the disease52. When symptoms and signs become evident, already probably 30% or more of the dopaminergic cells have been lost and endogenous striatal dopamine levels may be only 5-15% of normal39. Clinical symptoms consist of the classic trias of resting tremor, rigidity and bradykinesia. Next to these motor symptoms, also non-motor symptoms like depression, hallucinations and cognitive disorders exist. In chapter 1, a more extensive description of Parkinson’s disease can be found.Until so far, only symptomatic treatment is available. However, after some years, this treatment often becomes less effective. Unfortunately, there is no therapy available yet to stop or even delay the underlying process. Neuroprotection can be defined as an intervention that slows or stops the progression of neuronal degeneration, interfering with the underlying aetiology.

1.a.Pathophysiology in humansThe specific aetiology of Parkinson’s disease (PD) is not known yet, but it is likely to be the result of the cumulative effects of environmental and genetic factors. Main risk factors for developing PD include advancing age16 and positive family history24, 53, suggesting that it is an age-dependent genetic disorder, at least in a subset of patients. Epidemiological studies suggest some factors may increase the risk of developing PD. During the last

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decades several studies have been published, some suggesting environmental factors, others genetic factors to be the main cause of PD. Probably PD results from an interaction of (multiple) genetic mutations and environmental toxins: genetic factors may make an individual person more vulnerable to environmental factors. This is known as the double-hit hypothesis29.An increased risk for the development of PD has been found in association with environmental factors like exposure to well water drinking, herbicides (e.g. paraquat), pesticides (e.g. rotenone), industrial chemicals, farming and living in a rural environment71. Trace metals, cyanide, lacquer thinner, organic solvents, carbon monoxide and carbon disulphide have also been associated with an increased risk of developing PD72. However, post mortem analysis of brains of parkinsonian patients revealed no specific toxin. Special attention has been focused on the neurotoxin 1,2,3,6-methyl-phenyl-tetrahydroyridine (MPTP). MPTP, used in the drug scene, is a contaminate of the production of a synthetic meperidene derivate. After taking MPTP, drug addicts developed a syndrome, which clinically and pathologically closely resembled idiopathic PD40. Although many mechanisms have been proposed to be involved in the process of progressive cell death in Parkinson’s disease, like oxidative stress, glutamate excitotoxicity, free radical damage, mitochondrial (complex I) dysfunction and inflammatory processes or proteasomal dysfunction, the exact mechanisms responsible for the process are still unknown29, 39, 59. As we have studied the possible neuroprotective effect in PD of an anti-apoptotic agent, we will focus now on the role of apoptosis in the pathogenesis of Parkinson’s disease.

1.a.1. Apoptosis in pathogenesis of Parkinson’s diseaseParkinson’s disease is a slowly progressive disorder characterised by loss of dopaminergic neurons in the substantia nigra and degeneration of nigrostriatal pathways, resulting in a decrease of striatal endogenous dopamine concentration.Although the mechanisms of cell death in PD are still unknown, general belief is that the neuronal death in the pars compacta of the substantia nigra is apoptotic12, 29. However, the exact role of apoptosis remains unknown and necrosis may be involved as well. Apoptosis is a form of programmed cell death and is to a certain extent a normal physiologic process in dopaminergic neurons. It is characterised by nuclear chromatin condensation, intact cytoplasmic membranes, DNA fragmentation and cell shrinkage without inflammatory processes.Some studies have found evidence for apoptosis in PD, while others were not able to confirm this. In 1996 Mochizuki et al found, using in situ DNA end labelling, cells displaying DNA cleavage in the substantia nigra of 4 patients with PD, without apoptotic morphology, such as chromatin clumps50. However in situ DNA end labelling is prone to false-positive detection of apoptosis. One year later, Anglade detected typical features of

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apoptotic neurons in the substantia nigra of 3 patients with PD using electron microscopy, like condensed chromatin in the nucleus of neurons containing neuromelanin and intact cytoplasmic membranes4. Using fluorescent probes specific for both DNA cleavage and chromatin clumping, Tatton was able to confirm positive staining of melanized neurons in the pars compacta of the substantia nigra73. However, all of the studies mentioned are limited by the small numbers of patients studied and by the lack of suitable control material. By the absence of any data from age-matched normal brains, the significance of these findings for the aetiopathology of PD remains unclear. More recent studies have demonstrated increased numbers of cells that express markers of apoptosis, with individual nuclei staining positively for DNA fragmentation and chromatin clumping. Increased expression of cell signals associated with apoptosis, including BAX, caspase-3 and nuclear translocation of glyceraldehydes phosphate dehydrogenase have been detected in PD27,

59, 74. Increased expression of the pro-apoptotic factor p53 in nigral neurons of PD patients have been observed54. Those studies suggest that apoptosis could account for some of the cell death occurring in PD 59, 64. Apoptosis may be the primary form of cell death, but it cannot be excluded that it may be secondary to other forms of cell death which trigger apoptosis. By inhibiting apoptosis, the underlying progressive neurodegenerative processes of PD might thus be blocked, resulting in effective neuroprotection.

1.b. Animal modelsAfter the discovery of MPTP as a neurotoxin with particular affinity for the dopaminergic neurons, an animal model using this compound, has been developed. The MPTP-model is so far one of the best experimental model for the production of parkinsonism in animals and reproduces all its cardinal features of idiopathic Parkinson’s disease like rigidity, bradykinesia and sometimes tremor2, 9, 19, 40. A number of analogues of MPTP can cause similar damage to the nigrostriatal system26.Other agents used in animals to cause parkinsonism are 6-OHDA, paraquat, rotenone, lipopolysaccharide49 and proteasomal inhibitors like lactcystin and epoxomicin45. Also genetic models of Parkinson’s disease exist, in which animals have been genetically manipulated. All those compounds can be administered to various animals, but are most widely used in rodents and in non-human primates. The advantage of the latter above other animal species is that those animals are more similar to the human in both brain pathology and symptomatology. Therefore we used in our studies, the MPTP-treated monkey.

1.c. NeuroprotectionNeuroprotection can be defined as an intervention that slows or stops the progression of neuronal degeneration and seeks to interfere with the basic pathogenetic mechanism of nigral cell death. We have investigated the possible neuroprotective effect of TCH346 in MPTP-treated monkeys, see chapter 4.

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1.c.1. MAO- inhibitors: selegiline One of the first agents thought to be neuroprotective was the propargylamine selegiline (also known as Deprenyl), a selective irreversible inhibitor of mono-amine-oxidase, type B (MAO-B). Because of its ability to block the MAO-B metabolism of dopamine, selegiline was thought to increase striatal dopamine. In MPTP-treated animals, conversion of MPTP to MPP+ can be inhibited by blocking MAO-B, thus preventing the development of nigrostriatal degeneration and parkinsonian symptoms in experimental animals41. Thus MAO-B-inhibitors may prevent the conversion of a protoxin to a toxin, and could possible be neuroprotective. Knoll reported that selegiline extended lifespan in rats by 50% 35.Birkmayer et al tested selegiline in PD and reported modest antiparkinsonian benefits, a reduction in motor fluctuations, longer survival and less disability when selegiline was combined with levodopa7, 8. In 1987 a double-blinded, placebo-controlled, multicenter trial, the DATATOP-study (Deprenyl and Tocopherol Antioxidative Therapy Of Parkinsonism) was started to determine if deprenyl 10 mg/day or tocopherol 2000 IU/day, administered to untreated patients with early PD, would slow disease progression and prolong the time until levodopa therapy was necessary. In this study, 800 patients were enrolled1. According to this study, selegiline significantly delayed the development of disability requiring levodopa therapy. However, after drug withdrawal a worsening of the motor scores was observed. This indicates a symptomatic effect instead of a neuroprotective effect. Possible neuroprotective effects may have been masked by these symptomatic effects.There was no evidence of sustained advantages of selegiline, as measured by complications of L-dopa therapy or duration of life36, 58, 68. However, in the patients treated with selegiline, more cardiovascular adverse events and mortality occurred compared to the placebo-treated patients. This increase in adverse events can be explained by the amphetaminergic metabolites of selegiline. Because of the lack of proof for a neuroprotective effect and the amphetaminergic metabolites, selegiline is no longer eligible as a possible neuroprotective agent for Parkinson’s disease.

1.c.2. Apoptosis inhibitors: TCH346Another propargylamine without the toxic metabolites amphetamine and methamphetamine is CGP 3466B (dibenzo[b,f]oxepin-10-ylmethyl-methyl-prop-2-ynyl-amine), also known as TCH346. Unlike deprenyl, TCH346 does not inhibit MAO-B and cannot be metabolised into amphetamine and metamphetamine.TCH346 has been tested in a wide variety of cellular and animal models of PD and exhibits neurorescuing properties qualitatively similar to, but about 100-fold more potent than those of (-)-deprenyl79, 80. TCH346 does not inhibit MAO-B, but interacts with GAPDH (glyceraldehyde-3-phosphate dehydrogenase). GAPDH is a glycolytic enzyme. Beside its role as enzyme in the glycolysis, GAPDH has been found to play a critical

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role in apoptosis. Post-mortem analyses of brains of parkinsonian patients show nuclear accumulation of GAPDH in the substantia nigra (SN), suggesting that GAPDH plays a role in the neurodegenerative processes or apoptosis in PD. Normally GAPDH is confined to cytosol by RNA. During apoptotic death, GAPDH is overexpressed. This results in an increased amount of GAPDH, accumulating in the nucleus, accompanied by a decrease of affinity of GAPDH for RNA. Accumulation of GAPDH in the nucleus may result in malfunctioning of the cell, leading to increased glycolysis and eventually cell death. GAPDH exists in two forms: a dimeric and a tetrameric form. GAPDH can only exert its apoptotic function in its tetrameric form and not in its dimeric form. It is possible that GAPDH dimer cannot accumulate in the nucleus. TCH346 blocks the action of GAPDH by preventing accumulation of GAPDH in the nucleus of the cell and / or by converting GAPDH from its tetrameric form to a dimeric form13.

1.c.2-1 Animal studiesThe compound is not only able to rescue dopamine neurons in vitro from death induced by apoptotic stimuli37, but also has promising effects in rodent models of PD. Doses of 0.0014-1.4 mg/kg TCH346 given twice daily for 18 days have neuroprotective effects in mice. In addition, the compound prevents nigral degeneration and motor symptoms induced by low doses of 6-OHDA in the rat model of PD3. We have extended the rodent studies to an animal more similar to the human in both brain organisation and motor function, in order to analyse the neuroprotective effects of TCH346 in MPTP-treated rhesus monkeys (see chapter 4). The MPTP monkey model reproduces virtually all the classic behavioural, cognitive, biochemical and histological changes that occur in PD. We have also studied the correlation between behaviour parameters and degeneration of the nigrostriatal dopamine systems established with [18F]-DOPA positron emission tomograph (PET) scans in this animal model (see chapter 3).

1.c.2-2. Human studiesBecause of the positive results of the neuroprotective effect of TCH346 in MPTP-treated monkeys, in this thesis described in chapter 4, this compound has also been tested in the human setting. In a double-blind, randomised, placebo-controlled trial, around 300 patients with de novo PD with Hoehn and Yahr (H&Y) stage of 2 or less and disease duration of less than 1 year were included60. They were randomly assigned to TCH346 in 3 different doses or placebo. Time until symptomatic treatment was needed was the primary outcome measure and changes in Unified Parkinson’s Disease Rating Scale (UPDRS; total, part II and part III) and Parkinson’s Disease Questionnaire (PDQ) 39 formed the secondary outcome measures. Patients received the study drug for 12 to 18 months followed by a 4 week

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withdrawal period, or until symptomatic treatment was needed. However, in all the 4 treatment groups an equal percentage of patients needed symptomatic treatment. Also no significant difference was found in secondary outcome measures between the 4 treatment groups. So it can be concluded that TCH346 is not neuroprotective in patients with PD60.

1.c.2-3 Contradictory outcomesAlthough the animal studies showed a neuroprotective effect of TCH346 in several animal models, this could not be detected in the human setting. Several factors can be mentioned to explain this difference in efficacy (see also Chapter 8)60, 78. - the MPTP- treated animal model does not reflect the pathogenesis of PD correctly. - inaccurate doses of TCH346 have been administered to patients with PD. - PD had already progressed too far in the included patients - the follow-up period of 1 year was too short to be able to notice subtle neuroprotective effects - the chosen end-points were not sensitive enough to detect subtle changesThe contradictory outcomes between animal studies and human studies underscore the need for the development of a better progressive animal model, reflecting more accurately pathogenetic mechanisms of PD60, 65.

1.d. Early diagnosisIt is of interest to be able to diagnose PD in the early phases of the disease. Neuroprotective treatment, if available, should be given as soon as possible after the onset of signs and symptoms, as progression is probably fastest in the beginning of the disease52 and therefore neuroprotection is most effective in those early phases. In the beginning of the disease, diagnosing PD correctly on clinical signs and symptoms can be difficult. The motor symptoms can be subtle in the first phases of PD. More importantly, the clinical features of PD may occur in other neurodegenerative disorders as well, like Multi System Atrophy (MSA), Progressive Supranuclear Palsy (PSP) or Cortico Basal Degeneration (CBD). Differentiating idiopathic PD from these other forms of parkinsonism has therapeutic and prognostic impact. A non-movement disorder neurologist may misdiagnose up to 25% of cases with established parkinsonism of all sorts when compared with post-mortem pathology31, 48. Movement disorders specialists are more often able to diagnose idiopathic PD correctly when all the relevant clinical information is obtained: the positive predictive value of the clinical diagnosis PD was under those circumstances 98,6%30. These studies refer to advanced PD patients. No data are available to assess the situation in early, de novo, PD patients who on clinical grounds are likely to have indeed PD, but in whom this as yet could not be confirmed by response to medication or by the time course. Therefore, to facilitate

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diagnosing PD correctly in the above mentioned circumstances, auxiliary examinations are needed. Several non-motor signs and symptoms in PD have been tested for their utility in diagnosing PD in early (preclinical) phases, like olfactory dysfunction17, 18, 81, disturbances in mood and personality (e.g. depression)15, 28, 51, 84, visuomotor control abnormalities and subtle neurocognitive dysfunctions11, 14, 20. However, none of these signs and symptoms are sensitive nor specific for PD6, 83. Conventional imaging techniques of the brain, such as CT or MRI scans, are also not useful for diagnosing PD early, because the brain structure is usually not greatly altered in early PD and can not positively document dopaminergic biochemical activity.

2. Functional imagingAnother entrance for auxilliary methods in diagnosing PD is formed by visualising the underlying biochemical abnormalities in PD. At the time of diagnosis probably 30% or more of dopaminergic neurons are lost and endogenous striatal dopamine levels are estimated to be 5-15% of controls39. The preclinical phase is thought to be between 3.152 to 6.5 years57. This implies that patients can possibly be identified in a preclinical phase of the disease by assessing the biochemical changes. Striatal activity of aromatic aminodecarboxylase and the density of dopamine transporters is decreased in patients with PD. Radiotracer neuroimaging techniques using Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) can be helpful to visualise and measure striatal dopaminergic activity. Several radiotracers are developed to investigate the dopaminergic system and most of them can be divided in “presynaptic” and “postsynaptic” tracers. Presynaptic tracers are able to measure striatal activity of aromatic aminodecarboxylase (11C-DOPA, 18F-DOPA, 18F-FMT) or the density of dopamine transporters (11C-CFT, 11C-MP, 123I-FP-CIT or 123I-β-CIT). Postsynaptic tracers are able to quantify the postsynaptic D2 receptor binding: 11C-raclopride, 18F-NMSP, 18C-FLB457, 11C-SCH23390 or 123I- IBZM. Finally, the vesicular monoamine transporter can be quantified by the use of 11C-DTBZ. F-DOPA PET has been regarded as the gold standard for the assessment of presynaptic dopaminergic integrity in vivo, in part because it has been in use for many years and has been extensively studied55.F-DOPA PET was the first developed tracer to visualize the nigrostriatal dopaminergic system in vivo23. However, its use is limited by the restricted availability of PET instruments, the need for a cyclotron and the difficult production of F-DOPA. FP-CIT SPECT is easier available and can therefore form an alternative for F-DOPA PET. We compared the use of FP-CIT and F-DOPA by means of respectively SPECT and PET in patients with Parkinson’s disease and in healthy volunteers for diagnosing Parkinson’s disease in an early phase in chapter 6, and the correlation between uptake measures and clinical symptoms in chapter 5.

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2.a. Positron Emission TomographyBoth PET and SPECT use radioactive tracers: compounds with a radioactive atom attached to a molecule. Radioactive atoms decay, emitting gamma rays and nuclear particles. PET uses metabolically active compounds labelled with short-lived radioactive tracer isotopes, which decay by emitting a positron. After travelling up to a few millimetres, the positron encounters and annihilates with an electron, causing two gamma photons to be emitted in opposite directions. The tomographic image is formed by recording two 511 keV photons emitted in positron decay with a circumferential array of radiation detectors. Since these photons are simultaneous emitted at approximately 180 degrees to each other, it is possible to localize their source along a straight line of response. After processing these data through a conventional reconstruction algorithm, tomographic images of the tracer accumulation in the tissue can be formed.

2.b. Single Photon Emission Computed TomographySPECT is similar to PET in its use of radioactive tracer material and detection of gamma rays. In contrast with PET, however, the tracer used in SPECT emits gamma radiation which is measured directly. Also the level of energy of the emittors used in SPECT is different from those used in PET. 123I is used for FP-CIT SPECT which emits gamma rays of 159keV. SPECT imaging is performed by using a gamma camera to acquire multiple 2-D images from multiple angles. A computer is then used to apply a tomographic reconstruction algorithm to the multiple projections, yielding a 3-D dataset. This dataset may then be manipulated to show thin slices along any chosen axis of the body, similar to those obtained from other tomographic techniques. To acquire SPECT images, the gamma camera is rotated around the patient and projections are acquired at defined points during the rotations.

2.c. F-DOPA PETPET scans using 6-[18F]-fluoro-L-3,4-dihydroxyphenylalanine (F-DOPA) enable measurement of striatal levodopa decarboxylase activity, thereby estimating the rate of enzymatic decarboxylation of F-DOPA to F-dopamine, and trapping of F-dopamine in synaptic vesicles. Striatal F-DOPA uptake does not measure the endogenous dopamine concentration. It correlates with dopamine cell counts measured in post mortem specimens69. It is possible to discriminate patients with PD from the healthy population by means of F-DOPA PET. Several studies have reported a decrease in striatal F-DOPA uptake in PD compared to healthy controls, more pronounced in the putamen than in the caudate5, 22, 43, 44, 63. However, restricted availability of F-DOPA and PET instruments limit its use in routine clinical practice. It has been suggested that in the very beginning of this disease, levodopa decarboxylase activity is upregulated although this never has been proven. Such factors, however, may influence the sensitivity of this technique for diagnosing defects in the nigrostriatal system.

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2.d. FP-CIT SPECTUptake of tracers with a high affinity for the dopamine transporter (DAT) can be measured using PET and also SPECT. Dopamine transporters, localised on dopaminergic nerve endings, participate in the reuptake mechanism of dopamine into presynaptic terminals and are modulated by concentrations of endogenous dopamine25. Decrease of transporter density in the striatum has been associated with PD33, 56. DAT imaging can therefore be used as a marker for the relative degree of malfunction or loss of dopaminergic nerve endings. A selective and potent DAT imaging agent is [123I] N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) nortropane (FP-CIT). SPECT imaging with FP-CIT produces a high target to background ratio. Several studies have demonstrated that striatal FP-CIT uptake is reduced in patients with PD compared to healthy controls10, 32, 34, 46, 47, 66, 67, 75, 76. FP-CIT SPECT is, in contrast to F-DOPA PET, widely available and cheaper than F-DOPA PET. However, spatial resolution is lower in FP-CIT SPECT scans than in F-DOPA PET scans, resulting in poorer quality of the SPECT images. Another disadvantage of FP-CIT is the disability to continue medication known to interfere with DAT, like methylphenidate, benzatropine, bupropion, mazindol, phentanyl, sertraline and cocaine-derivates, before scanning, while it is not necessary to stop most medication before F-DOPA PET scanning. Only catechol-O-methyl-transferase (COMT)-inhibitors may influence the outcome of a F-DOPA PET scan. Finally, the duration of a FP-CIT SPECT scan with a regular double-headed gamma camera is longer (approximately 45 minutes) than the duration of a 3-D F-DOPA PET scan (approximately 6 minutes). This long duration of scanning can be a major disadvantage of FP-CIT SPECT scanning in parkinsonian patients, especially when they suffer from tremor or dyskinesia.

2.e. Other radiotracersNext to the above mentioned presynaptic dopaminergic tracer methods, postsynaptic dopaminergic tracers have been developed, like 11C-raclopride for PET or 123I- IBZM for SPECT. Decreased binding of postsynaptic dopaminergic tracers suggest the presence of other forms of neurodegenerative disease than Parkinson’s Disease. However, those tracers can not be used in clinical practice to confirm the diagnosis of Parkinson’s Disease. Another alternative is formed by FDG-PET scanning. This method is nowadays frequently used in oncology. By recognition of the pattern of brain metabolism, FDG-PET can be used in clinical practice for differentiation between different forms of parkinsonism, like MSA, PSP and CBD21. 99mTc-ethyl cysteinate dimer (ECD)-SPECT is sometimes used to differentiate between the different forms of neurodegenerative disorders as well by measuring regional cerebral blood flow85. However, the spatial resolution of SPECT is too low for an easy discrimination between the various forms of parkinsonism42.

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Outline of this thesis

In chapter 1 the pathophysiology of Parkinson’s disease in humans and in animal models is briefly described including the possible role of apoptosis. The motivation to explore possible neuroprotection by apoptosis inhibitors is explained. The need for diagnosing Parkinson’s disease in an early phase will become necessary if neuroprotective treatment really becomes available. The role of presynaptic striatal imaging in the diagnostic work-up of Parkinson’s disease in an early stage by means of F-DOPA PET and FP-CIT SPECT is described.

Chapter 2 gives an extensive description of Parkinson’s disease. Epidemiology, including mortality, regional, racial and sexual variation is described and sub classifications of Parkinson’s disease. An overview of motor and non-motor symptoms is given, followed by pathology findings. Several other movement disorders in the elderly and changes in gait with aging are described.Functional Neurobiology of Aging; Chapter 68. Edited by Hof and Mobbs.

Chapter 3 describes the correlation between motor behaviour and F-DOPA uptake in a well known animal model for Parkinson’s disease: the MPTP-treated monkey. Eight rhesus-monkeys received MPTP infusions and became parkinsonian to various degrees. Motor signs were rated regularly quantitatively and qualitatively and correlated with striatal uptake of [18F]-DOPA as measured with PET. Submitted.

In chapter 4 the neuroprotective effect of the compound TCH346 is evaluated in MPTP-treated monkeys. Eight rhesus monkeys received MPTP-infusions bilaterally by a two-step procedure, which induces a stable parkinsonian animal model. The effects of TCH346 on behaviour and striatal dopaminergic integrity were evaluated by means of quantitative and qualitative analyses of behaviour and striatal F-DOPA uptake. Post-mortem immunohistochemical analyses of the brains were performed. Neurobiol Dis 2003;14:205-17.

Chapter 5 describes the correlation between two radiotracers: FP-CIT SPECT and F-DOPA PET in patients with different stages of Parkinson’s disease. Also correlation between uptake of both tracers and motor scores are established and the ability to distinguish patients with advanced disease from de novo patients is described. In this study 13 de novo parkinsonian patients and 17 patients with advanced stage of disease underwent both FP-CIT SPECT scans and F-DOPA PET scans. Eur J Nucl Med Mol Imaging 2006;33:200-9.

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Chapter 6 focuses on the ability of FP-CIT SPECT and F-DOPA PET to distinguish de novo parkinsonian patients from the healthy population and establishes the sensitivity and specificity of both radiotracers in this. Ten healthy volunteers received a FP-CIT SPECT scan, 10 healthy volunteers an F-DOPA PET scan and a total of 28 parkinsonian patients had both a FP-CIT SPECT and a F-DOPA PET scan. Eur J Nucl Med Mol Imaging 2009;36:454-62.

Chapter 7 contains the summary of this thesis.

In chapter 8 discussion and future perspectives are discussed.

A summary of this thesis in Dutch including future perspectives is given in chapter 9.

In chapter 10 a list of abbreviations can be found.

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Reference List

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3. Andringa G, van Oosten RV, Unger W, et al: Systemic administration of the propargylamine CGP 3466B prevents behavioural and morphological deficits in rats with 6-hydroxydopamine-induced lesions in the substantia nigra. Eur J Neurosci 12:3033-3043, 2000

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8. Birkmayer W, Riederer P, Youdim MB, et al: The potentiation of the anti akinetic effect after L-dopa treatment by an inhibitor of MAO-B, Deprenil. J Neural Transm 36:303-326, 1975

9. Blum D, Torch S, Lambeng N, et al: Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease. Prog Neurobiol 65:135-172, 2001

10. Booij J, Tissingh G, Boer GJ, et al: [123I]FP-CIT SPECT shows a pronounced decline of striatal dopamine transporter labelling in early and advanced Parkinson’s disease. J Neurol Neurosurg Psychiatry 62:133-140, 1997

11. Brown RG, Marsden CD: Cognitive function in Parkinson’s disease: from description to theory. Trends Neurosci 13:21-29, 1990

12. Burke RE, Kholodilov NG: Programmed cell death: does it play a role in Parkinson’s disease? Ann Neurol 44:S126-S133, 1998

13. Carlile GW, Chalmers-Redman RM, Tatton NA, et al: Reduced apoptosis after nerve growth factor and serum withdrawal: conversion of tetrameric glyceraldehyde-3-phosphate dehydrogenase to a dimer. Mol Pharmacol 57:2-12, 2000

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16. de Rijk MC, Launer LJ, Berger K, et al: Prevalence of Parkinson’s disease in Europe: A collaborative study of population-based cohorts. Neurology 54:S21-S23, 2000

17. Doty RL, Deems DA, Stellar S: Olfactory dysfunction in parkinsonism: a general deficit unrelated to neurologic signs, disease stage, or disease duration. Neurology 38:1237-1244, 1988

18. Doty RL, Stern MB, Pfeiffer C, et al: Bilateral olfactory dysfunction in early stage treated and untreated idiopathic Parkinson’s disease. J Neurol Neurosurg Psychiatry 55:138-142, 1992

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4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and several of its analogs. Toxicology 49:493-501, 1988

27. Hirsch EC, Hunot S, Faucheux B, et al: Dopaminergic neurons degenerate by apoptosis in Parkinson’s disease. Mov Disord 14:383-385, 1999

28. Hoogendijk WJ, Sommer IE, Tissingh G, et al: Depression in Parkinson’s disease. The impact of symptom overlap on prevalence. Psychosomatics 39:416-421, 1998

29. Huang Z, Fuente-Fernandez R, Stoessl AJ: Etiology of Parkinson’s disease. Can J Neurol Sci 30 Suppl 1:S10-S18, 2003

30. Hughes AJ, Daniel SE, Ben Shlomo Y, et al: The accuracy of diagnosis of parkinsonian syndromes in a specialist movement disorder service. Brain 125:861-870, 2002

31. Hughes AJ, Daniel SE, Blankson S, et al: A Clinicopathological Study of 100 Cases of Parkinsons-Disease. Archives of Neurology 50:140-148, 1993

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32. Ishikawa T, Dhawan V, Kazumata K, et al: Comparative nigrostriatal dopaminergic imaging with iodine-123-beta CIT-FP/SPECT and fluorine-18-FDOPA/PET. J Nucl Med 37:1760-1765, 1996

33. Kaufman MJ, Madras BK: Severe depletion of cocaine recognition sites associated with the dopamine transporter in Parkinson’s-diseased striatum. Synapse 9:43-49, 1991

34. Kazumata K, Dhawan V, Chaly T, et al: Dopamine transporter imaging with fluorine-18-FPCIT and PET. J Nucl Med 39:1521-1530, 1998

35. Knoll J: The striatal dopamine dependency of life span in male rats. Longevity study with (-)deprenyl. Mech Ageing Dev 46:237-262, 1988

36. Koller WC: Selegiline monotherapy in the treatment of Parkinson’s disease. Neurology 47:S196-S199, 1996

37. Kragten E, Lalande I, Zimmermann K, et al: Glyceraldehyde-3-phosphate dehydrogenase, the putative target of the antiapoptotic compounds CGP 3466 and R-(-)-deprenyl. J Biol Chem 273:5821-5828, 1998

38. Kung MP, Stevenson DA, Plossl K, et al: [99mTc]TRODAT-1: a novel technetium-99m complex as a dopamine transporter imaging agent. Eur J Nucl Med 24:372-380, 1997

39. Lang AE, Lozano AM: Parkinson’s disease. First of two parts. N Engl J Med 339:1044-1053, 1998

40. Langston JW, Ballard P, Tetrud JW, et al: Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science 219:979-980, 1983

41. Langston JW, Irwin I, Langston EB, et al: Pargyline prevents MPTP-induced parkinsonism in primates. Science 225:1480-1482, 1984

42. Leenders KL: Significance of non-presynaptic SPECT tracer methods in Parkinson’s disease. Mov Disord 18 Suppl 7:S39-S42, 2003

43. Leenders KL, Palmer AJ, Quinn N, et al: Brain dopamine metabolism in patients with Parkinson’s disease measured with positron emission tomography. J Neurol Neurosurg Psychiatry 49:853-860, 1986

44. Leenders KL, Salmon EP, Tyrrell P, et al: The nigrostriatal dopaminergic system assessed in vivo by positron emission tomography in healthy volunteer subjects and patients with Parkinson’s disease. Arch Neurol 47:1290-1298, 1990

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46. Marek K, Innis R, van Dyck C, et al: [123I]beta-CIT SPECT imaging assessment of the rate of Parkinson’s disease progression. Neurology 57:2089-2094, 2001

47. Marek KL, Seibyl JP, Zoghbi SS, et al: [123I] beta-CIT/SPECT imaging demonstrates bilateral loss of dopamine transporters in hemi-Parkinson’s disease. Neurology 46:231-237, 1996

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48. Meara J, Bhowmick BK, Hobson P: Accuracy of diagnosis in patients with presumed Parkinson’s disease. Age Ageing 28:99-102, 1999

49. Meredith GE, Sonsalla PK, Chesselet MF: Animal models of Parkinson’s disease progression. Acta Neuropathol 115:385-398, 2008

50. Mochizuki H, Goto K, Mori H, et al: Histochemical detection of apoptosis in Parkinson’s disease. J Neurol Sci 137:120-123, 1996

51. Montgomery EB, Jr., Baker KB, Lyons K, et al: Abnormal performance on the PD test battery by asymptomatic first-degree relatives. Neurology 52:757-762, 1999

52. Morrish PK, Sawle GV, Brooks DJ: An [18F]dopa-PET and clinical study of the rate of progression in Parkinson’s disease. Brain 119 ( Pt 2):585-591, 1996

53. Mouradian MM: Recent advances in the genetics and pathogenesis of Parkinson disease. Neurology 58:179-185, 2002

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56. Niznik HB, Fogel EF, Fassos FF, et al: The dopamine transporter is absent in parkinsonian putamen and reduced in the caudate nucleus. J Neurochem 56:192-198, 1991

57. Nurmi E, Ruottinen HM, Bergman J, et al: Rate of progression in Parkinson’s disease: a 6-[18F]fluoro-L-dopa PET study. Mov Disord 16:608-615, 2001

58. Oakes D: Antiparkinson efficacy of deprenyl. DATATOP Steering Committee of Parkinson Study Group. Ann Neurol 34:634, 1993

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61. Rinne JO, Laihinen A, Nagren K, et al: PET examination of the monoamine transporter with [11C]beta-CIT and [11C]beta-CFT in early Parkinson’s disease. Synapse 21:97-103, 1995

62. Rinne JO, Laihinen A, Rinne UK, et al: PET study on striatal dopamine D2 receptor changes during the progression of early Parkinson’s disease. Mov Disord 8:134-138, 1993

63. Sawle GV, Playford ED, Burn DJ, et al: Separating Parkinson’s disease from normality. Discriminant function analysis of fluorodopa F 18 positron emission tomography data. Arch Neurol 51:237-243, 1994

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71. Tanner CM: Epidemiology of Parkinson’s disease. Neurol Clin 10:317-329, 1992 72. Tanner CM, Langston JW: Do environmental toxins cause Parkinson’s disease? A critical

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81. Ward CD, Hess WA, Calne DB: Olfactory impairment in Parkinson’s disease. Neurology 33:943-946, 1983

82. Wenning GK, Donnemiller E, Granata R, et al: 123I-beta-CIT and 123I-IBZM-SPECT scanning in levodopa-naive Parkinson’s disease. Mov Disord 13:438-445, 1998

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84. Wolters EC, Francot CM: Mental dysfunction in Parkinson’s disease. Parkinsonism Relat Disord 4:107-112, 1998

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2Parkinson’s disease: symptoms and age

dependency

S.A. Eshuis, K.L. LeendersDept of neurology, University Medical Center, Groningen, The Netherlands

Abbreviated according to chapter 68: “Parkinson’s disease: symptoms and age dependency” appeared in Functional Neurobiology of Aging; Edited by Patrick R. Hof

and Charles V. Mobbs, 2001Publisher: Academic Press, San Diego, CA, USA

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Parkinson’s disease: symptoms and age dependency

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Epidemiology of Parkinson’s Disease (PD)

Parkinson’s disease is one of the most frequent neurodegenerative diseases, which mainly affects the elderly. To estimate incidence and prevalence of PD, some problems need to be mentioned. No perfect ante mortem diagnostic test for PD exists and the most reliable diagnostic method is expert neurologic examination at regular time intervals. In autopsy studies, the diagnosis of PD before death has been found to be incorrect in about 24% of cases39. Essential tremor may account for 10% - 40% of the false-positive diagnoses of PD. In contrast PD may be misdiagnosed as depression, or in the very elderly, “normal” aging. Other neurodegenerative disorders, like progressive nuclear palsy (PSP) or multiple system atrophy (MSA) may not be distinguished easily from PD early in the course of the disease. Many “atypical” parkinsonian syndromes were only recognised in the past several decades and probably have been classified as PD in early reports.

Incidence and prevalence of Parkinson’s diseaseSeveral studies have been performed to estimate the frequency of PD. The overall incidence is estimated to be 20 / 100 000 per year, and raises to about 1% of persons over 50 years of age32, 78 and even higher when older9. Prevalence of PD varies from 10 to 405 per 100 000 population. This variation may be due to differences in case-finding procedures, in diagnostic criteria, in accessibility of medical services and in the age distribution of populations. Most frequently prevalence is about 100 - 187 per 100 00059. As for incidence, prevalence rises almost exponentially after age 50. By the eighth decade, prevalence in Europe and North America is estimated to be between 1000 and 3000 per 100 000 persons88. This is confirmed by the Rotterdam Study: prevalence figures were 0.3% for those aged 55 to 64 years, 1.0% for those aged 65 to 74 years, 3.1% for those aged 75 to 84 years and 4.3% for those aged 85 to 94 years. Among women aged 95 to 99 years, prevalence was 5.0%16. In some studies an apparent decrease in late life is seen. This is probably due to ascertainment and diagnosis difficulties in this population, rather than an actual decline in disease frequency.

Mortality In Hoehn and Yahr’s series mortality ratio in patients with primary parkinsonism was 2.9 times that expected in the age-matched population38. In a more recent study of parkinsonian patients the overall risk for death, adjusted for age and sex, was 2.0 times that of persons without parkinsonism4. Since the introduction of more effective antiparkinsonian medication, especially levodopa, mortality has decreased in younger parkinsonian patients. However in older parkinsonian patients an increase during the last few decades in mortality is found. In the United States between 1962 and 1984, mortality

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decreased for persons younger than age 70, but increased for persons of 75 years and older49. This is confirmed by Morens et al67, who found that after age 60, PD-associated mortality rates appeared to increase logarithmically. This increase in mortality rate was not attributable to age alone. Increased age-related PD mortality was associated with both absolute age and duration of illness longer than 10 years. Between the ages of 70 and 89 years, parkinsonian patients had a two- to three-fold increase in the risk of dying, corresponding to a mortality ratio of 2.567. In a study of Louis et al54 risk of mortality was higher in parkinsonian patients (rate ratio = 2,7) after adjusting for baseline age, years of education, sex, ethnicity and smoking status. It was even higher when PD was combined with dementia (rate ratio = 4.9). In fact dementia is a significant predictor of death in PD57. A high base-line score of extrapyramidal signs was most associated with increased risk of mortality among the patients with PD. After subanalysis of the different extrapyramidal signs, severe bradykinesia was the motor manifestation that most highly correlated with increased mortality54. The Sydney multicentre study found increased mortality risk among parkinsonian men, whereas this was not significantly different for women, compared to the general Australian population. Predictors of mortality, according to this study, are age at onset and progression rate. Several studies have shown that the effects of levodopa on mortality are apparent in the early years of the disease. In contrast, despite levodopa therapy, mortality is rising in a later stage of the disease12, 17. Data from the DATATOP cohort suggests that carefully selected patients with early PD without co-morbidity have normal life expectancy when adequately treated and frequently seen by consulting physicians1. This is confirmed by Tanner and Ben-Shlomo88. For persons with PD diagnosed before the age of 60, they found a relative survival similar to that of the general population, in contrast to people with an older age at diagnosis, who showed a lower relative survival88 . Pneumonia is the most common cause of death, probably due to immobility and increased risk of aspiration1. Death from cerebrovascular disease is increased as well35. Some studies have suggested a reduced risk of death from cancer in parkinsonian patients35, 42, but other studies did not confirm this91.

Regional and racial variationEstimates of prevalence vary widely depending on geographical location. A Northwest to Southeast gradient is suggested49 and PD prevalence appears to be highest in Europe and North America, whereas rates in Japan, China and Africa are markedly lower. In the USA PD prevalence is much lower among blacks. Already in 1972, Kessler found a higher frequency of PD for whites compared with blacks43, 44. In a survey of PD in New York, age-adjusted prevalence rates were lower for blacks than for whites and Hispanics. Surprisingly however incidence rates were highest among black men, but these incidence rates were otherwise comparable across sex and ethnic groups. By ethnic group, the


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cumulative incidence was higher for blacks than for whites and Hispanics, but more deaths occurred among incident black patients. These findings could result from a delay in diagnosis due to limited access to appropriate health services among these people60. Some studies however show similar rates for African-American, Asian-American and European-American subjects67. In a study among a cohort of American men of Japanese or Okinawan ancestry, epidemiological data are in general in accord with those from Europe and the United States. Incidence data are 5- to 10-fold higher at each age stratum than age-specific incidence figures from China. These findings most likely cannot be explained by methodological differences between Chinese and Western studies alone. However this possibility cannot be ruled out, as the Chinese data have not been verified yet by other studies67. Similar findings have been observed in a study in Mississippi, where black men and women have PD prevalence rates more like white men and women than black men and women in Nigeria79, 80. These data suggests that risk of developing PD is more a function of environmental factors than racial ones. So different distributions of PD causing factors across populations may contribute to geographic differences in epidemiological findings. These factors could be differences in exposure to causative and protective influences, but also genetic differences in susceptibility to disease. It could be that an environmental agent might only act in genetically susceptible people.

Gender differencesMales tend to have a modestly increased age-adjusted PD prevalence. Male-to-female ratio range from 0.86 (in Japan) to 3.7 (Chinese studies). In a study in New York, age-adjusted prevalence was lower for women compared with men across all ethnic groups. However in another study, age-adjusted incidence did not differ between men and women in all ethnic groups60. This is confirmed by the Rotterdam Study in which no significant gender differences in prevalence were found16.

Symptoms

Motor symptomsThe classic triad of symptoms of PD consists of tremor, rigidity and hypokinesia. Postural impairment has been called the fourth major symptom of PD. The disease is a slowly progressive disorder and signs and symptoms develop usually over several years. In the early stages of the disease the signs and symptoms may be vague and non-specific in such a way that a reliable diagnosis can not yet be made.The typical parkinsonian tremor is a 3-6-Hz distal resting tremor. It consists of alternate contractions of agonist and antagonist muscles, including flexors, extensors, pronators and

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supinators of the wrists and arms during rest. This may result in a pill rolling movement of the hand. Often the typical parkinsonian rest-tremor starts on one side of the body. In most patients the signs will develop in due course on both sides, but asymmetry will usually persist throughout the disease. The legs or lower jaw may also be involved. The tremor tends to disappear with action. Resting tremor is found in 79% - 90% of patients with PD in clinical studies and in 76% - 100 % in autopsy-proven studies. Some patients have little or no resting tremor but a predominant action or postural tremor. Tremor at rest may also be induced by neuroleptic agents.Rigidity can be present in all four limbs and in the trunk, but mainly affects the arms and is often of the cogwheel type. The increase in tone is fairly equal in flexors and extensors, but slightly more in flexors. It can be diagnosed in 89% - 99% of parkinsonian patients.Bradykinesia means slowness of movements, whereas hypokinesia stands for poverty of movement. Hypokinetic features include facial hypomimia, reduced eye blinking, hypophonic speech and micrographia. There is difficulty in initiating movements, resulting in start hesitation. Rapid repetitive movements are impaired. Bradykinesia is present in 77% to 98% of patients with PD, but it is not unique to PD. It can also occur as a result of other extrapyramidal disorders, such as PSP, MSA, CBD and normal aging. Symptoms can be gravitated by contralateral activation or concentration on mental or physical tasks. Each one of these features can be present for a long time, before others develop. Symptoms usually begin unilaterally or asymmetrical. Later they are bilateral or generalised.Gait is impaired in patients with PD as well. The patients walk slowly with small shuffling steps. Parkinsonian patients move in a rigid manner and turn en bloc. Their posture is stooped, because of flexion of the shoulders, neck and trunk. In PD the center of gravity is shifted forward. Walking can be hampered by stutter steps, resulting in start- and turn-hesitation, and sudden “freezing”. This phenomenon refers to the patient’s feet stuck to the ground while walking, rendering the patient unable to move with the lower body. It especially happens on turns and in elevators or doorways. Freezing and related phenomena are called motor blocks. It occurs in 32% of parkinsonian patients31. Retropulsion refers to the phenomenon that the standing patient if pushed backward, is only able to regain his balance slowly by small and slow steps or even fails to do so and falls. When walking, patients may have problems stopping and legs are preceded by the flexed trunk, resulting in frequent little short steps or propulsive gait. This pattern is characteristic of advanced PD. In the beginning of the disease symptoms are unilateral, affecting only one side. Patients appear to drag a leg when walking and arm swing is decreased at the affected side. In a later stage, when the opposite side is affected as well, steps are short and the feet barely clear the floor. Usually symptoms stay asymmetrical, in contrast to normal aging. In a later stage parkinsonian patients may experience problems of autonomic dysfunction such as constipation, incontinence, hypotension and impotence. If this occurs in an early stage of PD, the diagnosis should be questioned28 .


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Not only motor symptoms may occur. Another abnormality is for example an olfactory disorder. In PD there is an increase of the olfactory detection threshold70, which is probably due to the presence of Lewy bodies in the olfactory bulb and neuronal loss in the anterior olfactory nucleus14, 69. Other features include increased saliva production and increased sweating. None of the 3 major symptoms has enough sensitivity or specificity to diagnose PD. For this reason a scheme has been made for diagnostic classification: The UK Parkinson’s Disease Society Brain Bank clinical diagnostic criteria (see table 1: UK Parkinson’s Disease Society Brain Bank 1993)15.

Table 1

STEP 1 DIAGNOSIS OF PARKINSONIAN SYNDROME* bradykinesia (slowness of initiation of voluntary movement with progressive reduction in speed and amplitude of repetitive actions)* and at least one of the following: - 4 - 6 Hz rest tremor- rigidity- postural instability not caused by primary visual, vestibular, cerebellar or proprioceptive dysfunction STEP 2 EXCLUSION CRITERIA FOR PARKINSON’S DISEASE* history of repeated strokes with stepwise progression of parkinsonian features* history of repeated head injury* history of definite encephalitis* oculogyric crises* neuroleptic treatment at onset of symptoms* more than one affected relative* sustained remission* strictly unilateral features after 3 years* supranuclear gaze palsy* cerebellar signs* early severe autonomic involvement* early severe dementia with disturbances of memory, language and praxis* Babinski sign* presence of cerebral tumour or communicating hydrocephalus on CT scan* negative response to large doses of levodopa (if malabsorption excluded)* MPTP exposureSTEP 3 SUPPORTIVE PROSPECTIVE POSITIVE CRITERIA FOR PARKINSON’S DISEASE(three or more required for diagnosis of definite Parkinson’s disease)* unilateral onset* rest tremor present* progressive disorder* persistent asymmetry affecting side of onset most* excellent response (70 - 100%) to levodopa* severe levodopa-induced chorea* levodopa response for 5 years or more* clinical course of 10 years or more

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Non-motor symptomsIn PD there are several other nonmotor clinical features, like dementia, depression and psychotic features.

Dementia In 20 to 40% of patients with PD cognitive impairment develops and the risk of dementia in non-demented PD patients is almost twice that of age-matched non-demented elderly controls. Prevalence however is estimated to be 10.9%61. Here it needs to be considered that PD patients with dementia have a shorter life expectancy. Dementia in PD is characterized by a severe dysexecutive syndrome without instrumental disorders like aphasia, apraxia or agnosia. In several studies the influence of age at onset on the presentation and course of PD has been demonstrated. Young onset patients have little cognitive impairment even after a disease duration of over 20 years73. Dementia mainly develops in patients with onset after 70 years. In this cohort of patients prevalence of dementia is more than twice that of younger patients61. Usually it develops after several years of disease duration. This in contrast to diffuse Lewy body disease, where cognitive deficits occur very early (less than 2 years) or even precede motor symptoms19. Other risk factors for developing dementia in PD are lack of education and severe motor deficits (UPDRS motor scores above 24)56. Cognitive deficits however are usually less prominent than motor symptoms.

DepressionDepression is estimated to occur in 30 - 60% of patients with PD at some point during the disease. It has been suggested that there is a natural tendency for chronic, disabling diseases to induce depression. However it appears that the prevalence of depression in patients with PD is higher than in other chronic disorders. Besides, the depression is unrelated to the severity of motor symptoms and depression can continue despite improvement in motor symptoms after L-dopa therapy. It is possible that depression precedes the motor symptoms of PD. There are some similarities between clinical and biochemical changes in PD and depression. Clinical similarities include akinesia and psychomotor retardation, while biochemical similarities include dysfunction in dopaminergic, noradrenergic and serotonergic systems. Reduced serotonergic function is associated with psychomotor retardation, reduced noradrenergic function with bradyphrenia and reduced dopaminergic function with extrapyramidal symptoms, cognitive slowing and more severe symptoms of depression51.


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Psychotic featuresPsychotic symptoms may either be a manifestation of the disease itself, or it may be the result of therapy with dopaminergic agents25. Psychosis may also occur as a reaction to the disease and functional impairment (“reactive psychosis”) as well, although this is probably a rare condition. About 30% of parkinsonian patients, treated with levodopa, have experienced psychotic symptoms and the lifetime prevalence may approach 50%. Visual hallucinations are most common and the images are usually fully formed human or animal figures. Usually insight is preserved2.

Pathology findings

The most prominent lesion in PD is the degeneration of neuromelanin-containing neurons in the pars compacta of the substantia nigra, which at post-mortem inspection turns visibly pale. Also selected aminergic brain-stem nuclei (catecholaminergic and serotoninergic), the cholinergic nucleus basalis of Meynert, hypothalamic neurons, small cortical neurons (particularly in the cingulate gyrus and entorhinal cortex), olfactory bulb, sympathetic ganglia and parasympathetic neurons may be involved in this progressive degenerative process. Not all dopaminergic neurons are equally susceptible. Within the substantia nigra pars compacta, neuronal loss appears to be greatest in the ventrolateral part, followed by the medial part and the dorsal part24. This has been confirmed by Damier and coworkers13. It results in a regional loss of striatal dopamine50. The nigrostriatal dopaminergic neurons which project to the putamen are more affected than those which project to the caudate nucleus and nucleus accumbens. The latter one is believed to be responsible for akinesia and rigidity. This pattern of cell loss seems to be rather unique to Parkinson’s disease and is different of the pattern seen in normal aging. Neuronal loss of the medial nigral cells, with enhanced involvement of projections to the caudate nucleus, could result in more cognitive symptoms. Another possible clinical-pathological correlation may be based on degenerative changes of the olfactory bulb, causing anosmia. Autonomic dysfunction may be the result of lesions in the sympathetic and parasympathetic ganglia or degeneration in the intermediolateral columns of the spinal cord75. Some believe that dementia in PD may be the result of cell loss in the nucleus basalis of Meynert77.The surviving, but dying catecholaminergic neurons may contain Lewy bodies, an important pathological feature. Lewy bodies are spherical, eosinophylic cytoplasmic inclusions with a dense core and peripheral halos, found in pigmented cells. In PD the most important anatomical sites where these bodies are located are the substantia nigra and locus ceruleus. They are not specific to PD. They appear as well in some other neurodegenerative

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disorders, like Alzheimer’s disease (AD). AD however is associated with cortical Lewy bodies, particularly in frontal, temporal, anterior cingulate and insular regions, whereas PD is associated with subcortical Lewy bodies, mainly in the substantia nigra. Lewy bodies are also seen as an incidental finding in about 10% of people older than 60 years. Gibb and Lees30 discovered that the age-specific prevalence of Lewy bodies in the brains of persons without clinical PD increased from 3.8 to 12.8% between the sixth and ninth decade of life. It has been suggested that the presence of incidental Lewy-bodies constitutes actually a presymptomatic stage of PD30. McKeits however suggests a relationship between this “incidental” pathology and dementia with Lewy bodies62.The mechanisms of cell death in PD are still unknown. Several factors have been mentioned to play a role in neuronal degeneration in PD, like mitochondrial dysfunction, oxidative stress and excitotoxity and free radical production. It is believed that the neuronal death in the pars compacta of the substantia nigra is apoptotic6, 7, but this has not been universally accepted yet and necrosis has been suggested as well.

Other movement disorders in the elderly

Aging is a unequivocal risk factor for PD87. It is also a major risk factor for several other movement disorders and neurodegenerative diseases.There are many causes of movement or gait disorders in the elderly, of which some can easily be mistaken for PD. The many characteristics and patterns of gait disorders may look difficult and require special expertise, but simple observation of the patient and its gait may yield valuable information. The history from patients may reveal a stepwise progression, suggesting vascular disease. Pain with walking usually excludes a neurodegenerative cause of the gait disorder. Magnetic Resonance Imaging (MRI) may be used for screening for hydrocephalus or multiple infarcts. Positron emission tomography (PET) scans of the brain may for example help for diagnosing PD, MSA or PSP. Gait disorders in the elderly, irrespective their underlying pathology, contribute to the risk of falling and fractures89. The higher risk of falling limits the elderly in their mobility and independence, also because of the fear of falling itself. Imms and Edholm surveyed in 1981 a group of older people (mean age, 78 years) and found that half of them limited their activity because of their concerns about mobility40. Many signs of gait disorders, slowing down and stiffening up, are accepted as being part of aging. However most of these signs may be signals of an underlying disease. Critchley already warned in 1931 that “an abnormal gait in the aged is frequently the result of disease outside the nervous system” (Critchley, 1931). In many cases gait disorders are of orthopedic (osteoarthrosis, osteomalacia, unsuspected fractures), endocrinological


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(hypothyroidism), psychological (depressive state, fear of falling) or general (general muscle weakness) origin11. Circulatory or respiratory systems may play a role as well in determining gait velocity because of the need to minimize energy expenditure23. The incidence of subtle extrapyramidal signs on neurological examination of elderly persons with no known neurologic or psychiatric disorder is high. Bennett et al4 showed that 35% of people over 65 years old had these subtle changes, while around 3% in similarly aged persons had PD. In a community study in North Carolina, 15% of adults over 60 years of age had some degree of difficulty with ambulation18. In Western Europe 20-25% of people aged 80 or older, use mechanical aids for walking55. A review of the variety of conditions, which can cause gait disorders or may be confused with PD, will be given here.

Essential tremor (ET)One of the commonest causes of misdiagnosis of PD is essential tremor (also known as senile tremor, which is a misnomer). It is inherited as an autosomal dominant disorder with incomplete penetrance. A familial incidence is common in about one-third to one-half of cases. In the elderly however it is often sporadic32. The incidence of essential tremor increases with climbing age. Considering prevalence Rautakorpi’s study76 reported a prevalence of 12.5% in his population. In a study of a population aged 65 and older, Louis et al53 found a prevalence of 4%. A higher prevalence of 23% in a population of people older than 70 years has been observed by Elble22. Essential tremor is an action tremor with a frequency of 8- to 12-Hz (the resttremor in PD is 4- to 6-Hz). It may sometimes be present at rest as well, but generally increases with activity. The amplitude increases with age. According to Marttila and Rinne essential tremor accounted for 26% of cases of presumed PD58. This misdiagnosing can occur because of many reasons. Both conditions frequently occur in the elderly. Parkinsonian patients sometimes suffer from a postural action tremor of the hands, as seen in ET. Patients with ET may have some bradykinesia and rigidity, which could be normal in the elderly. Besides, some hallmarks of PD, like asymmetric manifestation and a resting component of the tremor, can also be found in cases of ET. However, there are also some striking differences between these two disorders. Essential tremor frequently involves the head in contrast to tremor in PD. Conversely, a resttremor of the leg or slow vertical jaw tremor is often seen in PD, but rarely in essential tremor. Also, hypokinesia may be present in PD, but not in ET.

Vascular parkinsonismThis results from multiple small cerebral infarcts, especially in the basal ganglia secondary to hypertensive cerebrovascular disease. Some patients with vascular parkinsonism present with a progressive gait disorder without a medical history of strokes83. Chronic

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hypertension leads to fibrinoid necrosis and occlusion of arterioles supplying the basal ganglia. An akinetic rigid syndrome with urinary incontinence, dysarthria, abulia and dementia may occur. The extrapyramidal signs may coexist with pyramidal dysfunction like weakness, hyperreflexia, spasticity, pseudobulbar palsy, emotional lability and Babinski signs. These pyramidal signs may be important in differentiating this disorder from PD. The patient is slow and walks wide-based (unlike in PD) with short, shuffling, somewhat irregular footsteps. Postural stability may be impaired. As in PD freezing and start hesitation may occur. However the upper body may show little or no parkinsonian features, thus no facial hypomimia may occur. Another term used for this disorder is lower body parkinsonism. Another striking difference with PD is usually the absence of tremor and no fatigue or decrement of rapid alternating movements. In vascular parkinsonism symptoms are symmetric in contrast to PD. However, if signs and symptoms are not clear cut, the differential diagnosis may be difficult.Computerised tomography shows multiple infarcts but sometimes misses small ones. Magnetic resonance imaging demonstrates infarctions in the deep gray matter structures and ischemic changes in periventricular white matter. According to Sudarsky, vascular parkinsonism accounts for 15 - 16% of the gait disorders among elderly patients83, 84.

Multiple system atrophy (MSA)MSA refers to a sporadic, gradually progressive, idiopathic neurodegenerative process of adult onset characterized by varying proportions of cerebellar dysfunction, autonomic failure, pyramidal signs and parkinsonism, that is poorly responsive to L-dopa therapy. Cell loss and gliosis (without Lewy bodies) are not only present in substantia nigra, but also in multiple other structures, like striatum, olives, pons, cerebellum, intermediolateral cell columns and Onuf’s nucleus in the spinal cord. Most commonly MSA begins in the early 50s, progresses more rapidly than PD and has a reduced life expectancy (median survival = 9.3 years), this in contrast to PD (see above). Parkinsonism occurs in 90% of patients with multiple system atrophy and is the dominant motor disorder is 80% of patients. In contrast to PD, the parkinsonism in MSA is usually bilateral. Unlike PD, tremor is often not the classical rest tremor, but an action tremor. Cerebellar dysfunction and pyramidal signs both occur in about half of patients71, 90. Cerebellar dysfunction as the dominant symptom occurs in about 20% of patients74. Autonomic failure appears in almost all patients with MSA. In PD patients it may occur as well, mostly in a late stage of disease, whereas in MSA it usually occurs earlier and more severe. Indeed, in MSA, autonomic failure may precede the motor symptoms by months or even years. Frequently, the first symptom is impotence in men and incontinence in both men and women (PD usually causes just frequency increase and urgency due to


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hyperreflexia of the detrusor). Postural hypotension is another common feature in MSA, which may appear in PD as well, though less severe. Inspiratory stridor is present in about 30% of patients with MSA. When combined with parkinsonism it is highly suggestive of the diagnosis MSA72. Other signs of autonomic failure are thermoregulation disturbances, gastro-intestinal problems and phenomena of Raynaud. The response to levodopa of patients with MSA is usually absent or poor. However, in about 30% of patients an initial response has been reported, usually temporary. Levodopa induced dyskinesias are usually absent in MSA, so high doses of levodopa can be administered.Differentiating MSA from PD may be very difficult, especially in an early stage of disease. There are some red flags suggesting non-idiopathic parkinsonism, like:

• Poor or no response to levodopa therapy• Cerebellar, pyramidal or autonomic signs• Symmetric start of symptoms• Absence of classic rest tremor• Early instability or falls• Rapid clinical progression71, 74, 90.

Some diagnostic tests may include external urethral (or anal) sphincter EMG, standard tests of cardiovascular autonomic function and imaging of the brain. Computed tomography or magnetic resonance imaging sometimes shows cerebellar or brainstem atrophy. Fluorodesoxyglucose positron emission tomography (PET) scanning may be useful as well20, 86.

Progressive supranuclear palsy (PSP)PSP, or Steele-Richardson-Olszewski syndrome, is an idiopathic degenerative disease, not uncommon in the elderly, which mimics PD. It occurs at a rate of 0.3 per 100.000 per year34 and its prevalence is 1.46 per 100.000. Pathologic investigation shows cell loss and neurofibrillary tangles, mainly in the brainstem, globus pallidus, nucleus subthalamicus and nucleus dentatus. The clinical picture includes the tetrad of supranuclear gaze paralysis, axial rigidity, dementia and pseudobulbar palsy. It is associated with bradykinesia, severe postural disorder and frequent falls. Supranuclear gaze paralysis affects vertical gaze more than horizontal. Voluntary downgaze is slow and usually incomplete, but when the oculocephalic reflex is performed, full down gaze is obtained. Pseudobulbar palsy is characterised by dysphagia and dysarthria. Dementia is progressive and consists of slowing of cognition, memory deficits and personality changes suggestive of frontal lobe dysfunction52. PSP may be distinguished from PD by the absence of rest tremor, the extended neck posture, rigidity more truncal than in limbs and abnormal eye movements. PSP patients have a stiff, broad-

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based gait with ataxia. In contrast to PD patients they do not turn en bloc, but pivot. During pivoting they also tend to fall backwards.

Cortico-basal degeneration (CBD)This disorder presents with a unique pattern of progressive impairment. It appears in mid- to late adult life, usually beginning after age 60. The duration of the illness until death is about 6 to 8 years. Pathologic and histologic evaluation reveal frontoparietal atrophy and neuronal loss, gliosis and swelling of the cell body with resistance to staining methods (achromasia)45. Prototypic findings are combined parkinsonian signs, other movement disorders and higher cortical dysfunction, with marked asymmetry of involvement. The most common extrapyramidal sign is rigidity, followed by bradykinesia. Sometimes tremor is present as well, but does not resemble the parkinsonian tremor. Tremor in CBD is more rapid (6-8Hz), is mainly during action, with varying amplitude. Other movement disorders include myoclonus and dystonia. Higher cortical dysfunction includes dyspraxia, involving the limbs, ocular and orofacial muscles, cortical sensory loss, dementia (which usually occurs late in disease) and aphasia. A striking feature of CBD is the “alien hand/limb” phenomenon81.

Normal pressure hydrocephalus (NPH)This disorder is often idiopathic, but has also been associated with many neurological diseases. The classic triad of symptoms includes frontal dementia, urinary incontinence and gait disorder with unsteadiness. It is associated with enlargement of the cerebral ventricles on CT or MRI and a CSF pressure of 180 mm H2O or less. A dynamic test is necessary to confirm the diagnosis of true (opposed to ex vacuo) hydrocephalus5. The removal of 50 ml of cerebrospinal fluid may improve the symptoms of gait disorder85. Mental dysfunction improves less than gait after a shunt. The patient walks wide-based with small steps, feet glued to the floor, marked imbalance and difficulty initiating walking. Postural instability with frequent falling may occur. Clinical signs may include hyperreflexia, extensor plantar responses and extrapyramidal signs, including hypokinesia and freezing during walking. Diagnosing NPH may be difficult since the three cardinal symptoms are common in the elderly. Besides gait disorder may precede other symptoms for several years and can be the only symptom for a long period. NPH is a common cause of gait disorders in the elderly, while in dementia it accounts for only 0 - 5% of persons with dementia26. It should account for 4 - 6.7% of the gait disorders in the elderly84, 85.


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Metabolic and endocrine disordersSome of these disorders may produce akinetic-rigid syndromes. Hypothyroidism may cause parkinsonism with motor slowing. Hypoparathyroidism, resulting in calcifications of the basal ganglia, may produce a clinical syndrome consisting of parkinsonism, chorea, cerebellar dysfunction or a mixed extrapyramidal-pyramidal syndrome, resembling the lacunar state48. Metabolic and toxic disorders causes gait disorders in 2,5 % of the elderly83, 84.

Drug-induced parkinsonismMany drugs commonly prescribed in the geriatric practice can affect gait. It can be caused by:

• drugs that deplete presynaptic dopamine stores, like reserpine or tetrabenazine• neuroleptic drugs, like phenothiazines (chlorpromazine), butyrophenones

(haloperidol), thioxanthines (flupenthixol) and substituted benzamides (sulpiride) • metoclopramide for gastrointestinal symptoms or migraine• the atypical calcium blocking drug cinnarizine and flunarizine for vestibular

disorders or hypertension• others, like fluoxetine and rarely valproate

Drug-induced parkinsonism results in bradykinesia and rigidity with facial amimia, dysarthria and diminished or disappeared arm swing. Tremor is less common, but can be identical to the classic rest tremor of PD. Moreover symptoms of drug-induced parkinsonism usually are, just like in PD, asymmetrical. Drug-induced parkinsonism often resolves quickly within weeks after stopping those drugs. Sometimes this will take months, especially after depot neuroleptic medications.

Subclassification of PD

The variance in expression of the clinical syndrome of PD is large and suggests the existence of subtypes with distinct clinical patterns, especially concerning the age of symptom onset, rate of disease progression and clinical manifestations. Probably this clinical heterogeneity reflects a broad spectrum of manifestations of one pathological disorder: the loss of pigmented neurons in the substantia nigra pars compacta and the presence of Lewy bodies (intracytoplasmic inclusions). Several studies have tried to define clinical subgroups on the basis of distinguishing features, like family history of PD, variable progression and age of onset of symptoms of disease.

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Young-onset versus old -onset of PDFriedman has compared clinical expression of patients with onset age of symptoms above 70 years with patients with onset age below 45 years27. In this study there were some striking differences between these groups. The DATATOP (deprenyl and tocopherol antioxidative therapy of parkinsonism)- study has investigated the variability in clinical expression by comparing several factors, like early versus late onset of disease, benign versus malign status, H/Y stage I versus H/Y stage II and tremor versus postural instability and gait disorder (PIGD) type41. This study suggests that there are probably 2 different subtypes of PD: early-onset and late-onset of disease. Patients with early onset of PD progress at a slower rate, which has been confirmed by others33. Patients with young-onset of disease are more sensitive to levodopa and to levodopa-induced dyskinesias, and dyskinesias seem to appear earlier in those patients. Not only will they develop focal dystonia early in the course of illness, but also end-of-dose phenomenon is more often observed in patients with young-onset of disease29, 73. Similarly, motor fluctuations, such as the wearing-off effect, tend to occur earlier in young-onset patients47. Initial symptom in this group is usually tremor. This is in contrast to Friedmans results27 who found that young-onset patients usually start with paraesthesia and have bradykinesia as dominant symptom. They tend to perform better on neuropsychological tests, but this may be explained by their younger age. Patients with late-onset of disease have a more aggressive form of PD with faster progression and greater motor disability. They have as initial symptoms bradykinesia, postural instability, rigidity and less often tremor, according to the DATATOP-study. This study also suggests that motor deterioration in PD does not necessarily parallel cognitive decline and it is postulated that cognitive impairment in PD results mainly from a nondopaminergic deficit. According to Friedman27 old-onset patients with PD more often have tremor as both presenting and dominant symptom. Old-onset parkinsonian patients more frequently develop psychotic complications and less frequent dyskinesia as complication of levodopa treatment than young-onset ones. Friedman found a striking difference in symptomatology of psychotic complications. Whereas old-onset patients used to have simple, mostly visual hallucinations with preserved insight, young-onset ones tend to develop paranoid behaviour without preserved insight. The number of young-onset patients with psychotic complications however was very low in this study. There was no striking difference in overall functioning based on activities of daily living (ADL) between those two groups. According to Friedman27, the lesion in young-onset PD concerns predominantly the dopaminergic system. This monosystemic lesion may explain the greater susceptibility to dyskinesia (and the fact that bradykinesia is the dominant symptom of this disease). Lower susceptibility to dyskinesia in old-onset parkinsonian patients could be the result of age-related decline in the number of dopaminergic receptors in striatum. In contrast old-onset patients are more susceptible to developing psychotic complications, probably due to more widespread lesions of the


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central nervous system resulting from ageing. Older people are in general more likely to develop psychotic reactions to various external stimuli, e.g. infections, intoxications, etc 46. Age-related brain atrophy involving also other neurotransmitter systems is probably the reason for the different symptoms in PD between old-onset and young-onset patients27. Some studies have observed a higher frequency of a first-degree relative with PD in young-onset patients3, 82, although other studies found no difference in the number of affected relatives among young- and old-onset cases29, 73. Others have noted an increased exposure to farming and rural living in patients with young-onset of disease. Despite the differences in clinical manifestation, the cellular morphology and frequency of Lewy bodies in the substantia nigra are identical in young-and old-onset cases29.

Other subtypes of PDSeveral other studies have investigated the existence of possible subgroups of PD, including young onset, tremor dominant, postural instability/gait disorder predominant, benign and malignant forms. Comparing tremor-dominant versus postural instability/gait disorder-dominant PD (PIGD), greater motor disability has been found in the latter group. Patients with tremor as dominant symptom usually did not only have more severe tremor at rest, but also more severe action- and postural tremor. PIGD patients had, in addition to their greater postural and gait difficulties, more severe bradykinesia and rigidity41. Some authors have divided patients into benign or malignant groups based on duration of symptoms and stage of disease. Patients were arbitrarily considered to have a benign form of PD if they were Hoehn and Yahr stage 2 or less, combined with parkinsonian symptoms for 4 years or less. In contrast, patients with malignant PD were those with early postural imbalance, while disease duration is less than 1 year. Patients with a benign form of PD usually were younger at onset of disease and had tremor as dominant symptom. Indeed, benign tremulous parkinsonism is considered to be a well-recognized subgroup of patients with a relatively nonprogressive, long-term course of disease. The benign group had an earlier onset than the malignant one and the latter one performed slightly worse on the Mini Mental State Examination, although this difference was not statistically significant after adjusting for age. To distinguish the benign form from the malignant one, other factors may be important as well, like response to medication, genetic factors and cognitive impairment82. Hely et al suggest that patients with a benign form of PD have mild dyskinesia, if present, and that none of these patients had dementia or hallucinations. All of them are responsive to drugs37.Graham and Sagar have investigated the heterogeneity in PD, using a data-driven approach36. They suggested the existence of three distinct subtypes:

• a motor only subtype, without intellectual impairment• a motor and cognitive subtype• a rapid progressive subtype

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The latter group is characterized by an older age of disease onset than the other subtypes, rapidly progressive motor and cognitive disability and more orthostasis. Because of the rapid progression patients belonging to this subtype have a shorter life expectancy than the other groups. This is confirmed by Louis et al54, who found that mortality increases with climbing of the years, the development of dementia and the severity of motor disability.Gender may be associated with a different progression of PD. According to the Sydney multicentre study of Parkinson’s Disease, dyskinesia develops earlier in women during the first 10 years of disease. After 10 years there is no significant difference in the prevalence of dyskinesia, but women tend to have a higher score in Hoehn and Yahr stage37.

Brain metabolism in aging and Parkinson’s Disease

During development and aging, the brain will change anatomically and physiologically to support the behaviour of normal adults. Chugani et al8 have investigated the maturation of the brain during the first years of life by using positron emission tomography (PET) and 2-deoxy-[18F]fluoro-D-glucose (FDG). They discovered that in the first 4 weeks of life (the neonatal period), the most important region of metabolic activity is the primary sensorimotor area, while high activity was also found in the thalamus, brainstem and cerebellar vermis. The second postnatal month a small increase in metabolism in calcarine and temporal cortices can be seen. Approximately 3 months after birth, considerable rises in metabolic activity in the anterior parietal, temporal and calcarine cortices as well as in basal ganglia and cerebellar cortex were found. By 1 year, the metabolic pattern resembled that in adults, but absolute values were lower than adult ones. Adult rates were reached by 2 years of age. Metabolic rates continued to rise until 3 - 4 years, so values exceeded those of adults by a factor of approximately 2. These high values were maintained until age 8 - 9. At this age, they begin to decline and by the end of the second decade adult values are reached. The highest increases in metabolic activity were observed in the cerebral cortices8.Aging is associated with the degeneration of specific neural systems. Normal aging is predominantly characterized by metabolic changes in the prefrontal cortex. By using FDG-PET scans metabolism of these systems can be investigated. Moeller and coworkers65 have tried to explore the metabolic topography of aging. They found various topographic profiles. One was characterized by relative frontal hypometabolism associated with covariate metabolic increases in the parietooccipital association areas, basal ganglia, mid-brain and cerebellum. Another one revealed relative basal ganglia hypermetabolism associated with covariate decreases in frontal premotor cortex65. Mielke et al also found a decline of regional cerebral glucose metabolism in frontal areas63.


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Although the most important lesion in PD is located in the substantia nigra and its dopaminergic projections, lesions of the presynaptic nigrostriatal dopamine system result in widespread abnormalities in regional brain metabolism. To define the metabolic topography of parkinsonism the same strategy as above has been used. According to the results of Eidelberg and coworkers21 the metabolic profile of PD is characterized by increased activity in the lentiform nucleus, thalamus, pons and cerebellum whereas activity was decreased in the lateral frontal, paracentral and parietal association areas. Increase in activity in the lentiform nucleus and thalamus in PD is consistent with experimental animal studies64, 68. Increased metabolism in the ventral thalamus has been confirmed by others10, 68. The subject scores for the metabolic profile in PD correlated with the individual Hoehn and Yahr score and with rigidity and bradykinesia ratings, but not with tremor21,

68. The reproducibility of this unique pattern of regional metabolic covariation in patients with PD has been assessed by Moeller et al66, 68. This topography appears to be highly reproducible across patient populations and tomographs.

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Acta Neurol Scand 92:55-58, 1995

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3Motor behavior correlates with striatal [18F]-DOPA

uptake in MPTP-lesioned primates

S.A. Eshuis, R. Kortekaas, G. Andringa, A. Cools, K.L. Leenders, R.P. Maguire

Submitted

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Abstract

The MPTP-lesioned monkey is a well-known, animal model for Parkinson’s disease (PD). MPTP causes damage to dopaminergic cell groups resulting in motor dysfunction similar to PD. PET scans using [18F]-DOPA are applied to determine presynaptic striatal dopaminergic activity. In a patient with PD, striatal uptake of [18F]-DOPA is decreased and striatal uptake values correlate inversely with motor scores. We have correlated uptake values of [18F]-DOPA to motor impairment in MPTP-lesioned monkeys in various stages of neuronal degeneration. Eight rhesus monkeys received MPTP infusions. Motor signs were rated regularly and correlated to striatal uptake of [18F]-DOPA as measured with PET. MPTP caused the expected parkinsonian motor signs which were accompanied by reduced striatal uptake of [18F]-DOPA. There were significant correlations between the two endpoints.In conclusion, striatal [18F]-DOPA uptake correlates inversely with the severity of motor impairment in MPTP-lesioned non-human primates.

Keywords: MPTP, Parkinson’s disease, primates, [18F]-DOPA, PET, movement disorders

Motor behavior correlates with striatal [18F]-DOPA uptake in MPTP-lesioned primates

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Introduction

Parkinson’s disease (PD) is a movement disorder, associated with degeneration of dopaminergic neurons from the substantia nigra pars compacta. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) destroys monoaminergic neurons. As dopaminergic neurons are especially sensitive to this neurotoxin, MPTP induces depletion of dopamine in the striatum. Brain damage induced by MPTP in monkeys induces many of the behavioural and biochemical features of PD3, 7, 8, 28, 29. Behavioural deficits include akinesia, rigidity and flexed posture and sometimes resting tremor. MPTP-lesioned non-human primates are a widely used animal model for PD3, 5, 10, 24. In vivo measurements with positron emission tomography (PET) or single-photon emission computed tomography (SPECT) in this primate model provide an opportunity to study the pathophysiology and biochemistry of the dopaminergic system in time, in humans as well as in animal models5, 10, 14, 25, 26, 33. [18F]-DOPA (6-[18F]-fluoro-L-3,4-dihydroxyphenylalanine) is a fluoro analogue of the naturally occurring amino acid derivative and dopamine precursor dihydroxyphenylalanine (DOPA). It is metabolised into [18F]-fluoro-dopamine by the enzyme Amino Acid Decarboxylase (AADC). Several human studies have shown that dopaminergic function, measured by [18F]-DOPA PET scans is negatively associated with clinical severity in patients with PD4, 21, 25, 27. Also, the degree of clinical asymmetry is correlated with asymmetry in [18F]-DOPA uptake in the putamen of patients with PD4. [18F]-DOPA uptake is correlated with post mortem determined dopamine cell counts and dopamine levels in humans32. Both SPECT and PET scanning with respectively [123I]-β-CIT ([123I]-2-β-carbomethoxy-3-β-(4-iodophenyl)-tropane, imaging the dopamine transporter) and with [18F]-DOPA or [18F]-FMT (6-[18F]-fluoro-L-m-tyrosine, analogue of L-dopa) have been applied to investigate the dopaminergic system in vivo in MPTP-lesioned monkeys15-18, 28. MPTP-treated monkeys display motor deficits in combination with reduced uptake of the mentioned radiolabeled dopaminergic markers. Some studies have analysed the correlation between neuroimaging measures and behavioural measures in these monkeys. Uptake of [123I]-β-CIT and [18F]-FMT both correlated well with motor symptoms in MPTP-lesioned monkeys16, 17, 19, 28. [18F]-DOPA uptake in the striatum of MPTP-lesioned parkinsonian monkeys is also altered12-14. So far, a systematic analysis of correlation between behaviour and presynaptic dopaminergic imaging has not yet been reported despite the extensive use of [18F]-DOPA in the study of PD.Our aim in this study was to study the correlation between motor symptoms and striatal uptake of [18F]-DOPA PET in MPTP-lesioned monkeys in a quantitative manner.

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Materials and methods

SubjectsA total of eight male, right-handed rhesus monkeys (Macaca mulatta) were studied under an approved protocol that met all institutional guidelines and requirements stated in the ‘Principles of Laboratory Care’ (NIH publication No. 85-23, revised 1985). The monkeys were over the age of 7 years, weighing between 6 and 12 kg and were individually housed in cages under standard conditions. Diet consisted of lab chow supplemented with fruit while water was ad libitum available. The monkeys participated in a drug trial investigating a potentially neuroprotective substance, namely CGP 3466B. See for detailed results2, 30. This study was approved by the local ethics committee for animals of the University Medical Center Groningen, Groningen, The Netherlands.

MPTP treatmentA two-phase bilateral MPTP lesion approach was used to generate bilateral parkinsonism. This treatment induces moderate to severe parkinsonian symptoms with a significant reduction in limb movements and decreased striatal [18F]-DOPA uptake3, 31.Under total anaesthesia a first dose of 2.5 mg MPTP was administered into the carotid artery contralateral to the dominant limb (unilateral stage) and, eight weeks later, a second dose of 1.25 mg MPTP was infused into the other carotid artery (bilateral stage). The second dose was lower to limit incapacitation of the animals. This procedure induces a stable PD model with little recovery6. Four out of the eight monkeys received a potentially neuroprotective agent, the anti-apoptotic compound CGP 3466B (also known as TCH346), at several dose levels for the next fourteen days after the second injection. CGP 3466B blocks glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an enzyme involved in the apoptotic pathway. The other four monkeys received saline. It was assumed that while this compound may retard neurodegeneration in this model, it does not alter the relationship between dopaminergic function and behaviour.

PET proceduresAll monkeys were scanned in the healthy stage (1 week prior to the first MPTP lesion), between the first and the second lesion (7 weeks after the first lesion) and ca 5 weeks after the second lesion.Animals were fasted overnight prior to the PET scan. During handling and transportation they were sedated with 3.1 mg/kg (±)-ketamine (Nimatek, Eurovet, Bladel, the Netherlands) intramuscular (IM), which also functioned as anaesthetic induction (4.7 mg/kg). An intravenous (IV) cannula was positioned into the vena saphena magna for


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administration of drugs and radiotracer. Atropine (0.06-0.09 mg/kg IM; Centrafarm, Etten-Leur, The Netherlands) and sodium pentobarbital (8-11 mg/kg IV; Sanofi, Maassluis, The Netherlands) were administered, after which the trachea was intubated, and the monkey was ventilated with 1-2% (v/v) vaporised isoflurane (Abbott, Kent, UK) in a carrier gas of O2:N2O (50:50). The peripheral decarboxylase inhibitor carbidopa was administered orally (3 mg/kg) 30 minutes before [18F]-DOPA administration.The monkeys’ heads were fixated in a stereotactic frame and positioned centrally in the field of view of a Siemens ECAT Exact HR+ scanner (Siemens, Munich/Erlangen, Germany). This tomograph has a resolution of 5 mm full width at half maximum in the centre of the field of view and an axial field of view of 15.5 cm. A transmission scan with 68Ge was performed for attenuation correction. An average of 98.2 MBq (range: 22 to 167) of [18F]-DOPA in six ml of physiological saline was injected IV over one minute. Scanning was initiated immediately and 21 frames were acquired (10 x 30, 3 x 300, 4 x 600, 4 x 900 sec, totalling 120 minutes). One monkey’s PET scan failed in the unilateral phase due to a technical problem.Heart rate, electrocardiogram, respiration rate and O2 saturation were monitored throughout the experiment.

PET data analysisPET data were reconstructed to a 128x128x63 matrix with a plane separation of 0.2425 cm and a bin size of 0.2250 cm.Using the Clinical Applications Programming Package (Siemens, Munich/Erlangen, Germany), regions of interest (ROIs) were placed by hand on PET data collected from 60 – 120 minutes. Elliptical ROIs were placed over the left and right striatum, and as a reference, on the posterior half of the brain. The reference region included both cortical and white matter and was of a deliberately large size to minimise intersubject variability in placement. The rate of specific uptake into the striatum (Kref values) relating tracer uptake to the reference brain region, was calculated9. In our case, instead of the occipital lobe, the back of brain is used as a reference region. Mean striatal uptake was calculated as the average of left striatal uptake and right striatal uptake.

Assessment of motor symptomsMonkeys were observed and videotaped for 1 hour each test day: at 10 and 3 days prior to the first MPTP treatment (naive stage), 6 and 7 weeks after the first MPTP treatment (unilateral stage) and 3, 7, 14, 21, 28 and 35 days after the second MPTP treatment (bilateral stage). Given the lack of significant differences, the several time points in each stage were averaged per stage.

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A single, trained, ‘blind’ investigator (GA) evaluated motor symptoms using two different rating scales. The first scale was a qualitative assessment of parkinsonian symptoms using a rating scale for non-human primates23, with minor modifications for bilaterally treated MPTP monkeys31, 34. This clinically oriented rating scale included the items: tremor, gait, akinesia/bradykinesia, balance, rigidity/posture and food intake. The total parkinsonian symptoms score was obtained as the sum of all items.The second rating scale was a quantitative assessment of goal-directed limb movements3,

34. Decreased values indicate increased parkinsonian signs. Lateralised limb movements consisted of the collection of all limb movements on either of both body halves.

StatisticsFor the statistical analysis, the three measurements in the eight subjects were treated as independent because there had always been a new lesion between any two measurements, thereby violating the assumption of dependence within subjects. Because one PET measurement was missing, this resulted in a total of 23 observations. Because data were not normally distributed according to the Shapiro Wilk test, we calculated Spearman’s rho, two-tailed. A p value < 0.05 was considered significant.

Results

[18F]-DOPA uptakeBefore injection of MPTP, mean [18F]-DOPA uptake for left and right striatum was 0.00208 ±0.00025 (Kref ± st.dev., see table 1). An example of a transaxial slice through the striatum is shown in figure 1.Unilateral administration of MPTP resulted in a significant decrease of left [18F]-DOPA striatal uptake, see table 1 (p = 0.01; paired t-test, two-tailed). In contrast, [18F]-DOPA uptake in the right striatum was increased, but not significant (p = 0.16; paired t-test, two-tailed). After administration of MPTP into the right carotid artery, [18F]-DOPA uptake in the right striatum was decreased in the four monkeys receiving saline, although not as much as after the first application of MPTP. [18F]-DOPA uptake in the right striatum stayed within the normal range in the CGP 3466B treated group. No effect of the second application of MPTP was noticed on [18F]-DOPA uptake in the left striatum.


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Figure 1: Transaxial slice through the striatum in the non-lesioned state.

Table 1: Striatal uptake of [18F]-DOPA (mean of all monkeys ± standard deviation) for the three experimental stages. F-DOPA uptake (Kref) in mean of all monkeys ± standard deviation Lesion left right meanNone 0.00211 ± 0.00039 0.00204 ± 0.00025 0.00208± 0.00025 Left 0.00055 ± 0.00097 0.00271 ± 0.00098 0.00163± 0.00059Bilateral 0.00035 ± 0.00041 0.00175 ± 0.00130 0.00118± 0.00066

Mean uptake is the average of left and right uptake

Motor scoresBefore injection of MPTP, all monkeys displayed normal motor behaviour. No signs of parkinsonism were detected and likewise mean parkinsonian symptoms scores were 0. Monkeys spent more time on right than on left limb movements (see table 2).Administration of MPTP into the left carotid artery induced right-sided parkinsonism in all monkeys as described previously2, resulting in an increase of mean parkinsonian symptoms scores. The amount of limb movements on the right body half decreased, while the amount of left sided limb movements remained in the same range as in the non-lesioned stage.In the saline treated group the second infusion of MPTP into the right carotid artery induced parkinsonism on the left side of the body, thus increasing the mean parkinsonian symptoms scores. In the four monkeys receiving CGP 3466B, no parkinsonism developed on the left side of the body, and mean parkinsonian symptoms scores remained unchanged. These effects have been described in detail elsewhere1, 2. The severity of parkinsonian signs on the right body side was unaffected by the second MPTP treatment. Time spent on left limb movements was reduced in the saline group, but not in the CGP 3466B treated group. Also, no effect was noticed of the second infusion of MPTP on right limb movements.

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Table 2: Overview of behavioural data (mean ± standard deviation). Limb movements Total parkinsonian symptomsleft right

Lesion Mean ± sd Mean ± sd Mean ± sd None 10.28 ±5.7 18.18 ±9.7 0 ± 0 Left 10.11 ±4.7 3.94 ±3.4 1.69 ± 1.9 Bilateral 6.26 ±6.0 3.76 ±2.5 9.87 ± 5.9

The higher the value of parkinsonian symptoms, the worse the condition. Concerning the limb movements: the higher the value, the better the condition.Sd = standard deviation;

Correlation between motor symptoms and [18F]-DOPA uptakeMean parkinsonian symptoms scores were significantly correlated with mean striatal uptake of [18F]-DOPA, according to Spearman’s rho (see figure 1). A significant relationship was also found between left striatal [18F]-DOPA uptake and right limb movements (see figure 2) and between right striatal [18F]-DOPA uptake and left limb movements (see figure 3 and table 3).

Figure 2: Correlation between mean striatal [18F]-DOPA uptake (Kref) and total parkinsonian symptoms

scores.

　

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Figure 3: Correlation between left striatal [18F]-DOPA uptake (Kref) and right limb movements.

Table 3: Correlation between striatal [18F]-DOPA uptake (Kref) and behavioural data. Observations were pooled over all experimental stages.

ρ (p)Mean striatal [18F]-DOPA uptake -0.521 (0.01)vs. total parkinsonian symptoms scoresLeft striatal [18F]-DOPA uptake 0.499 (0.05)vs. right limb movementsRight striatal [18F]-DOPA uptake 0.418 (0.05)vs. left limb movements

ρ = Spearman’s Rho

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Figure 4: Correlation between right striatal [18F]-DOPA uptake (Kref) and left limb movements.

Discussion

This study attempted to correlate motor symptoms with striatal [18F]-DOPA uptake in the MPTP lesioned monkey in a systematic and quantitative fashion.We found significant correlations between: (i) total parkinsonian symptoms scores and mean striatal [18F]-DOPA uptake; (ii) lateralised limb movements and contralateral [18F]-DOPA uptake in MPTP lesioned monkeys. This finding of significant correlations between motor scores and dopamine function in MPTP-lesioned monkeys is in agreement with previous studies using [123I]-β-CIT SPECT16 and [18F]-FMT PET17, 18. In the non-lesioned phase we found a mean Kref of 0.002. This is in the same range as the results of others11, 22. Differences of absolute or relative striatal tracer uptake values between our data and those of others may be explained by different doses of premedication given, or different time points of data collection and ROI size20. Some of our Kref values were negative. As Kref is a random variable, the ‘spot estimate’ can be negative if the true Kref is 0 or very low.Because all monkeys were right-handed, right limb movements were higher than left limb movements in the naïve phase. In the bilateral phase [18F]-DOPA uptake in the right striatum was decreased in the four monkeys receiving saline, although not as much as after the first application of MPTP into the left carotid artery. This can be explained by the lower dose given the second time.

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A wide range in motor dysfunction and [18F]-DOPA uptake is advantageous for a regression analysis between these parameters. The two different treatment regimes of the monkeys resulted in a different degree of the MPTP lesions and therefore, different decreases in striatal [18F]-DOPA uptake and different degrees of parkinsonian motor signs. Besides, severity of parkinsonism after the first lesion was variable between monkeys, suggesting large variation in individual susceptibility to the neurotoxic effects of MPTP. As time progressed, more variability occurred in motor symptoms scores and [18F]-DOPA uptake, partly being induced by the administration of CGP 3466B to a subset of monkeys. As we have shown previously2, administration of CGP 3466B may influence the degree of nigral damage after injection of MPTP. Thus the CGP 3466B treatments increase the range of PET and behavioural parameters. By those treatment regimes, we were able to reach a wide range in motor dysfunction and [18F]-DOPA uptake.We assumed that CGP 3466B does not alter the relationship between striatal dopaminergic uptake and behaviour, as both are altered in a consistent manner. This is supported by the finding that the correlations between motor scores and striatal uptake were similar in all treatment groups. However, this assumption has not been tested by experimental data and a possible effect of CGP 3466 B on the mutual relation between the two parameters cannot completely be ruled out.In our statistical analyses, we considered all data points as independent observations, although these are not completely independent from each other. However, the internal relationship disappears after administration of MPTP. The inter-animal and inter-treatment variances have been treated as random error.In this study we found a significant correlation between total parkinsonian symptoms scores and mean striatal [18F]-DOPA uptake and between lateralised limb movements and contralateral [18F]-DOPA uptake in a valid animal model for PD: MPTP lesioned monkeys. This correlation between striatal [18F]-DOPA uptake and behavioural measures was consistent in different degrees of severity of MPTP-induced parkinsonism.Therefore, the present data show that striatal [18F]-DOPA uptake correlates with behavioural measures in MPTP-treated monkeys.

AcknowledgementsThe authors are grateful to T. Peters, M. Faassen and A. Hanssen for professional and able technical support and to the staff at the Dept. Nuclear Medicine and Molecular Imaging at the University Medical Centre Groningen. This research was partially supported by a grant to K.L.L. from the School for Behavioral and Cognitive Neurosciences, Groningen, The Netherlands.

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23. Kurlan R, Kim MH, Gash DM: Oral Levodopa Dose-Response Study in Mptp-Induced Hemiparkinsonian Monkeys - Assessment with A New Rating-Scale for Monkey Parkinsonism. Movement Disorders 6:111-118, 1991

24. Langston JW, Ballard P: Parkinsonism Induced by 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (Mptp) - Implications for Treatment and the Pathogenesis of Parkinsons-Disease. Canadian Journal of Neurological Sciences 11:160-165, 1984


26. Melega WP, Raleigh MJ, Stout DB, et al: Longitudinal behavioral and 6-[F-18]fluoro-L-DOPA-PET assessment in MPTP-hemiparkinsonian monkeys. Experimental Neurology 141:318-329, 1996

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27. Nagasawa H, Saito H, Kogure K, et al: 6-[18F]fluorodopa metabolism in patients with hemiparkinsonism studied by positron emission tomography. J Neurol Sci 115:136-143, 1993

28. Oiwa Y, Eberling JL, Nagy D, et al: Overlesioned hemiparkinsonian non human primate model: correlation between clinical, neurochemical and histochemical changes. Front Biosci 8:a155-a166, 2003

29. Pate BD, Kawamata T, Yamada T, et al: Correlation of striatal fluorodopa uptake in the MPTP monkey with dopaminergic indices. Ann Neurol 34:331-338, 1993

30. Perentes E, Andringa G, Waldmeier PC, et al: Neuroprotective effects of TCH346 in bilaterally MPTP-treated rhesus monkeys. Journal of Neuropathology and Experimental Neurology 61:461, 2002

31. Smith RD, Zhang Z, Kurlan R, et al: Developing A Stable Bilateral Model of Parkinsonism in Rhesus-Monkeys. Neuroscience 52:7-16, 1993


33. Vingerhoets FJG, Snow BJ, Tetrud JW, et al: Positron Emission Tomographic Evidence for Progression of Human Mptp-Induced Dopaminergic Lesions. Annals of Neurology 36:765-770, 1994


4TCH346 prevents motor symptoms and loss of

striatal F-DOPA uptake in bilaterally MPTP-treated

primates

G. Andringa, S.A. Eshuis, E. Perentez, R.P. Maguire, D Roth, M Ibrahim, K.L. Leenders, A.R. Cools

Neurobiology of disease 2003; 14: 205-217

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Abstract

The neuroprotective efficacy of the propargylamine TCH346 was studied in the primate model of Parkinson’s disease, the bilaterally MPTP-treated monkey.

Male rhesus monkeys received 2.5 mg MPTP into the left carotid artery and, 8 weeks later, 1.25 mg MPTP into the right carotid artery. Starting 2 h after the second MPTP infusion, either 0.014 mg/kg TCH346 or its solvent was subcutaneously injected twice per day for 14 days.

The first MPTP treatment induced mild Parkinson symptoms, reduced right limb movements, and reduced F-DOPA uptake in the left striatum. The second MPTP treatment made Parkinson symptoms worse, reduced left limb movements, and reduced F-DOPA uptake in the right striatum of solvent-treated monkeys. In contrast, the second MPTP treatment did not further worsen motor symptoms and did not decrease F-DOPA uptake in the right striatum of TCH346-treated monkeys. Although the effects of the second MPTP treatment were largely prevented, the effects of the first MPTP treatment were not reversed by TCH346. Immunohistochemical examination confirmed the dramatic loss of dopamine cells in vehicle-treated monkeys and the preservation of these neurons in the right brain side of the TCH346-treated animals.

In conclusion, systemic administration of TCH346 prevented motor symptoms and nigrostriatal degeneration induced by MPTP in primates.

Keywords: Parkinson’s disease; MPTP; F-DOPA; Propargylamine; TCH346; Primate; CGP 3466B; PET; Immunohistochemistry; Motor behavior

TCH346 prevents motor symptoms and loss of F-DOPA uptake in MPTP-treated primates

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Introduction

Parkinson’s disease (PD) is a common neurodegenerative disorder affecting 1% of the population over age of 50. The disease is characterized by the death of dopaminergic neurons29 preferentially in the substantia nigra pars compacta. The disease is progressive and further loss of nigrostriatal function contributes to disability and development of levodopa-related motor response complications15. Several pathogenic factors possibly contributing to the selective neuronal loss have been identified28, 30, 37. Despite the fact that these factors offer clues to potential therapeutic strategies that could halt or slow disease progression, such strategies are not yet available in clinical practice. Indeed, developing and evaluating neuroprotective drugs is essential for improving the present day therapy of PD. The MAO-B inhibitor deprenyl is the most extensively explored drug in this respect. The compound exerts neuroprotective effects in a wide variety of cellular and rodent models for PD23, 32, 43. Interestingly, an increasing number of studies reports neuroprotective effects of deprenyl in nondopaminergic cells, which indicates that MAO-B inhibition does not account for this7, 33, 35, 49, 50. Clinical effects, however, are disappointing: a double-blinded, placebo-controlled, multicenter clinical trial, i.e., the DATATOP-study (Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism) showed that, although deprenyl significantly delayed the development of disability requiring levodopa therapy, a worsening of motor scores was observed after drug withdrawal1. This indicates a symptomatic effect and a limited, if any, neuroprotective effect. Conversion of deprenyl into the toxic metabolites amphetamine and methamphetamine may explain the lack of neuroprotection35 and also its cardiovascular adverse effects17. Indeed, propargylamines that lack these metabolic complications may be more effective in PD. The deprenyl analogues rasagiline and TCH346 (dibenzo[b,f]oxepin-10-yl-methyl-methyl-prop-2-ynylamine; labeled previously CGP 3466B) are excellent candidates in this respect. These drugs are not metabolized into (meth)amphetamine22, 52 and provide neuroprotection in cellular and rodent models of PD. Rasagiline protects dopaminergic SH-SY5Y cells from 6-hydroxydopamine-induced apoptosis36 and is more effective than deprenyl in reducing oxygen/glucose deprivation damage in PC 12 cells2, 53. The s-isomer of rasagiline, TVP1022, is neuroprotective in mice with closed head injuries2. Unlike rasagiline, TVP1022 does not inhibit MAO-B. Thus, similarly to deprenyl, MAO-B inhibition is not a prerequisite for neuroprotection by rasagiline. Further clinical studies are necessary to evaluate the neuroprotective efficacy of rasagiline in PD.TCH346 has been tested in a wide variety of cellular and animal models of PD and exhibits neurorescuing properties qualitatively similar to, but about 100-fold more potent, than those of deprenyl41, 44, 52. The compound is not only able to rescue dopamine neurons in vitro from death induced by apoptotic stimuli, but also has promising effects in rodent models of PD. Doses of 0.0014 – 1.4 mg/kg TCH346 given twice daily for

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18 days have neuroprotective effects in mice and, in addition, the compound prevents nigral degeneration and motor symptoms induced by low doses of 6-hydroxydopamine in the rat model of PD5, 52. In our previous study3 and in the present study, we extend the evaluation of the neuroprotective effects of TCH346 to an animal more similar to the human disease in both brain pathology and symptomatology, namely the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-hydrochloride (MPTP)-treated rhesus monkey. Although MPTP primate models have allowed well-founded appraisal of a vast range of symptomatic treatments8, 9, 39, not all MPTP primate models are suitable for assessment of the efficacy of neuroprotective drugs. Recovery from MPTP-induced deficits21, 34 and high interindividual variability in susceptibility to the neurotoxin20 are confounding factors that prevent adequate analysis of putative neuroprotective effects. In order to circumvent this, we used a two-phase intracarotid lesion approach4, 45; not only does this approach induce a stable syndrome with little recovery over time11, it offers the opportunity to estimate the sensitivity of each individual monkey to MPTP, prior to studying the effects of putative neuroprotective compounds such as of TCH346.Our previous study has shown that TCH346 is able to prevent Parkinson symptoms in this primate model of PD, as assessed qualitatively with a nonhuman primate clinical rating scale3. Here, we extended the behavioral evaluation of this compound with a quantitative analysis of motor symptoms by scoring goal-directed limb movements. Moreover, we investigated the in vivo efficacy of TCH346 to spare the integrity of nigro-striatal neurons using the 6-[18F]fluoro-L-dopa (F-DOPA) PET scanning technique. Finally, the in vivo data were confirmed postmortem by light and confocal microscopical examination.


SubjectsPreceding MPTP exposure, the dominant limb was selected as described earlier4. Eight male Rhesus monkeys (Macaca mulatta) over the age of 7 years, weighing between 6 and 12 kg, were studied under an approved protocol that met all institutional guidelines and requirements stated in the Principles of Laboratory Care (NIH publication no. 85–23, revised 1985). The animals were individually housed in cages (90 x 180 x 200 cm) equipped with a climbing bar, toys, and a “squeeze cage” (60 x 60 x 70). Diet consisted of lab chow supplemented with fruit; water was available ad libitum. Monkeys were kept under stable room conditions (temperature = 22 ± 2°C) and exposed to a 12-h light–dark cycle (light on 7:00 a.m. to 7.00 p.m). This study was approved by the local ethics committee for animals of the University Medical Center Groningen, Groningen, The Netherlands.


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MPTP treatmentA two-step bilateral MPTP lesion was created as first described by Smith et al.4, 45; see Andringa et al., for details). In short, under total anesthesia a first dose of 2.5 mg MPTP was administered into the carotid artery contralateral to the dominant limb (being the right limb in all monkeys) and, 8 weeks later, a second dose of 1.25 mg MPTP was infused into the other carotid artery. The second dose was kept lower to reduce the risk of severe incapacitation or even death of the animals. This MPTP treatment procedure induces a stable model with little recovery, according to [123I]FP-CIT SPECT-experiments11.

Administration of TCH346Monkeys received their first subcutaneous injection with TCH346 (0.014 mg/kg, generous gift of Novartis, Basel, Switzerland) or its solvent, saline, 2 h after the second MPTP infusion. During the consecutive 13 days, the monkeys were injected twice daily. The selected dose of 0.014 mg/kg was based on dose–range studies of TCH346 in rats5 and pilot studies in monkeys. These studies show bell-shaped dose–response curves, with an optimum around 0.014–0.14 mg/kg sc. The lowest effective dose (0.014 mg/kg) was chosen for evaluation in primates.

Assessment of motor symptomsMonkeys were videotaped for 1 h each test day. Evaluation occurred 10 and 3 days prior to the first MPTP treatment (naive stage), 6 and 7 weeks after the first MPTP treatment (unilateral stage), and 3, 7, 14, 21, 28, and 35 days after the second MPTP treatment (bilateral stage). Given the lack of a significant difference, the two sets of data collected in the naive stage were averaged; the same held true for the two sets of data collected in the unilateral stage. The motor symptoms induced by the initial MPTP treatment were used to estimate the sensitivity of each monkey to MPTP: the unilaterally treated animals were then divided among the TCH346 and control group so that the mean Parkinson scores of the group was approximately equal, prior to infusing the second dose of MPTP in the opposite artery and starting the TCH346 or saline treatment. Motor symptoms were evaluated using a quantitative assessment of goaldirected limb movements4. Evaluation occurred in a blinded fashion and by a single trained investigator (GA). Animals were housed, treated with MPTP and TCH346, behaviorally assessed, and euthanized at the Central Animal Facility of the University of Nijmegen, The Netherlands.

F-DOPA PET scansF-DOPA PET scans can be used to assess, in vivo, the status of the striatal dopaminergic system. Scans were conducted approximately 1 week prior to the first MPTP treatment

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(naive stage), approximately 7 weeks after the first MPTP treatment (unilateral stage), and approximately 5 weeks after the second MPTP treatment (bilateral stage).Doudet and colleagues18 have shown that the interanimal variability of F-DOPA measures is small (COV~14%) and thus appropriate to examine dopamine uptake repeatedly within the same animal. Animals were fasted overnight prior to each F-DOPA PET scan. Anesthesia of the monkeys was initiated with 7 mg/kg impentobarbital and 0.05 mg/kg imatropine. The animals were maintained under light anesthesia with isofluorane gas (1.5 %) for the remainder of the scan. The peripheral decarboxylase inhibitor carbidopa was administered orally (3 mg/kg) approximately 30 min before F-DOPA administration. A venous access line was inserted and the monkeys were intubated. The monkey’s head was always fixated in the same position with a modified stereotactic apparatus as head holder. Monkeys were placed in the camera in such a way that the center of the field of view was positioned just above the eyes. PET scans were performed on the Siemens ECAT Exact HR + scanner (Siemens, Munich/Erlangen, Germany). This tomograph has a resolution of 5 mm full width at half maximum in the center of the field of view and an axial field of view of 15.5 cm. Twenty minutes before the scanning, a transmission scan, using a 68Ge-ring, was performed for attenuation correction. F-DOPA was injected intravenously as a bolus in a volume of 6 ml saline over a period of 1 min. The injected dose ranged from 54 to 152 MBq, with a mean of 78.5 MBq and a standard deviation of 17.7 MBq. Scanning consisted of 21 frames starting directly after the tracer infusion and proceeding at gradual increasing intervals up to a total scan duration of 120 min.Three regions of interest (left and right striatum and a reference region of nonspecific uptake) were defined. The size of the regions of interest was fixed in a standard template, which was used for all the scans. This approach avoids possible bias introduced by resizing regions to fit the apparent size of the striatum in an image, which may be affected by changes in function. The reference region included both cortical and white matter regions and its large size also reduced possible intersubject biases in placement. The estimate for each region was an average of separate measurements made on each of three planes. The center of each region was repositioned separately on each plane. Indices for comparing uptake in striatum with uptake in reference regions were calculated as uptake in striatum minus uptake in reference region, divided by uptake in reference region for both the left and right side, and referred to as Striatum reference index (SRI). Indices were calculated using the data between 75 and 105 min scanning time. F-DOPA PET scans were performed at the Groningen University Hospital PET Center, Groningen, The Netherlands.

Histology and immunohistochemistrySeven weeks after the last TCH346 injection animals were euthanized with an overdose of sodium pentobarbital and transcardially perfused with saline, followed by


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4% paraformaldehyde in 0.1 M phosphate buffer. Animal 4 did not wake up from the isofluorane anesthesia after the third PET scan. Pathological examination did not reveal any abnormalities and anesthetic oversensitivity was concluded to be the most probable cause of death. This animal was not included in the neuropathological examination. Histological and immunohistochemical examinations were performed at the Preclinical Safety Assessment Department, Novartis Pharma AG, Basel, Switzerland. Hemispheres of each brain were sectioned together into 5- to 8-mm-thick coronal slices; similar tissue slices were obtained by performing cross-sections through the brainstem and cerebellum. Specimens were embedded in paraffin wax and 5-µm-thick sections were obtained. Sections from three anteroposterior levels of the striatum and from the upper, mid, and lower levels of the midbrain were stained with hematoxylin and eosin or immunostained either for TH, the rate-limiting enzyme in catecholamine synthesis, or for GFAP, the major constituent of glial cytoplasmic filaments42. Immunohistochemical procedures were performed as described previously38. Dopaminergic integrity and astrogliosis, as assessed by TH- and GFAP-immunoreactive cells and processes, respectively, were evaluated in substantia nigra and striatum for each animal. The relative number of dopaminergic neurons was evaluated in three sections of three levels (upper, mid, and lower) in the left and right substantia nigra pars compacta. The number of nigral neurons present in a midpower field (20 x objective) was categorized as follows: low number (<15), moderate number (15–30), and high number (>30). TH immunoreactivity in the striatum, as well as GFAP immunoreactivity in the substantia nigra pars compacta and striatum, were semiquantitatively analyzed. For confocal microscopy analysis, paraffin sections from three anteroposterior levels of the left and right caudate and putamen were immunofluorescently labeled for TH (1:4000) followed by Alexa Fluor 488 goat anti-mouse IgG (1:250; Molecular Probes, The Netherlands) and counterstained with propidium iodide. Labeling was examined with a Leica TCS-NT (Leica Microsystems Ltd) confocal laserscanning microscope. Using a 63 x water immersion objective and 0.284-µm optical sections, the upper and lower limits of the TH-labeled fibers were imaged. Confocal settings were kept the same for all analyzed slides. Raw data were imported to a Silicon Graphics (SGI) workstation. The density of the TH-labeled fibers (green channel) was quantified using Voxelshop Pro software (Bitplane AG, Switzerland), by dividing the calculated volume of fibers by the total volume of the tissue section confocaled. The fiber density was pooled and averaged. Propidium iodide-labeled structures were excluded from the calculation.

StatisticsAll data are expressed as means ± SEM. The effects of the first MPTP treatment were analyzed by comparing the scores of the solvent and TCH346-treated group in the naive stage with those in the unilateral stage, using the Wilcoxon signed rank test for

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the Parkinson scores and the paired t-test for limb movements and PET scores. The effects of the second MPTP treatment were analyzed by comparing the scores of the solvent-treated group in the unilateral stage with those of the solvent-treated group in the bilateral stage, using the nonparametric Friedman test for Parkinson scores, a one-way ANOVA for limb movements followed by Dunnett’s posthoc tests where appropriate, and a paired t-test for the PET scores. The ability of TCH346 to prevent MPTP-induced behavioral and neurochemical deficits was analyzed by comparing Parkinson scores, limb movements, and F-DOPA uptake of the solventtreated monkeys in the bilateral stage with those of the TCH346-treated monkeys in the bilateral stage using a twoway ANOVA with repeated measures. Posthoc t-tests were done where appropriate. A p value < 0.05 was considered significant.

Results

Naive stageIn the naive stage animals spent an average time of 10.3 and 18.2 % on left and right limb movements, respectively (Fig. 1A B). Furthermore, all animals scored 0 on the Parkinson rating scale, which indicates that they all displayed normal motor behavior before MPTP administration (Table 1). Consistent with the behavioral data, F-DOPA uptake in the right and left striatum was within the normal range in all animals (Figs. 2A and 3 Table 2 ).

A


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Figure 1: Percentage of time spent on left (A) and right (B) goal-directed limb movements in rhesus

monkeys prior to MPTP treatment (naive stage), after initial infusion of 2.5 mg MPTP into the left

carotid artery (unilateral stage), and after a second infusion of 1.25 mg MPTP into the right carotid

artery, 8 weeks later (bilateral stage). Monkeys received saline (n = 4) or TCH346 (n =4) twice per

day for 2 weeks, starting 2 h after the second MPTP infusion. Data are expressed as mean + SEM.

Table 1: SRI of F-DOPA uptake in the left and right striatum of naïve, unilaterally and bilaterally MPTP-treated rhesus monkeys.Vehicle Naïve Unilateral Bilateral

Day 3 Day 7 Day 14 Day 21 Day 28 Day 350 1.75±1.03 14.50±3.60 15.75±3.45 13.75±3.90 14.50±3.77 14.25±3.22 13.50±3.66 TCH346Naïve Unilateral Bilateral

Day 3 Day 7 Day 14 Day 21 Day 28 Day 350 1.63±0.97 6.25±0.63 7.75±1.03 5.25±1.03 4.75±1.03 5.00±0.91 4.25±0.48

Note: F-DOPA uptake depicted as SRI in the left and right striatum in rhesus monkeys prior to MPTP treatment (naïve stage), after initial infusion of 2.5 mg MPTP into the left carotid artery (unilateral stage) and after a second infusion of 1.25 mg MPTP into the right carotid artery, 8 weeks later (bilateral stage). Monkeys received saline (n = 4) or TCH346 (n=4) twice per day for 2 weeks, starting 2 h after the second MPTP infusion. Data are expressed as means ± SEM.

B

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Figure 2: (A) F-DOPA uptake in the left and right striatum in rhesus monkeys prior to MPTP treatment

(naive stage) and after initial infusion of 2.5 mg MPTP into the left carotid artery (unilateral stage).

SRI was calculated as F-DOPA uptake in striatum minus uptake in reference region, divided by uptake

in reference region. Indices were calculated using the data between 75 and 105 min scanning time.

Individual data and average SRI are depicted.

(B) F-DOPA uptake in the left and right striatum in rhesus monkeys after initial infusion of 2.5 mg MPTP

into the left carotid artery (unilateral stage) and after a second infusion of 1.25 mg MPTP into the

right carotid artery, 8 weeks later (bilateral stage). Monkeys received saline (n= 4) or TCH346 (n = 4)

twice per day for 2 weeks, starting 2 h after the second MPTP infusion. SRI was calculated as F-DOPA

uptake in striatum minus uptake in reference region, divided by uptake in reference region. Indices

were calculated using the data between 75 and 105 min scanning time. Individual data and average

SRI are depicted. Open circles, saline-treated primates; closed circles, TCH346-treated primates.

A

B


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Table 2: SRI of F-DOPA uptake in the left and right striatum of naïve, unilaterally and bilaterally MPTP-treated rhesus monkeys.

Naïve Unilateral BilateralLeft Right Left Right Left Right

Saline1 0.487 0.446 0.250 0.649 0.183 0.2882 0.459 0.531 0.135 1.018 0.220 0.0953 0.386 0.417 0 0.432 0.060 0.4734 0.531 0.498 0.082 0.566 0 0.315Mean ± SEM 0.466±0.026 0.473±0.022 0.109±0.05 0.666±0.109 0.098±0.057 0.293±0.067TCH3465 0.512 0.463 0 0.622 0.126 0.8146 0.306 0.331 0.388 0.335 0.352 0.4407 0.539 0.458 no data no data 0.120 0.7638 0.446 0.404 0.308 0.873 0.185 0.493Mean ±SEM 0.451±0.045 0.414±0.027 0.231±0.084 0.610±0.110 0.196±0.047 0.628±0.082Mean±SEM 0.451±0.028 0.444±0.022 0.161±0.056 0.642±0.084 0.147±0.043(n=8)Mean±SEM 0.451±0.017 0.402±0.079(n=16)

Note: F-DOPA uptake depicted as SRI in the left and right striatum in rhesus monkeys prior to MPTP treatment (naïve stage), after initial infusion of 2.5 mg MPTP into the left carotid artery (unilateral stage) and after a second infusion of 1.25 mg MPTP into the right carotid artery, 8 weeks later (bilateral stage). Monkeys received saline (n = 4) or TCH346 (n=4) twice per day for 2 weeks, starting 2 h after the second MPTP infusion. Data are expressed as means ± SEM.

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3

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Figure 3: Representative images of F-DOPA labeling in coronal sections of a vehicle (A–C) and

TCH346-treated (D–F) rhesus monkey brain prior to MPTP treatment (naive stage; A and D), after

initial infusion of 2.5 mg MPTP into the left carotid artery (unilateral stage; B and E) and after a

second infusion of 1.25 mg MPTP into the right carotid artery, 8 weeks later (bilateral stage; C and

F). Monkeys received saline (n = 4) or TCH346 (n = 4) twice per day for 2 weeks, starting 2 h after

the second MPTP infusion.

Figure 4: (A and B) TH immunoreactivity in transverse sections through the midbrain of a vehicle-

(A) and a TCH346-treated (B) monkey. Five-micrometer sections were immunostained with TH using

the streptavidin/peroxidase method and counterstained with hematoxylin. Note the absence of TH

immunoreactivity in the left side of the substantia nigra in the both animals. In the vehicle-treated

monkey, a dramatic loss of TH immunoreactivity was also observed in the right substantia nigra

contrasting with the intensely immunostained right substantia nigra of the TCH346-treated monkey. In

the latter, only the ventrolateral zona compacta appears partly affected. Original magnification, 2.5x.

Bar represents 0.4 mm. (C and D) Hematoxylin and eosin-stained sections through the left (C) and

right (D) substantia nigra of a TCH346-treated monkey. Nigral neurons are virtually absent in the left

substantia nigra and glial proliferation, characterized by an increase in small cell nuclei, is obvious.

In the right substantia nigra, numerous dopaminergic neurons are present. Original magnification,

200x. Bar represents 50 µm. (E and F) TH immunoreactivity in semisuccessive sections to C and D,

respectively, in a TCH346-treated monkey. Five-micrometer sections were immunostained with TH

using the streptavidin/peroxidase method. Note the dramatic loss of TH-positive neurons and of their

dopaminergic fiber network in the left midbrain (E). The neurons appear dystrophic with pyknotic

perikarya and short interrupted processes (E). In the contralateral substantia nigra, the numerous

neurons and dopaminergic fiber network are intensely immunostained for TH. Original magnification,

200x. Bar represents 50 µm. (G and H). TH immunoreactivity in 5-µm coronal sections through the

paraventricular part of the left (G) and right (H) caudate nucleus of a TCH346-treated monkey; the

ependymal layer of the left (G, right) and right (H, left) lateral ventricle is present. Sections were

immunostained with TH using the streptavidin/peroxidase method. Scattered TH-positive dopaminergic

fibers are present in the left nucleus caudate (G, arrows) and a dense dopaminergic fiber network is

preserved in the right caudate nucleus (H). Original magnification, 200x. Bar represents 50 µm. (I and

J) GFAP immunoreactivity in coronal 5-µm sections through the paraventricular part of the left (I) and

right (J) caudate nucleus of a TCH346-treated monkey; same areas as in G and H. The left caudate

nucleus displays astroglial proliferation (I), while in the right caudate nucleus, GFAP immunoreactivity

is almost exclusively expressed in the ependymal/subependymal layer (J). Streptavidin/peroxidase

method for GFAP, hematoxylin counterstaining. Original magnification, 200x. Bar represents 50 µm.

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Fig. 5. Confocal photomicrographs showing TH immunofluorescence of dopaminergic fibers in

the left (A) and right (B) caudate nucleus of a TCH346-treated animal. Five-micrometer sections

were immunofluorescently labeled with TH followed by Alexa Fluor 488 goat anti-mouse IgG and

counterstained with propidium iodide. Note the dramatic loss of TH-positive fibers in the left caudate

nucleus (A), contrasting with the dense dopaminergic fiber network present in the right caudate

nucleus (B). Original magnification, 630x. Bar represents 20 µm.

Unilateral stage: effects of unilateral MPTP administrationThe initial MPTP infusion into the left carotid artery significantly reduced the percentage of time spent on goal directed right limb movements [naive stage vs unilateral stage t(14) = 3.92, p < 0.001; paired t-test, Fig. 1B], but had no effect on the time spent on left limb movements [naive stage vs unilateral stage t(14) = 0.92, p < 0.05; paired t-test; Fig. 1A].Following unilateral MPTP administration, all monkeys displayed Parkinson symptoms that ranged from very mild (score = 1) to mild (score = 5) [naive stage vs unilateral stage: t(7) = 10.87, p < 0.01: Wilcoxon; Table 1]. Six of eight monkeys displayed symptoms that were restricted to the side contralateral to the MPTP infusion (in all monkeys being the right side of the body), while two monkeys exhibited bilateral symptoms. In line with the behavioral data, F-DOPA uptake was strongly reduced in the left striatum 7 weeks after MPTP injection into the left carotid artery [naive stage vs unilateral stage: t(6) = 3.65, p < 0.01; Figs. 2A and 3, paired t-test] In contrast, F-DOPA uptake in the right striatum was increased [naive stage vs unilateral stage: t(6) = 2.62, p < 0.04; Figs. 2A and 3, Table 2, paired t-test].

Bilateral stage: protective effects of TCH346 on MPTP-induced deficitsInfusion of a second dose of MPTP into the right carotid artery strongly reduced the time spent on left limb movements in the saline-treated group [saline groups: unilateral stage vs bilateral stage: F(6,27) = 2.60, p < 0.05; Fig. 1A, one-way ANOVA] but did not further decrease the time spent on right limb movements [saline groups: unilateral stage vs bilateral stage: F(6,27) = 1.00, p < 0.05; Fig. 1B, one-way ANOVA]. In contrast to its effects in saline-treated monkeys, the second MPTP injection did not reduce the time spent on left limb movements in TCH346-treated animals [TCH346 groups: unilateral stage vs bilateral stage: F(6,27) = 0.19, p < 0.05; Fig. 1A]. Indeed, the percentage of time spent on left limb movements was significantly lower in the animals that had received saline than in the animals that had received TCH346 [saline; bilateral stage vs TCH346, bilateral stage: F(1,36) = 16.06, p < 0.001; Fig. 1A; two-way ANOVA]. In the TCH346-treated group, right limb movements were not affected by the second MPTP treatment [TCH346 unilateral stage vs TCH346 bilateral stage: F(26) = 0.52, p < 0.05; Fig. 1B; two-way ANOVA].


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The second MPTP injection significantly worsened the Parkinson score in the saline-treated monkeys, including the monkey that exhibited bilateral symptoms after the initial MPTP treatment [saline group: unilateral stage vs bilateral stage: F(6,27) = 78.45, p < 0.0007; Friedman; Table 1]. Parkinson scores were very similar over all test days, which indicates that the MPTP-induced syndrome was stable in time. There was a positive correlation between the Parkinson score of the saline-treated animals in the unilateral and bilateral phase (F(4,1) = 17.24, r = 0.95, p < 0.05, data not shown), indicating that the severity of the parkinsonian symptoms after the first MPTP injection is a good predictor for the effects of the second MPTP treatment. The second MPTP injection only mildly affected the parkinsonian symptoms in the monkeys that had received TCH346, including the monkey that already exhibited bilateral symptoms after the initial MPTP treatment [total score, unilateral vs bilateral stage: F(6,27) = 34.82, p < 0.001; Friedman; Table 1]. The severity of Parkinsonian symptoms was much lower in the bilaterally MPTP-treated primates that had received TCH346 than in the animals that had received the solvent of TCH346 [F(1,36) = 930.20, p < 0.0001, twoway ANOVA, factor treatment]. The score between the saline and TCH346-treated group was significantly different on all test days.In the saline group, the second MPTP treatment into the right carotid artery tended to decrease F-DOPA uptake in the right striatum. Compared to the first MPTP lesion on the left side, damage was less severe, in concordance with the reduced amount of MPTP given [unilateral stage vs bilateral stage: t(3) = 1.85, p< 0.08; Figs. 2B and 3, Table 2].F-DOPA uptake in the left striatum was not altered by the second MPTP treatment (Table 2). In the group that had received TCH346, F-DOPA uptake in the right striatum remained unaffected after the second MPTP treatment. In line with this, F-DOPA uptake in the right striatum showed a tendency to be higher in the TCH346 group compared to the control group [F(2,0.14) = 2.92, p < 0.067, two-way ANOVA, factors treatment x stage; Figs. 2B and 3, Table 2], suggesting that TCH346 prevented the reduction in F-DOPA induced by the second MPTP administration. However, similarly to the saline group, F-DOPA uptake in the left striatum was unaltered, illustrating that TCH346 was not able to reverse the reduction in F-DOPA uptake that had been established by the first MPTP administration, 8 weeks before the start of the treatment with TCH346.The main neuropathological findings are summarized in Table 3. All vehicle-treated monkeys showed a dramatic loss of the pigmented TH-positive neurons and of their dopaminergic network in the left substantia nigra (Fig. 4A). Two of these animals (1 and 2) exhibited a similar picture in the right side of the midbrain (Fig. 4A), while the third monkey (3) displayed a relatively preserved right substantia nigra, with moderate numbers of dopaminergic neurons. Increased astroglial proliferation was observed in the same areas. TH immunoreactivity was dramatically decreased in the caudate nucleus and the putamen, in both sides, in two of the vehicle-treated animals (1 and 2), while moderate

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TH immunoreactivity was present in the right striatum of the third control monkey (3). Mild gliosis was observed in the striatum (bilaterally) of two control animals (1 and 2). In TCH346-treated monkeys, small (Fig. 4B, C, and E; monkeys 5 and 7) and moderate (monkeys 6 and 8) numbers of dopaminergic neurons were found in the left substantia nigra, where slight (monkey 6) to moderate (monkeys 5, 7, and 8) gliosis was present. All four animals exhibited moderate (monkey 8) and high (monkeys 5–7) numbers of neurons in the right substantia nigra (Fig. 4D and F), although the ventrolateral zona compacta appeared partly affected (Fig. 4B). Intense TH immunoreactivity (Fig. 4H), without gliosis (Fig. 4J), was observed in the right striatum of all four monkeys, while minimal (Fig. 4G; monkeys 5 and 7), slight to moderate (monkey 8), and moderate (monkey 6) TH immunoreactivity and slight astroglial proliferation (Fig. 4I) were found in the left striatum. Confocal microscopy analysis of TH labeling in the striatum (Figs. 5 and 6 ) enabled a direct comparison of dopaminergic fiber density between vehicle- and TCH346-treated groups and between individual animals. In all vehicle-treated animals, density of the TH-labeled fibers in the left striatum was very low (0.003–0.006). In three (monkeys 5, 7, and 8) of four animals treated with TCH346, fiber densities in the left striatum (Fig. 4I) corresponded to those recorded in the vehicle-treated monkeys (0.002–0.08), with one primate exhibiting higher fiber densities in the left striatum. In two of the vehicle-treated monkeys (1 and 2), there was a low density of TH-labeled fibers in the right striatum. In the third vehicle-treated animal (monkey 3), however, the right striatum showed higher fiber densities, averaging 0.32. In contrast, in the right striatum of the all TCH346-treated monkeys, TH-labeled fibers were much greater in density ranging from 0.34 to 0.41 (Figs. 5B and 6). Overall, different techniques used to analyze nigrostriatal integrity, i.e., F-DOPA PET scanning, TH light microscopy, and TH confocal microscopy rendered very similar outcomes in each individual animal.


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Table 3: Neuropathological examination of vehicle and TCH346-treated MPTP monkeysTreatment Vehicle TCH346Animal no. 1 2 3 5 6 7 8Left

TH-positive neurons in SN + + + + ++ + ++Gliosis in SN ++ ++ ++ ++ + ++ ++TH-positive fibers in striatum (-) (-) + + ++ + ++Gliosis in striatum + + (-) (-) (-) (-) (-)

RightTH-positive neurons in SN + + ++ +++ +++ +++ ++Gliosis in SN ++ ++ + + + + +TH-positive fibers in striatum (-) + +++ +++ +++ +++ +++Gliosis in striatum + + (-) (-) (-) (-) (-)

Note: overview of TH and GFAP immunohistochemistry in the left and right substantia nigra and

striatum of vehicle and TCH346-treated primates. Monkeys were perfused 7 weeks after the last

TCH346 or vehicle injection. Five-micrometer coronal sections were immunostained for TH or GFAP

and analyzed using light microscopy. The terms gliosis refers to GFAP-positive cells and cell processes.

(-) almost absent

+ low/low number

++ moderate/moderate number

+++ high / high number

Fig. 6. Confocal microscope analysis of dopaminergic fiber density in the left and right striatum of

monkeys treated with either TCH346 or vehicle. Density is calculated as the volume of fibers divided

by the total volume of the examined tissue.

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Discussion

The present data provide neurochemical, neuropathological, and behavioral evidence to demonstrate that TCH346 prevents the death of nigrostriatal dopaminergic neurons and concomitant motor symptoms in an animal model that most closely resembles the pathophysiology and symptomatology of PD, namely the bilaterally MPTP-treated monkey. The 14-day administration of TCH346 starting 2 h after the second 1.25 mg MPTP infusion was able to largely prevent motor deficits induced by such an MPTP treatment in saline-treated primates. Not only Parkinson scores were lower in the TCH346-treated group than in the solvent-treated group, TCH346-treated monkeys also did not show any reduction in the percentage of time spent on left limb movements. In accordance with this, the F-DOPA uptake in the right striatum appeared greater in the group that had received TCH346 than in the group that had received saline. Moreover, in three of four TCH346-treated monkeys, semiquantitative and quantitative analysis of TH and GFAP immunoreactivity in the substantia nigra and striatum confirmed the preservation of the nigrostriatal system in the right hemisphere. Our data are consistent with findings of Pate and colleagues40, which show that a reduction in F-DOPA uptake correlates well with a reduction in the amount of dopamine nerve terminals in the striatum. Therefore, it is concluded that TCH346 was able to prevent the MPTP-induced degeneration of dopamine neurons in these primates.Although TCH346 almost completely prevented the reduction in F-DOPA uptake in the right striatum induced by the administration MPTP 2 h earlier, as anticipated, it did not reverse the Parkinson symptoms or reduced F-DOPA uptake induced by the initial MPTP treatment 8 weeks earlier. The majority of neurodegeneration is established within a week after intracarotid administration of MPTP, thus indicating that the degenerative process had mostly ended by the time of TCH346 administration 8 weeks later.Our data are in line with the accumulating evidence that TCH346 can interfere with the process of dopaminergic cell death whereas neurotrophic effects on surviving dopaminergic neurons have not been established.A two-phase lesion approach was used to induce a bilateral Parkinsonian syndrome in rhesus monkeys4, 45. This MPTP treatment induced a significant reduction in limb movements and moderate to severe parkinsonian symptoms, as well as decreased striatal F-DOPA uptake. Our data confirm earlier reports45 that both motor symptoms and neurochemical effects are stable with little compensation in time: limb use and F-DOPA uptake remained constant from day 3 to day 35 in the bilateral phase. In the present model, the second lesion is produced with a lower concentration of MPTP, to more closely mimic the bilateral lesion progression seen in the disease. This allowed us to study the neuroprotective effects of TCH346 in slowing the disease process on the side, which is relatively spared, as might be the case in the clinical setting. Due to the lesser amount of MPTP, the difference between


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the saline-treated group and the TCH346-treated group of monkeys was small and less than the difference between the results of the naive group versus the unilateral group. This small difference lowered statistical power. Statistical power was also decreased by missing data in the TCH346-treated group of animals.As anticipated, the interindividual susceptibility to the MPTP treatment varied markedly: whereas in six out of eight animals the initial MPTP treatment dramatically decreased F-DOPA uptake and striatal fiber density, the dopamine system remained partially intact on both sides in one TCH346-treated primate and on the right side in one saline-treated primate. Therefore dividing animals among the different treatment groups in order to obtain comparable groups appears essential for obtaining reliable data on putative neuroprotective compounds.We observed a small but significant increase in the SRI between the naive and unilateral stages. Similar small increases in the contralateral striatal function after MPTP lesion have been observed24, 31. The high capacity of dopaminergic neurons for functional compensation may underlie this effect46. Indeed, the increased F-DOPA uptake is consistent with the reported upregulation of high-affinity dopamine uptake in the nonlesioned side of the MPTP-treated animals14. In a further study, which showed evidence of early effects after MPTP lesions19, the authors argue convincingly that these effects are consistent with the wider body of literature. The fact that two of the animals showed bilateral Parkinson symptoms at the unilateral stage supports the idea that the ipsilateral substantia nigra may have been affected to some small degree during the unilateral lesioning.In contrast to many propargylamines, including rasagiline and deprenyl, TCH346 does not induce symptomatic effects by increasing synaptic dopamine through an inhibitory effect on MAO-B. Indeed, the indication that the effects of TCH346 were not symptomatic is underlined by the observation that the animals not only displayed low Parkinson scores during the 14-day TCH346 administration period, but also as long as 3 weeks after its last administration. Distribution studies have shown that midbrain levels of MPTP peak within 2 h of administration26. Since TCH346 was administered 2 h after the MPTP infusion, it is unlikely that TCH346 interfered with the uptake of MPTP.An increasing number of studies suggest that TCH346 can prevent dopamine cells from dying by blocking apoptotic pathways13, 33, 52. Therefore it appears feasible that TCH346 prevented the Parkinson syndrome in our nonhuman primates by blocking MPTP-induced apoptosis of dopamine cells. A potential target mediating the antiapoptotic effects of TCH346 is glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Besides its role as enzyme in glycolysis, GAPDH has been found to be one of the key factors in apoptosis16. TCH346 prevents neuronal apoptosis induced by GAPDH overexpression in vitro52, possibly by preventing nuclear accumulation of GAPDH and/or by converting GAPDH from its usual tetrameric form to a dimeric structure13.

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There is no question that apoptotic cell death can occur in nigral dopamine cells: these neurons die from apoptosis in MPTP and 6-hydroxydopamine models of PD10, 27, 54. Although not all postmortem studies report the morphological characteristics of apoptosis in PD6,

12, 48, various executioners of apoptosis, including GAPDH, are activated in remaining dopaminergic substantia nigra neurons25, 47, 51. The enzyme accumulates in the nucleus of the latter neurons, suggesting that the proapoptotic, nuclear translocation of GAPDH is not limited to cultured cells but actually plays a role in the pathogenesis of PD47. Accordingly, it implicates that the neuroprotective actions of TCH346 through GAPDH could prove significant for protecting dopamine neurons in PD. In conclusion, the data show that TCH346 prevented MPTP-induced Parkinson symptoms in the best animal model of PD, possibly through its inhibitory effect on GAPDH. This compound may prove useful for inhibiting the progression of dopaminergic degeneration in patients with PD.

AcknowledgmentsThe authors are thankful to M. Faassen and T. Arts for excellent technical assistance in the lesion procedure. The staff of the PET Center, Groningen University Hospital, is gratefully acknowledged for technical assistance in performing the F-DOPA PET scans.


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13. Carlile GW, Chalmers-Redman RM, Tatton NA, et al: Reduced apoptosis after nerve growth factor and serum withdrawal: conversion of tetrameric glyceraldehyde-3-phosphate dehydrogenase to a dimer. Mol Pharmacol 57:2-12, 2000

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14. Cass WA, Gerhardt GA, Zhang Z, et al: Increased dopamine clearance in the non-lesioned striatum of rhesus monkeys with unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) striatal lesions. Neurosci Lett 185:52-55, 1995

15. Chase TN, Ng LK: Central monoamine metabolism in Parkinson’s disease. Arch Neurol 27:486-491, 1972

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17. Churchyard A, Mathias CJ, Lees AJ: Selegiline-induced postural hypotension in Parkinson’s disease: a longitudinal study on the effects of drug withdrawal. Mov Disord 14:246-251, 1999

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19. Eberling JL, Bankiewicz KS, Jordan S, et al: PET studies of functional compensation in a primate model of Parkinson’s disease. Neuroreport 8:2727-2733, 1997

20. Elsworth JD, Deutch AY, Redmond DE, Jr., et al: Symptomatic and asymptomatic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated primates: biochemical changes in striatal regions. Neuroscience 33:323-331, 1989

21. Elsworth JD, Taylor JR, Sladek JR, Jr., et al: Striatal dopaminergic correlates of stable parkinsonism and degree of recovery in old-world primates one year after MPTP treatment. Neuroscience 95:399-408, 2000

22. Finberg JP, Lamensdorf I, Commissiong JW, et al: Pharmacology and neuroprotective properties of rasagiline. J Neural Transm Suppl 48:95-101, 1996

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24. Guttman M, Yong VW, Kim SU, et al: Asymptomatic striatal dopamine depletion: PET scans in unilateral MPTP monkeys. Synapse 2:469-473, 1988

25. Hartmann A, Hunot S, Michel PP, et al: Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. Proc Natl Acad Sci U S A 97:2875-2880, 2000

26. Hartvig P, Lindquist NG, Aquilonius SM, et al: Distribution of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in experimental animals studied by postiron emission tomography and whole body autoradiography. Life Sci 38:89-97, 1986

27. He Y, Lee T, Leong SK: 6-Hydroxydopamine induced apoptosis of dopaminergic cells in the rat substantia nigra. Brain Res 858:163-166, 2000

28. Hirsch EC, Hunot S, Hartmann A: Mechanism of cell death in experimental models of Parkinson’s disease. Funct Neurol 15:229-237, 2000


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31. Jordan S, Eberling JL, Bankiewicz KS, et al: 6-[18F]fluoro-L-m-tyrosine: metabolism, positron emission tomography kinetics, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine lesions in primates. Brain Res 750:264-276, 1997

32. Koutsilieri E, Chen TS, Rausch WD, et al: Selegiline is neuroprotective in primary brain cultures treated with 1-methyl-4-phenylpyridinium. Eur J Pharmacol 306:181-186, 1996

33. Kragten E, Lalande I, Zimmermann K, et al: Glyceraldehyde-3-phosphate dehydrogenase, the putative target of the antiapoptotic compounds CGP 3466 and R-(-)-deprenyl. J Biol Chem 273:5821-5828, 1998

34. Kurlan R, Kim MH, Gash DM: The time course and magnitude of spontaneous recovery of parkinsonism produced by intracarotid administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to monkeys. Ann Neurol 29:677-679, 1991

35. Magyar K, Szende B, Lengyel J, et al: The pharmacology of B-type selective monoamine oxidase inhibitors; milestones in (-)-deprenyl research. J Neural Transm Suppl 48:29-43, 1996

36. Maruyama W, Youdim MB, Naoi M: Antiapoptotic properties of rasagiline, N-propargylamine-1(R)-aminoindan, and its optical (S)-isomer, TV1022. Ann N Y Acad Sci 939:320-329, 2001

37. Mattson MP, Pedersen WA, Duan W, et al: Cellular and molecular mechanisms underlying perturbed energy metabolism and neuronal degeneration in Alzheimer’s and Parkinson’s diseases. Ann N Y Acad Sci 893:154-175, 1999

38. Pace V, Perentes E, Germann PG: Pheochromocytomas and ganglioneuromas in the aging rats: morphological and immunohistochemical characterization. Toxicol Pathol 30:492-500, 2002

39. Papa SM, Chase TN: Levodopa-induced dyskinesias improved by a glutamate antagonist in Parkinsonian monkeys. Ann Neurol 39:574-578, 1996


41. Paterson IA, Tatton WG: Antiapoptotic actions of monoamine oxidase B inhibitors. Adv Pharmacol 42:312-315, 1998

42. Perentes E, Rubinstein LJ: Recent applications of immunoperoxidase histochemistry in human neuro-oncology. An update. Arch Pathol Lab Med 111:796-812, 1987

43. Roy E, Bedard PJ: Deprenyl increases survival of rat foetal nigral neurones in culture. Neuroreport 4:1183-1186, 1993

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44. Sagot Y, Toni N, Perrelet D, et al: An orally active anti-apoptotic molecule (CGP 3466B) preserves mitochondria and enhances survival in an animal model of motoneuron disease. Br J Pharmacol 131:721-728, 2000

45. Smith RD, Zhang Z, Kurlan R, et al: Developing A Stable Bilateral Model of Parkinsonism in Rhesus-Monkeys. Neuroscience 52:7-16, 1993

46. Song DD, Haber SN: Striatal responses to partial dopaminergic lesion: evidence for compensatory sprouting. J Neurosci 20:5102-5114, 2000

47. Tatton NA: Increased caspase 3 and Bax immunoreactivity accompany nuclear GAPDH translocation and neuronal apoptosis in Parkinson’s disease. Exp Neurol 166:29-43, 2000

48. Tatton NA, Maclean-Fraser A, Tatton WG, et al: A fluorescent double-labeling method to detect and confirm apoptotic nuclei in Parkinson’s disease. Ann Neurol 44:S142-S148, 1998

49. Tatton WG, Chalmers-Redman RM, Ju WY, et al: Apoptosis in neurodegenerative disorders: potential for therapy by modifying gene transcription. J Neural Transm Suppl 49:245-268, 1997

50. Tatton WG, Wadia JS, Ju WY, et al: (-)-Deprenyl reduces neuronal apoptosis and facilitates neuronal outgrowth by altering protein synthesis without inhibiting monoamine oxidase. J Neural Transm Suppl 48:45-59, 1996

51. Vila M, Jackson-Lewis V, Vukosavic S, et al: Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease. Proc Natl Acad Sci U S A 98:2837-2842, 2001


53. Youdim MB, Wadia A, Tatton W, et al: The anti-Parkinson drug rasagiline and its cholinesterase inhibitor derivatives exert neuroprotection unrelated to MAO inhibition in cell culture and in vivo. Ann N Y Acad Sci 939:450-458, 2001

54. Zuch CL, Nordstroem VK, Briedrick LA, et al: Time course of degenerative alterations in nigral dopaminergic neurons following a 6-hydroxydopamine lesion. J Comp Neurol 427:440-454, 2000

5Comparison of FP-CIT SPECT and F-DOPA PET in

patients with de novo and advanced Parkinson’s

disease

S.A. Eshuis, R.P. Maguire, K.L. Leenders, S. Jonkman, P.L. Jager

Eur J Nucl Med Mol Imaging 2006; 33: 200-209

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Abstract

Purpose: Diagnosis of Parkinson’s disease (PD) can be difficult. F-DOPA PET is able to quantify striatal dopa decarboxylase activity and storage capacity of F-dopamine, but is expensive and not generally available. FP-CIT binds to the dopamine transporter, and FP-CIT SPECT is cheaper and more widely available, but has a lower resolution. The aim of this study was to compare these two methods in the same patients with different stages of PD to assess their power in demonstrating deficits of the striatal dopaminergic system.

Thirteen patients with de novo PD and 17 patients with advanced PD underwent FP-CIT SPECT and static F-DOPA PET. After data transfer to standard stereotactic space, a template with regions of interest was used to sample values of the caudate, putamen and an occipital reference region. The outcome value was striato-occipital ratios. Patients were clinically examined in the “off state” (UPDRSIII and H&Y stage).

Good correlations were found between striatal F-DOPA uptake and striatal FP-CIT uptake (r=0.78) and between putaminal F-DOPA uptake and putaminal FP-CIT uptake (r=0.84, both p<0.0001). Both striatal uptake of FPCIT and that of F-DOPA correlated moderately with H&Y stage (ρ=−0.52 for both techniques), UPDRS-III (ρ=−0.38 for F-DOPA; ρ=−0.45 for FP-CIT) and disease duration (ρ=−0.59 for F-DOPA; ρ=−0.49 for FP-CIT, all p<0.05).

FP-CIT values correlate well with F-DOPAvalues. Both methods correlate moderately with motor scores and are equally able to distinguish patients with advanced PD from patients with de novo PD.

Keywords: F-DOPA PET – FP-CIT SPECT – Parkinson’s disease – Dopamine transporter

Comparison of FP-CIT and F-DOPA in patients with de novo and advanced PD

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Introduction

Parkinson’s disease (PD) is a slowly progressive disorder, characterised by progressive degeneration of dopaminergic neurones in the substantia nigra. The classic triad of clinical symptoms of PD consists of bradykinesia, rigidity and resting tremor. In the early phases of disease, these signs may be subtle. Also, PD may present heterogeneously and may also be confused with parkinsonism caused by other disorders like multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration or essential tremor. Assessment of the severity and progression of PD can be done by examination of motor symptoms and application of standardised rating scales. However, the clinical heterogeneity of PD, inter-rater variability and the influence of symptomatic medication may complicate the clinical evaluation. Examining the patient in the “off state”, i.e. after 12 h without any symptomatic antiparkinsonian medication, may only partially overcome the masking effect of antiparkinsonian treatment.The difficulties in primary diagnosis and assessment of disease progression are illustrated by findings from autopsy studies, where the diagnosis of PD before death was found to be incorrect in about a quarter of cases14. In addition, even neurologists associated with a clinic specialising in movement disorders incorrectly diagnose PD in about 10% of cases13. Against this background, objective in vivo markers of dopaminergic degeneration, such as neuroimaging studies, are important for the detection of PD, especially in the early stages of disease, for the assessment of disease severity, and for the monitoring of disease progression.Neuroimaging techniques as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) enable visualisation and measurement of striatal dopaminergic functioning. PET scans with 6-[18F]-fluoro-L-3,4-dihydroxyphenylalanine (F-DOPA) allow quantification of striatal dopa decarboxylase activity and storage capacity of F-dopamine. However, high costs, restricted availability of PET instruments and the difficult production of F-DOPA limit its use.Another neuro-imaging technique is SPECT using [123I] N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) nortropane (FP-CIT). This tracer belongs to a group of compounds derived from cocaine, which has a high affinity for the dopamine transporter (DAT). SPECT scans using the ligand FP-CIT (DaTscan) are cheaper and much more widely available. FP-CIT is a selective and potent DAT imaging agent with a high target to background ratio and rapid clearance from the cerebellum and cortical brain regions. Several studies have shown a reduction in striatal FP-CIT uptake in PD 4, 7, 17, 24, 32, 33, 36, 37compared with healthy controls. Although the uptake mechanism of the two tracers is quite different, FP-CIT SPECT may be a good alternative to F-DOPA PET. To the best of our knowledge, only one study has compared these two imaging modalities in the same subjects. Ishikawa et al. performed

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both FP-CIT SPECT and F-DOPA PET scans in healthy controls and patients with mild PD, but not in patients with an advanced stage of disease15.Therefore, the aim of this study was to compare FP-CIT SPECT with F-DOPA PET in the same group of patients with different stages of PD, in order to study the degree of correlation of the two methods, the feasibility of separating de novo from advanced disease stage, and the relation between tracer uptake and clinical parameters such as motor scores.


PatientsA total of 30 consecutive PD patients were recruited between 2002 and 2003 from the Movement Disorders outpatient clinic of the University Hospital of Groningen, which has a tertiary referral function. Patients were recruited based on their clinical inclusion requirements.The subjects were divided into two distinct groups: de novo PD patients (n=13) and patients in an advanced stage of disease. The de novo group included patients with strong diagnostic evidence of PD based on two of the three cardinal symptoms (rest tremor, bradykinesia and rigidity), a disease duration of less than 3 years, no use of antiparkinsonian medication and a Hoehn and Yahr (H&Y) stage of less than 2.0. The advanced group consisted of PD patients with two of the three cardinal symptoms, with disease duration of 5–15 years, use of antiparkinsonian medication and a H&Y stage of 2.0–4.0. Patients with atypical signs, psychiatric disorders, signs of severe cognitive deterioration or severe cardiovascular co-morbidity, or on medication known to interfere with the dopamine transporter, were excluded from participation.The motor score of patients was determined in the “off” state (after 12 h without any symptomatic antiparkinsonian medication) to avoid confounding effects on the clinical examination. Clinical examination included the motor score of the Unified Parkinson’s Disease Rating Scale (UPDRS-III), which is composed of subscores for speech, facial expression, tremor, rigidity, bradykinesia and axial symptoms (total motor score ranges from 0 to 108). H&Y stage was also determined, which includes several stages of disease severity varying from ‘no signs of disease’ to ‘wheelchair bound or bedridden unless aided’ (1– 5).Each subject underwent a [123I]FP-CIT SPECT and an [18F]F-DOPA PET scan at a mean interval of 61 days (±35 days). Written informed consent was obtained according to the Helsinki convention and the study was approved by the local medical ethics committee.


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F-DOPA PETPatients fasted for at least 4 h before the start of the scan. Patients were allowed to continue their antiparkinsonian medication. Patients taking other medication potentially affecting the dopaminergic system were excluded (see above). On arrival, patients were given carbidopa (2.5 mg/kg) orally. Sixty minutes after the carbidopa dose, 200 MBq of [18F]F-DOPA was administered intravenously. [18F]F-DOPA was prepared in the radiochemical laboratory of the University Hospital Groningen as described elsewhere9. Ninety minutes after administration of the tracer, the subject was positioned in the PET camera in a comfortable head holder, with their orbito-meatal line in a transverse plane. One static 3D acquisition of 6 min was performed with a Siemens HR+PET camera (Siemens, Erlangen, Germany), according to the standard operating procedures protocol of the University Hospital Groningen. In daily clinical practice, static scans are preferred to dynamic scans in patients with PD as dynamic scans take a very long time to perform and are therefore too inconvenient. For estimation of the diagnostic value in this patient group, it was therefore decided to perform static and not dynamic scans. Furthermore, it is strongly suggested that the striatal–occipital ratio (SOR) determined from a static scan can be as accurate as kinetic parameters (such as binding constant Kocc) from a dynamic scan10. Scan data were reconstructed using iterative methods (ordered subsets expectation maximisation) and were corrected for attenuation using a separate ellipse algorithm. An example of an F-DOPA PET scan is shown in Fig. 1a.

FP-CIT SPECTPatients were allowed to continue all medication, including antiparkinsonian medication. Patients were injected with 185 MBq FP-CIT (DaTscan, commercially obtained from Amersham Health, Eindhoven, The Netherlands). No thyroid blocking was given, according to our local operating procedure, as thyroid blocking will not interfere with striatal uptake. After 180 min a SPECT acquisition was performed using a dual-headed gamma camera (Multispect 2, Siemens, Hoffman Estates, IL, USA) with a low-energy high-resolution collimator, 128×128 image matrix, zoom factor 1.23, 40 s per view and 2×64 views. Data acquisition was in agreement with the Dutch National Guidelines and with the guidelines from the manufacturer. Acquisition time was approximately 45 min. Images were acquired in a symmetrical 15% energy window around the photopeak of 123I at 159 keV. System resolution was 12mm full-width at half-maximum at 10 cm. Patients had been carefully positioned in the gamma camera, with their meato-orbito axis in a transverse plane to avoid reorientation during reconstruction, in a special head-holder that allowed a minimal rotation distance. Image data were reconstructed using filtered back-projection and a Butterworth (0.50/6) filter. No attenuation correction was performed (as advised by the manufacturer at the time of the study). An example of an FP-CIT SPECT scan is shown in Fig. 1b.

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Figure 1: Transaxial slices through the striatum in a patient with advanced PD.

Upper row: F-DOPA PET

Lower row: FP-CIT SPECT

Images show comparable activity, but the ipsilateral dysfunction (left) is slightly more severe on SPECT

than on PET.

Image analysisReconstructed PET and SPECT data were both realigned3 to the common co-ordinate system of the stereotactic brain atlas of Talairach and Tournoux. This realignment was performed using standard linear brain normalisation algorithms (SPM software, FIL London, UK). An image analysis algorithm was used to remove skin uptake. A standard set of regions of interest (ROIs) was used to sample both the caudate and putamen and a non-specific reference region in the occipital cortex. Occipital activity was assumed to represent non-specific radioactivity. Ratios of specific to non-specific binding (SORs) were calculated by dividing striatal count density by occipital count density. ROI size-weighted average uptake values of caudate and putamen were used to calculate mean whole striatal binding. Uptake values on the same half of the body as the dominant (and generally initial) side of motor symptoms were called ipsilateral uptake values and those opposite to that side were called contralateral uptake values. Asymmetry indices were used to quantify the degree of asymmetry. These indices were calculated by subtraction of ipsilateral values from contralateral values, divided by the sum of the values of both sides: (contralateral−ipsilateral)/(contralateral+ipsilateral). The two groups of patients were lumped for correlation of clinical scores with uptake values.


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Statistical analysisParametric or non-parametric correlation coefficients were calculated for all correlations of SPECT SOR vs PET SOR as appropriate. The Shapiro-Wilk test was used to examine the presence of a normal distribution of datasets. Sensitivity and specificity values were calculated for PET and SPECT, after performing receiver operator characteristic (ROC) analysis of both SPECT and PET data in both groups. The optimal cut-off levels were derived from the ROC data. If data were not normally distributed, Spearman’s rho was used for correlations. In addition to correlation, linear regression analysis was performed and Bland-Altman plots were constructed. Spearman’s rho was also used to correlate the SOR values of the PET and SPECT studies with the UPDRS-III and H&Y scores and disease duration. SPECT and PET values were entered into separate ANOVA analysis to determine whether there is a significant difference between the distinct clinical stages using each method.

Results

PatientsThirteen patients with de novo PD and 17 patients with advanced stage of disease were included. Table 1 shows the clinical profile of all patients included in the study. More male than female patients participated, but the male to female ratio was similar in both groups (chi-square p=0.41). Mean age in the de novo PD group was 54 years [standard deviation (SD) =11] years, with mean disease duration of 1.3 (SD=0.5) years. Mean UPDRS-III score was 20 (SD=8) and the median H&Y stage was 1.5. In the group with advanced stage of disease, mean age was 64 (SD=6) years and mean disease duration was 9.4 (SD=3.1) years. In this group, mean UPDRS-III score was 33 (SD=11) and median H&Y stage was 2.5. One de novo PD patient and one patient with advanced stage of disease refused clinical examination in the “off state”. As might have been expected, H&Y stage, UPDRS-III score and disease duration were significantly lower in the de novo PD group than in the advanced PD group (p=0.003 for mean age; p<0.0001 for H&Y stage, p=0.002 for UPDRS-III and p<0.0001 for disease duration).

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Table 1: Clinical profile of all patients

Patient Sex Stage Disease H&Y UPDRS-III Age (yr)duration (yr)

1 M De novo 2 2 25 612 M De novo 2 1.5 21 503 F De novo 1 1.5 14 414 M De novo 1 2.5 33 615 M De novo 1 2 27 696 M De novo 1 5 19 327 M De novo 2 1.5 23 548 M De novo 1 2 27 479 M De novo 2 1 8 6610 F De novo 1 1 NA 6111 F De novo 1 1.5 21 6712 M De novo 3 1 16 4113 M De novo 1 1 6 5314 F Advanced 7 2.5 30 6015 M Advanced 7 NA NA 6916 M Advanced 10 2.5 24 6517 M Advanced 5 2 22 6718 M Advanced 11 2.5 38 6719 F Advanced 7 4 42 5520 M Advanced 13 3 37 6521 M Advanced 10 3 40 5522 M Advanced 13 2 11 5823 M Advanced 11 3 58 7124 M Advanced 14 2.5 24 7125 M Advanced 8 3 29 5926 M Advanced 14 3 31 7527 M Advanced 6 3 45 6428 M Advanced 11 2.5 31 5629 M Advanced 6 2 39 7030 M Advanced 6 2 27 70

M = male F = femaleNA = not available

Correlation of F-DOPA PET and FP-CIT SPECTStriatal SORFP-CIT ranged from 1.32 to 2.50 and striatal SORF-DOPA ranged from 1.66 to 2.51. Mean striatal SORFP-CIT was 1.95 for the total group, 2.12 for the de novo patients and 1.81 for the patients with an advanced stage of disease. The area under the ROC curve for FP-CIT SPECT was 0.79, and the optimal cut-off SOR to separate the groups was determined to be 1.95. Mean striatal SORF-DOPA of the total group was 2.05. For the de novo patients, striatal SORF-DOPA was 2.20 and for the advanced patients it was


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1.94. The area under the ROC curve for F-DOPA PET was 0.86, and the optimal cut-off SOR to separate the groups was determined to be 2.05. In general, uptake values of FP-CIT show a greater variability than uptake values of F-DOPA. Striatal SORF-DOPA in healthy volunteers has previously been reported as above 2.41, 22. Normal values for striatal SORFP-

CIT are less well known, but appear to vary around 3–3.5, also depending on the method of acquisition and processing. For the whole group together and for the whole striatum, SORF-DOPA correlated well with SORFP-CIT (Fig. 2): r=0.78, p<0.0001. Also SORF-DOPA and SORFP-CIT for putamen and caudate separately were highly correlated (r=0.84, p<0.0001 for putaminal uptake and r=0.74, p<0.0001 for caudate uptake).Also, in the subgroups of de novo and advanced PD patients, whole striatal uptake on the two scans correlated significantly (r=0.77, p=0.0009 for de novo PD patients and r=0.57, p=0.016 for advanced PD patients). When comparing ipsilateral and contralateral striatal SORs of the two scanning methods we found good correlations (r=0.77, p<0.0001 for ipsilateral uptake values and r=0.76, p<0.0001 for contralateral uptake values).The mean difference between SORF-DOPA and SORFP-CIT was 0.11. However, the difference between F-DOPA uptake and corresponding FP-CIT uptake was not constant across the range of measured values, but decreased as F-DOPA uptake and FP-CIT uptake increased (Bland-Altman plot: Fig. 3).

Figure 2: Correlation between striatal FP-CIT uptake and striatal F-DOPA uptake in all 30 patients

(r = 0.78, p < 0.0001).

Regression line: F-Dopa mean striatum = 1.13 + 0.48 FP-CIT mean striatum

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Figure 3: Bland-Altman plot of FP-CIT SPECT and F-DOPA PET. At low uptake values (in advanced

disease), F-DOPA is more positive. At higher uptake values (in early disease), FP-CIT is more positive.

Regression line: F-Dopa – FP-CIT = 1.16 - 0.53 (F-Dopa + FP-CIT) / 2

Correlation of SOR and disease severity

H&Y stageBoth striatal uptake of F-DOPA and striatal uptake of FP-CIT correlated moderately with H&Y stage (Fig. 4a,b): Spearman’s rho=0.52, p=0.004 for both scanning methods. Putaminal and caudate uptake showed a significant relationship with H&Y stage, although caudate SOR correlated less closely with H&Y stage than did putaminal SOR.

UPDRS-IIIUPDRS-III score correlated moderately with striatal uptake of F-DOPA and FP-CIT (Fig. 4c,d): Spearman’s rho=−0.38 for F-DOPA, p=0.05 and Spearman’s rho=−0.45 for FP-CIT, p=0.02.Subanalyses of putaminal and caudate uptake of F-DOPA and FP-CIT showed similar results to whole striatal uptake for correlation between uptake and UPDRS-III scores. Both correlation and significance level increased slightly when using asymmetry indices: for correlation between asymmetry index UPDRS-III and asymmetry index striatal F-DOPA uptake, Spearman’s rho=−0.58, p=0.002 and for correlation between asymmetry index UPDRS-III and asymmetry index striatal FP-CIT uptake, Spearman’s rho=−0.49, p=0.012.


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Figure 4: Correlation between motor scores (H&Y stage and UPDRS-III) and putaminal uptake for PET

and SPECT in all 30 PD patients. Ellipses form the 95% confidence interval.

Disease durationSignificant but moderate correlations were found between disease duration and striatal F-DOPA uptake (Spearman’s rho=−0.59, p=0.001) and between disease duration and striatal FP-CIT uptake (Spearman’s rho=−0.49, p=0.01) (Fig. 5). From the whole dataset, we calculated a mean annual decrease in uptake, which proved equal for striatum, putamen and caudate and for both F-DOPA and FP-CIT values at a value of 3% decrease per year. The results are also presented in Table 2.

Figure 4a: H&Y stage vs F-DOPA uptake:

Spearman’s rho = -0.52, p = 0.004

Figure 4b: H&Y stage vs FP-CIT uptake:

Spearman’s rho = -0.52, p = 0.004

Figure 4c: UPDRS-III vs F-DOPA uptake:

Spearman’s rho ρ = -0.38, p = 0.05

Figure 4d: UPDRS-III vs FP-CIT uptake:

Spearman’s rho ρ = -0.45, p = 0.02

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Figure 5: Correlation between disease duration and striatal uptake. Ellipses form the 95% confidence

interval.

Figure 5a: disease duration vs striatal F-DOPA uptake: Spearman’s rho ρ = -0.59, p = 0.001

Figure 5b: disease duration vs striatal FP-CIT uptake: Spearman’s rho ρ = -0.49, p = 0.01


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Table 2: Overview of relationship between motor scores and uptake valuesScan ROI Motor evaluationmethod UPDRS-III (p) H&Y (p) Disease

duration (p)FP-CIT Striatum −0.45 (0.02) −0.52 (0.004) −0.49 (0.01)

Putamen −0.45 (0.02) −0.51 (0.004) −0.48 (0.008)Caudate −0.47 (0.01) −0.49 (0.007) −0.33 (0.07)Striatum ipsilateral −0.48 (0.01) −0.55 (0.002) −0.47 (0.009)Striatum contralateral −0.34 (0.07) −0.37 (0.05) −0.29 (0.13)Putamen ipsilateral −0.48 (0.01) −0.54 (0.002) −0.44 (0.02)Putamen contralateral −0.26 (0.18) −0.34 (0.07) −0.38 (0.04)Caudate ipsilateral −0.43 (0.02) −0.51 (0.005) −0.45 (0.020)Caudate contralateral −0.38 (0.05) −0.35 (0.07) −0.06 (0.77)

F-DOPA Striatum −0.38 (0.05) −0.52 (0.004) −0.59 (0.009)Putamen −0.36 (0.06) −0.54 (0.003) −0.70 (0.0003)Caudate −0.35 (0.07) −0.44 (0.02) −0.41 (0.03)Striatum ipsilateral −0.47 (0.01) −0.62 (0.0003) −0.65 (0.0001)Striatum contralateral −0.24 (0.22) −0.34 (0.07) −0.49 (0.006)Putamen ipsilateral −0.46 (0.01) −0.62 (0.0003) −0.74 (0.0001)Putamen contralateral −0.12 (0.54) −0.25 (0.18) −0.49 (0.008)Caudate ipsilateral −0.43 (0.02) −0.53 (0.003) −0.42 (0.02)Caudate contralateral −0.15 (0.44) −0.25 (0.18) −0.38 (0.04)

ROI = region of interest

Discrimination between patients with de novo and patients with advanced PDPatients with de novo PD could be discriminated from patients with more severe stages of the disease with both methods (p=0.0002 for F-DOPA and p=0.01 for FP-CIT) (Fig. 6). Sensitivity of FP-CIT SPECT for discrimination between de novo and advanced PD patients was 0.88, while specificity was 0.70. For F-DOPA PET, sensitivity for discrimination between the two patient groups was 0.88 and specificity, 0.77. The differences between PET and SPECT were not significant. When putaminal values were used instead of striatal values, sensitivity and specificity of both methods remained approximately the same (sensitivity and specificity for FP-CIT=0.77 and 0.71 respectively and sensitivity and specificity for F-DOPA=0.76 and 0.85 respectively).

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Figure 6: Discrimination between the two groups of patients by means of F-DOPA PET and FP-CIT

SPECT

Figure 6a: ANOVA analysis: F-DOPA PET

Figure 6b: ANOVA analysis: FP-CIT SPECT


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Discussion

In this study, we have compared FP-CIT SPECT and F-DOPA PET in terms of their ability to quantify the activity of the striatal dopaminergic system in patients with different stages of PD. We found that FP-CIT uptake and F-DOPA uptake correlate highly with each other and that both scanning methods correlate reasonably with motor scores and disease duration. The two tracers were equally able to discriminate de novo from advanced PD patients with a high sensitivity and specificity.Both tracers are in vivo markers of the presynaptic dopaminergic system. However, the two scanning methods are based on different biochemical processes. F-DOPA PET uptake reflects the activity of the decarboxylating enzyme AADC and the storage capacity of F-dopamine in the nerve terminals21, 22, whereas FP-CIT SPECT measures the activity of DAT12. This results in a different approach to assessment of the functioning of the presynaptic dopaminergic system, and the question arises as to whether it is permissible to compare these methods with each other. Lee et al. applied three tracer methods in vivo in patients with PD20. In that study they compared striatal PET measurements using [11C]dihydrotetrabenazine (labelling the vesicular monoamine transporter type 2), [11C]methylphenidate (labelling the plasma membrane DA transporter, in a similar way to FP-CIT) and [18F]DOPA in patients with PD. Striatal F-DOPA uptake was higher in parkinsonian patients than the uptake of methylphenidate. It was postulated that this difference in tracer uptake may be due to an upregulation of AADC and thereby of F-DOPA uptake, and a downregulation of DAT and thus of FP-CIT uptake, although no direct proof was provided for this. Experimental animal studies support this contention and suggest that loss of DA neurones is functionally compensated by an increase in dopamine release and by down-regulation of DA reuptake in an attempt to maintain dopamine levels42. Also, β-CIT binding and L-DOPA uptake decrease in parallel with decrease in dopamine neurones in several stages of PD. However, the decrease in β-CIT binding mirrors more closely the reduction in dopaminergic neurones than does the decrease in L-DOPA uptake, suggesting that β-CIT binding is a superior indicator of dopaminergic neurone loss16. These different reactions of the two tracers in response to a reduction in dopamine imply a different degree of decrease in striatal uptake of the two tracers, as striatal FP-CIT uptake will be reduced in an earlier phase of disease than will F-DOPA uptake. This is also suggested by our findings. Our results are also in agreement with the results described by Ishikawa et al., who found a correlation coefficient of 0.77 (p<0.0001) for the correlation between striatal FP-CIT uptake and striatal F-DOPA uptake15. Although our study demonstrates a significant correlation between F-DOPA PET measures and corresponding FP-CIT SPECT measures, differences are still observed between the uptake values. Striatal FP-CIT shows a greater variability in uptake than does striatal F-DOPA (Fig. 1). As indicated by the Bland-Altman plot (Fig.

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2), at low values of FP-CIT uptake, F-DOPA values are higher than FP-CIT uptake values, whereas at high values of FP-CIT uptake, F-DOPA uptake values are lower than those of FP-CIT. Furthermore, when specific uptake of FP-CIT equals aspecific uptake (SOR=1), F-DOPA still shows specific uptake. These findings may be due to technical problems such as lower resolution of SPECT scans compared with PET scans, differences in signal to noise ratio and scattered radiation, but they may also be the result of the above-mentioned different biochemical mechanisms of the two tracer uptake methods.This study demonstrates that the uptake values for the two tracers show a similar but moderate correlation with UPDRS motor scores (Fig. 4c,d). After reducing interindividual variability by calculating asymmetry indices, this correlation increases but remains moderate. Many others have found a statistically significant correlation between UPDRS motor scores and SORF-DOPA and SORFP-CIT, but with higher correlation coefficients varying from 0.51 to 0.812,

4, 10, 15, 26, 29, 36, 41. However, other investigators did not find any statistically significant correlation between UPDRS-III and striatal uptake of CIT7, 37. This discrepancy in findings may be due to methodological differences in data processing. Patients were clinically examined 12 h after withdrawal of antiparkinsonian medication. It is assumed that any therapeutic effect of the medication is washed out at that time. Nevertheless, long-lasting effects of antiparkinsonian therapy on motor signs should not be ignored, and it is possible that such effects, and their interindividual variation, also partially explain the differences in correlation between clinical measures and striatal uptake. The moderate correlation between motor scores and tracer uptake further underlines the difficulties in clinical assessment even when it is standardised. This increases the value of imaging studies in general. We did find a good correlation between H&Y stage and striatal uptake, with a higher correlation coefficient than for the correlation between SOR and the motor part of the UPDRS. This was also found by others7, 26, 33. Our data show that the uptake values of the two tracers correlated well in a similar way with disease duration (Fig. 5) and that the different patient groups could be discriminated with both methods (Fig. 6). In addition, correlation coefficients were highest for ipsilateral SOR values, as expected because the disease starts on the ipsilateral side.Many studies suggest a significant age-dependent decline in SORFP-CIT

15, 17, 19, 35, 38, 40. On the other hand, it has been suggested that adjusting SORFP-CIT for age barely alters accuracy in the assessment of nigrostriatal functioning in PD. Decline with age in SORFP-

CIT is therefore not sufficiently large as to require a specific correction in the assessment of parkinsonism15, 27, or such correction may result in only a minimal improvement in the correlation of striatal DAT binding with UPDRS-III26. A few authors mention an ageing effect with F-DOPA uptake as well39, but many others have not been able to confirm this11,

15, 31. The absence of an ageing effect on striatal F-DOPA uptake may be explained by up regulation of AADC activity as dopaminergic neurones decline in normal ageing. This is


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supported by post-mortem studies showing little or no decrease in striatal AADC activity in normal senescence18.Some studies suggest gender effects, as striatal FP-CIT binding ratios have been found to be significantly higher in females than in males. We also looked for a gender effect in our data but, due to the small number of women participating in the study, we were not able to find any significant effect.The annual decrease in uptake in PD patients is, according to our data, 3% per year. This is in line with the results of others19, 28, 30. Morrish et al. reported a progression rate of 4.7% of the normal mean per year in a group of patients with PD with a mean disease duration of 39 months, using F-DOPA PET scans25. An annual decrease of 11.2% was found by Marek et al. using β-CIT SPECT scans23. Pirker and co-workers reported an annual reduction of striatal β-CIT SPECT binding of 3–4.5%27. An annual decline of 3% in striatal 123I-IPT binding was found in 11 PD cases followed over 12 months by Tatsch et al34. It has been suggested that progression will be faster in those regions which are less affected in the beginning, like the caudate nucleus8. We were not able to confirm this, as annual decrease was equal in caudate and putamen. Our data show that FP-CIT SPECT scans have a high sensitivity and specificity for discriminating de novo parkinsonian patients from patients with advanced stage of disease. Sensitivity and specificity are at levels comparable to those of F-DOPA PET scans, based on our method of separation of patients into the de novo and advanced groups on clinical grounds, which is notoriously difficult. In this study we used the H&Y stage as well as the duration of symptoms. However, it cannot be concluded from our data whether it is also possible to discriminate parkinsonian patients from healthy persons. This is important for early detection of patients with PD. Several studies have demonstrated the utility of FP-CIT SPECT scans for discriminating patients with PD from healthy controls5-7,

17, 24, 32, 33, 36, 37. Ishikawa et al. showed that FP-CIT SPECT scans and F-DOPA PET scans were able to discriminate PD patients from healthy controls with comparable accuracy, even when the analysis was restricted to patients with H&Y stage 115. Although it has been widely demonstrated that parkinsonian patients can be discriminated from healthy controls with either of these scanning methods, this has only been investigated in patients with an obvious clinical diagnosis, and no studies have been performed in patients with a questionable diagnosis of PD. In daily clinical practice, helping to differentiate between PD and a healthy state will be especially interesting in patients with debatable clinical symptoms. The high sensitivity and specificity of FP-CIT SPECT scans in discriminating between different stages of PD justify extension of the study to include healthy controls in order to allow comparison of the ability of the two scanning methods to discriminate patients with PD from patients with suspicious symptoms but without PD.

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ConclusionBoth FP-CIT SPECT and F-DOPA PET can be used to measure the presynaptic dopaminergic system in vivo, and they show equally good ability to separate the early from the advanced stage of PD. In our study the good correlation between F-DOPA uptake values and FP-CIT uptake values, as well as the moderate correlation between striatal uptake and clinical findings, underscores this conclusion. It remains to be seen whether, in daily clinical practice, either method will allow similarly good discrimination between patients with early PD and patients with suspicious symptoms but without PD.

AcknowledgementsWe thank GE Health for financial support and John P Seibyl, MD (Molecular Neuro Imaging, New Haven, CT, USA) for valuable advice.The experiments comply with the current laws of the Netherlands, including approval of the local medical ethics committee.

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27. Pirker W, Holler I, Gerschlager W, et al: Measuring the rate of progression of Parkinson’s disease over a 5-year period with beta-CIT SPECT. Movement Disorders 18:1266-1272, 2003

28. Pohjalainen T, Rinne JO, Nagren K, et al: Sex differences in the striatal dopamine D2 receptor binding characteristics in vivo. Am J Psychiatry 155:768-773, 1998

29. Ribeiro MJ, Vidailhet M, Loc’h C, et al: Dopaminergic function and dopamine transporter binding assessed with positron emission tomography in Parkinson disease. Arch Neurol 59:580-586, 2002

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6Direct comparison of FP-CIT SPECT and F-DOPA PET

in patients with Parkinson’s Disease and healthy

controls

S.A. Eshuis, P.L. Jager, R.P. Maguire, S.Jonkman, R.A. Dierckx, K.L. Leenders

Eur J Nucl Med Mol Imaging 2009;36:454-462

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Abstract

Diagnosing Parkinson’s disease (PD) on clinical grounds may be difficult, especially in the early stages ofthe disease. F-DOPA PET and FP-CIT SPECT scans are able to determine presynaptic dopaminergic activity in different ways. The aim of this study was to determine and compare the sensitivity and specificity of the two methods in the detection of striatal dopaminergic deficits in the same cohort of PD patients and healthy controls.

Movement disorder specialists recruited 11 patients with early-stage PD and 17 patients with advanced PD. The patients underwent both an FP-CIT SPECT scan and an F-DOPA PET scan. In addition, 10 FP-CIT SPECT scans or 10 F-DOPA PET scans were performed in 20 healthy controls. A template with regions of interest was used to sample tracer activity of the caudate, putamen and a reference region in the brain. The outcome parameter was the striatooccipital ratio (SOR). Normal SOR values were determined in the controls. The sensitivity and specificity of both scanning methods were calculated.

FP-CIT SPECT and F-DOPA PET scans were both able to discriminate PD patients from healthy controls. For the early phases of the disease, sensitivity and specificity of the contralateral striatal and putaminal uptake of FP-CIT and F-DOPA was 100%. When only caudate uptake was considered, the specificities were 100% and 90% for FPCIT and F-DOPA, respectively, while the sensitivity was 91% for both scanning techniques.

FP-CIT SPECT and F-DOPA PET scans are both able to diagnose presynaptic dopaminergic deficits in early phases of PD with excellent sensitivity and specificity.

Keywords FP-CIT SPECT. F-DOPA PET.Parkinson’s disease . Diagnosis . Movement disorders

Comparison of FP-CIT and F-DOPA in patients with PD and healthy controls

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Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder, characterized by progressive loss of dopaminergic neurones in the substantia nigra. In the early stages of the disease it may be difficult to diagnose PD on the basis of clinical signs and symptoms. Diagnosing PD can also be hampered by the existence of nonneurodegenerative disorders, in which the presynaptic dopaminergic system is still intact, but which may mimic parkinsonism, such as essential tremor, vascular parkinsonism or drug-induced parkinsonism. A nonmovement disorder neurologist may misdiagnose up to 25% of patients with established parkinsonism of all sorts when compared with post-mortem pathology16, 32. In contrast, movement disorder specialists can almost always correctly diagnose idiopathic PD when all the relevant clinical information is obtained. In such circumstances, the positive predictive value of the clinical diagnosis PD has been found to be 98.6%15. These studies relate to patients with advanced PD. No data are available to assess the situation in patients with early undiagnosed PD who on clinical grounds are indeed likely to have PD, but in whom this cannot yet be confirmed by response to medication or by the time course. This question is of even more interest for those patients in whom the clinician is not yet sure about the diagnosis, but would like to know whether a striatal dopaminergic defect is present or not15. Therefore, to facilitate diagnosing PD in the above-mentioned circumstances, auxiliary examinations are needed. For this purpose, conventional imaging techniques of the brain, such as CT and MRI scans, are not useful, because the brain structure is usually not greatly altered in early PD and dopaminergic biochemical activity cannot be positively documented by these techniques. Radiotracer neuroimaging techniques using positron emission tomography (PET) or single photon emission computed tomography (SPECT) can be helpful in visualizing and measuring striatal dopaminergic activity27, 28. PET scans using 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (F-DOPA) enable measurement of striatal levodopa decarboxylase activity and trapping of F-dopamine in synaptic vesicles, which are decreased in PD1, 11, 27,

28, 39. It has been suggested that in the early stages of this disease, levodopa decarboxylase activity is upregulated19, 26, although this has not yet been proved. Such factors, however, may influence the sensitivity of this technique for diagnosing defects in the nigrostriatal system.Using SPECT, the uptake of tracers with a high affinity for the dopamine transporter (DAT) can be measured. DATs, located on dopaminergic nerve endings, participate in the reuptake mechanism of dopamine into presynaptic terminals and are modulated by concentrations of endogenous dopamine12. A decrease in DAT density in the striatum has been associated with PD21, 34. DAT imaging can therefore be used as a marker for the degree of malfunction or loss of dopaminergic nerve endings. A selective and potent DAT imaging agent is [123I]

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N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) nortropane (FP-CIT). SPECT imaging with FP-CIT produces a high target to background ratio. Several studies have demonstrated that striatal FP-CIT uptake is reduced in patients with PD compared to healthy controls5, 18, 22,

29, 30, 40, 41, 43, 44. It has been shown that striatal uptake values of FP-CIT SPECT and F-DOPA PET correlate well with each other in patients with different stages of PD9, 18.It has been suggested that DATs are downregulated as an early response to reduction of the amount of endogenous dopamine. This would result in a decreased striatal binding of FP-CIT in early phases of PD. Therefore FP-CIT might be more sensitive than F-DOPA for detecting early striatal dopaminergic deficits.The aim of this study was to determine and to compare intraindividually the sensitivity and specificity of the two tracer methods in the detection of striatal dopaminergic nerve terminal malfunctioning in the same cohort of PD patients and healthy controls.


PatientsA total of 29 consecutive patients with strong clinical evidence of having PD were recruited between 2002 and 2003 from the Movement Disorders outpatient clinic of the University Hospital of Groningen, which has a tertiary referral function. Enrolment and clinical evaluation of participating patients was standardized and carried out by experienced movement disorder specialists. At clinical follow-up after more than 1 year after the two scans, 28 of the patients were still diagnosed with PD, confirmed by a documented response on symptomatic antiparkinson medication or, in drug-naive patients at follow up, worsening of extrapyramidal signs without any other symptoms. One patient did not respond to symptomatic antiparkinson medication and did not show any worsening of extrapyramidal signs after a period of 3 years of clinical follow-up. This patient was therefore excluded from further analyses.The patients were divided into two groups: 11 patients (8 men and 3 women) with early-stage PD and 17 patients (15 men and 2 women) with advanced PD. The 11 patients with early-stage PD showed strong clinical evidence of PD based on two of the three cardinal symptoms (rest tremor, bradykinesia and rigidity), had a disease duration of less than 3 years, were not using antiparkinson medication, and had a Hoehn & Yahr (H&Y) stage of less than 2.0. The 17 patients with advanced PD showed two of the three cardinal symptoms, had a disease duration of 5–15 years, were using antiparkinson medication with a documented response, and had a H&Y stage of 2.0–4.0. Patients with atypical signs, psychiatric disorders, signs of severe cognitive deterioration, severe


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cardiovascular comorbidity or on medication known to interfere with the DAT or catechol O-methyltransferase inhibitors, were excluded from the study.A total of 20 healthy controls, 11 men and 9 women, in the same age range as the patients, were recruited. Subjects with psychiatric disorders, signs of severe cognitive deterioration, severe cardiovascular comorbidity or on medication known to interfere with the DAT, were excluded from the study.Each patient with PD underwent a [123I]FP-CIT SPECT scan and a one-time-frame static F-DOPA PET scan. Ten healthy controls underwent a [123I]FP-CIT SPECT scan and ten others underwent a static F-DOPA PET scan. Written informed consent was obtained according to the Helsinki Convention and the study was approved by the local medical ethics committee. The ethics committee did not allow performance of both F-DOPA and FP-CIT scans in the same healthy control subjects.

F-DOPA PET scansSubjects fasted for at least 4 h before the start of the scan. Patients were allowed to continue their antiparkinson medication. On arrival subjects were given carbidopa (2.5 mg/kg) orally, and 60 min after the carbidopa dose, received 200 MBq of F-DOPA intravenously. F-DOPA was prepared in the radiochemical laboratory of the University Medical Center Groningen as described elsewhere6. After a further 90 min from administration of the tracer, the subject was positioned in the PET camera (Siemens HR+; Erlangen, Germany) in a comfortable head holder with the orbitomeatal line in a transverse plane and a one-time-frame 3-D acquisition of 6 min duration was performed according to the standard operating procedures protocol of the University Medical Center Groningen. In daily clinical practice, static scans are preferred to dynamic scans in patients with PD as dynamic scans take a long time to perform and are therefore inconvenient. For estimation of the diagnostic value in this patient group it was therefore decided to perform static and not dynamic scans. Furthermore, static F-DOPA PET scans are preferred for a correct comparison with (static) FP-CIT SPECT scans. Finally, it is suggested that the striatal-occipital ratio (SOR) determined from a static scan can be as accurate as kinetic parameters, such as the binding constant Kocc, from a dynamic scan7. Scan data were reconstructed using iterative methods (ordered subsets expectation maximization) and were corrected for attenuation using a separate ellipse algorithm, according to the standard operating procedure in our centre for clinical PET scans.

FP-CIT SPECT scansPatients were allowed to continue all medication, including antiparkinson medication. Subjects were injected with 185 MBq FP-CIT (DaTscan, obtained commercially from GE, Eindhoven, The Netherlands). No thyroid blocking was given. After 180 min a SPECT

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acquisition was performed using a dual head gamma camera (Multispect 2; Siemens, Hoffman Estates, IL) with a LEHR (low-energy high-resolution) collimator, 128×128 image matrix, zoom factor 1.23, 40 s per view and 2×64 views. Data acquisition was in agreement with the Dutch National Guidelines and with the guidelines from the manufacturer. Acquisition time was approximately 45 min. Images were acquired in a symmetric 15% energy window around the photopeak of 123I at 159 keV. The system resolution was 12 mm FWHM at 10 cm. Subjects were carefully positioned in the gamma camera in a special head-holder which allowed minimal rotational distance with the orbitomeatal axis in a transverse plane to avoid reorientation during reconstruction. Image data were reconstructed using filtered back-projection and a Butterworth (0.50/6) filter. No attenuation correction was performed. As the aim of our study was to compare the two scanning methods in daily clinical practice, reconstruction of data were performed according to the standard operating procedures in our centre.

Image analysisReconstructed PET and SPECT data were realigned2 to the common coordinate system of the Stereotactic Brain Atlas of Talairach and Tournoux. This realignment was performed using standard linear brain normalization algorithms (SPM software, FIL, London, UK). An image analysis algorithm was used to remove skin uptake. A standard set of regions of interest (ROIs) was used to sample both the caudate and putamen and a non-specific reference region in the occipital cortex. Ratios of specific to non-specific binding (SOR) were calculated by dividing the striatal count density by the occipital count density. ROI size-weighted average uptake values of the caudate and putamen were used to calculate mean whole striatal binding. In patients, uptake values in the same half of the body as the dominant (and generally initial) side of motor symptoms were called ipsilateral uptake values and those opposite to that side were called contralateral uptake values.

Statistical analysisShapiro-Wilk test was used to examine the normality of the distributions of the parameters of the different datasets. Normal values were calculated as the mean of all healthy controls ±2 standard deviations (SD). Mean values in subgroups were compared using Student’s t test, and t tests were also used to determine if uptake values in male controls differed significantly from those in female controls. If values were not normally distributed, Wilcoxon’s rank sum was used. One-sided p values of less than 0.05 were considered statistical significant. The results of the FP-CIT SPECT and F-DOPA PET scans were initially considered abnormal when the uptake values were less than the mean values minus 2 SD of the healthy controls. Next receiver operating curves (ROC) were produced and the sensitivity and specificity of the two scanning techniques for discriminating patients with


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early stage PD from healthy controls were calculated. Sensitivity and specificity of F-DOPA and FP-CIT scans were compared using McNemar test. For determining the correlation between uptake values and age, Pearson’s product was used if the data were normally distributed and Spearman’s rho if the data were not normally distributed.

Results

Group characteristicsThe mean age of the healthy controls was 60.8 years (SD 8.4, range 40–74 years), the mean age of the patients with early-stage PD was 51.3 years (SD 10.1, range 33–66 years), and the mean age of the patients with advanced PD was 64.5 years (SD 6.3, range 46–71 years; see Table 1). The ages of the two groups of healthy controls were similar (t test, p=0.78). The age of the healthy controls was not significantly different from the ages of the patients in both groups (t test, p=0.42). However, as expected, the age of patients with advanced disease was higher than the age of patients with early-stage disease (t test, p=0.001). There was no significant difference in age between patients with advanced disease and healthy controls (t test, p=0.36).The male:female ratio was similar in the two patient groups (chi-squared test; p=0.30). However, the male: female ratio was higher in patients than in controls (chi-squared test; p=0.04; see Table 1).

Table 1: Subject characteristics FP-CIT

controls de novo advancedn 10 11 17Age 60.2 (8.7) 52.3 (11.4) 63.9 (7.5)M: F 5:5 8:3 15:2

F-DOPA

controls de novo advancedn 10 11 17Age 61.4 (8.5) 52.3 (11.4) 63.9 (7.5)M: F 5:5 8:3 15:2

n = number of participants

Age in mean years (standard deviation)

M: F = male: female ratio

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Group resultsIn the control subjects, the mean SOR values for the caudate, putamen and striatum, respectively, were 3.16 (range 2.70– 3.86, SD 0.38), 2.65 (range 2.39–3.06, SD 0.21) and 2.90 (range 2.74–3.28, SD 0.26) for FP-CIT, and 2.69 (range 2.32–3.17, SD 0.25), 2.70 (range 2.29–3.42, SD 0.30) and 2.69 (range 2.31–3.30, SD 0.27) for F-DOPA (see Table 2).SORFP-CIT and SORF-DOPA for the left versus right caudate and the left versus right putamen were identical in the control subjects (all p>0.2). Examples of F-DOPA PET scans and FP-CIT SPECT scans are shown in Fig. 1.

Table 2: Uptake values of healthy controls and PD patientsFP-CIT

Patient group Region Mean SD 2 SD-intervalControls (n=2x10) Mean caudate 3.16 0.31 2.54 - 3.78

Mean putamen 2.65 0.23 2.19 - 3.11Mean striatum 2.90 0.20 2.50 - 3.10

De novo (n=11) Contralateral caudate 2.22 0.28 1.66 - 2.78Contralateral putamen 1.82 0.16 1.50 - 2.14Contralateral striatum 2.02 0.17 1.68 - 2.36

Advanced (n=17) Contralateral caudate 1.91 0.47 0.97 - 2.85Contralateral putamen 1.60 0.21 1.18 - 2.02Contralateral striatum 1.76 0.32 1.12 - 2.40

F-DOPA

Patient group Region Mean SD 2 SD-interval

Controls (n=2x10) Mean caudate 2.69 0.25 2.19 - 3.19Mean putamen 2.70 0.30 2.10 - 3.30Mean striatum 2.69 0.27 2.15 - 3.23

De novo (n=11) Contralateral caudate 2.27 0.15 1.97 - 2.57Contralateral putamen 1.91 0.15 1.61 - 2.21Contralateral striatum 2.09 0.14 1.81 - 2.37

Advanced (n=17) Contralateral caudate 2.05 0.21 1.63 - 2.47Contralateral putamen 1.76 0.11 1.54 - 1.98Contralateral striatum 1.91 0.15 1.61 - 2.21

n = number of participantsSD = standard deviation

Figure 1a: F-DOPA PET scan healthy control

Figure 1c: FP-CIT SPECT scan healthy control


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Figure 1: F-DOPA PET scans of healthy controls and de novo PD patients (respectively figure 1a en

1b) and FP-CIT SPECT scans of healthy controls and de novo PD patients (respectively figure 1c en 1d)

Disease discriminationContralateral values The SORFPCIT and SORF-DOPA values for the caudate, putamen and striatum contralateral to the symptomatic side were significantly reduced in patients with early-stage PD compared with controls (t test; p<0.0002 for all comparisons; see also Fig. 2). Ipsilateral uptake values were also significantly reduced (t test; SORFP-CIT p<0.001 for all comparisons). The measured uptake values of both tracers for the contralateral putamen and striatum did not show any overlap between patients and controls. However, these groups could not be completely separated by means of the calculated 2×SD interval (above and below the mean) of SORF-DOPA for the contralateral putamen and striatum (Table 2).In patients with early-stage PD, sensitivity and specificity based on contralateral putaminal and striatal SOR was 100% for FP-CIT SPECT and for F-DOPA PET (see Table 3). Sensitivity based on contralateral caudate uptake values was the same (91%) for FP-CIT and F-DOPA,

A

C

B

D

Figure 1a: F-DOPA PET scan healthy control Figure 1b: F-DOPA PET scan de novo PD

Figure 1c: FP-CIT SPECT scan healthy control Figure 1d: FP-CIT SPECT scan de novo PD

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while specificity was 90% for F-DOPA and 100% for FP-CIT. The ROC showed area under the curve (AUC) values of 0.98, 1.00 and 1.00 for the contralateral caudate, putaminal and striatal values, respectively, of FP-CIT, and AUC values of 0.95, 1.00 and 1.00 for the contralateral caudate, putaminal and striatal values, respectively, of F-DOPA. For the whole group of patients with early and advanced stages of disease, the specificity and sensitivity were 1.00 for both contralateral putaminal and striatal SORFP-CIT and SORF-DOPA (see Table 3). Also the AUC values of the ROC curves were equal for FP-CIT and for F-DOPA uptake for the contralateral putamen and striatum. There were no significant differences in sensitivity and specificity between the two scanning techniques (McNemar test).

Table 3: Sensitivity and specificity based on uptake valuesDe novo versus controls

FP-CIT F-DOPARegion Sensitivity Specificity AUC Sensitivity Specificity AUC

CaudateContralateral 91% 100% 0.98 91% 90% 0.95Ipsilateral 82% 90% 0.86 64% 90% 0.80PutamenContralateral 100% 100% 1.00 100% 100% 1.00Ipsilateral 100% 100% 1.00 91% 90% 0.95StriatumContralateral 100% 100% 1.00 100% 100% 1.00Ipsilateral 73% 100% 0.95 82% 90% 0.92

All patients versus controls

FP-CIT F-DOPARegion Sensitivity Specificity AUC Sensitivity Specificity AUC

CaudateContralateral 100% 91% 0.98 90% 96% 0.98Ipsilateral 100% 82% 0.94 90% 86% 0.91PutamenContralateral 100% 100% 1.00 100% 100% 1.00Ipsilateral 100% 96% 0.99 100% 89% 0.98StriatumContralateral 100% 100% 1.00 100% 100% 1.00Ipsilateral 100% 86% 0.98 90% 93% 0.97

AUC = area under curve in receiver operating characteristics curve


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Figure 2: Contralateral uptake values in healthy controls and de novo PD patients for FP-CIT (figure

2a) and F-DOPA (figure 2b).

Figure 2a: FP-CIT uptake values in HC and de novo PD patients (contralateral values)

Closed diamonds are healthy controls, open triangles are de novo PD patients.

Figure 2b: F-DOPA uptake values in HC and de novo PD patients (contralateral values)

Closed diamonds are healthy controls, open triangles are de novo PD patients.

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Ipsilateral valuesWhen the analysis of disease discrimination was restricted to patients with H&Y stage 1 (i.e. one-sided parkinsonism without axial problems; n=4) and controls, ipsilateral uptake values were also significantly reduced in patients, albeit to a lesser degree (t test: SORFP-CIT p=0.0007 for the caudate, p=0.0002 for the putamen and p<0.0001 for the striatum; SORF-DOPA p=0.0141 for the caudate, p=0.0097 for the putamen and p=0.0091 for the striatum).

Normal ageingIn the group of 20 controls (n=10 for each tracer), we found a significant age-dependent decline in SORFP-CIT of 4.6% per decade for the mean putamen and of 3.9% per decade for the mean striatum (p=0.04 and 0.05, respectively), but not for the mean caudate (p=0.33). No significant effect of ageing was found for uptake values of F-DOPA (p=0.75 for the mean caudate, p=0.65 for the mean putamen and p=0.92 for the mean striatum).

Gender differencesIn the group with healthy controls we found no significant effect of gender on striatal uptake of FP-CIT (t test: p=0.77 for the caudate, p=0.08 for the putamen and p=0.50 for the striatum). For F-DOPA, females tended to have slightly lower striatal uptake values than males. However, this difference did not reach statistical significance (t test: p= 0.45 for the caudate, p=0.12 for the putamen and p=0.23 for the striatum).

Discussion

This study demonstrates that both FP-CIT SPECT and F-DOPA PET scans are equally able to distinguish patients with PD patients from healthy controls. Our data show that scans with either tracer are able with 100% specificity and sensitivity to detect nigrostriatal damage in a group of patients with typical early-phase PD when considering tracer uptake in the contralateral putamen or striatum. To our knowledge only one previous study has compared FP-CIT SPECT scans with F-DOPA PET scans in patients with mild PD and healthy volunteers18. With each method separately, they were able to identify correctly all 15 healthy controls and 11 out of 12 PD patients. Others reported a sensitivity of 95% to 97% for FP-CIT SPECT3, 35. Morrish et al. were able to separate patients with PD from the normal group completely by F-DOPA PET33. Our findings emphasize the specificity of the applied methods. In our earlier study we demonstrated that both FP-CIT SPECT and F-DOPA PET can be used to measure the presynaptic dopaminergic system in vivo and that they both show equally good ability to separate the early from the advanced stage of PD.


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Uptake values of F-DOPA correlated well with FP-CIT uptake values9. Both tracers are in vivo markers of the presynaptic dopaminergic system, but reflect different underlying biochemical mechanisms. F-DOPA uptake reflects the activity of aromatic L-amino acid decarboxylase (AADC) in the nerve terminals of the nigrostriatal dopaminergic pathway and the storage of F-dopamine in these nerve terminals27, 28. FP-CIT binds to DATs, involved in the reuptake of dopamine from the synaptic cleft12. This difference between the two tracers results in a different method for examining the presynaptic dopaminergic system.In daily practice FP-CIT SPECT scanning is readily applicable, in contrast to F-DOPA PET, whose use is limited by restricted availability of PET instruments and related tracer production. FP-CIT SPECT scanning, assuming that a normal dataset is available as reference for this technique, may therefore be the only realistic option to obtain relevant in vivo data about striatal dopaminergic activity. We found that in patients with likely early PD, a dopaminergic defect can be detected with FP-CIT SPECT scans as well as with F-DOPA PET scans. The finding of defects in the presynaptic dopaminergic system can be helpful for the physician to diagnose parkinsonian syndromes correctly in patients with doubtful clinical extrapyramidal symptoms.Lee et al. 26 have examined the uptake of different tracers in patients with PD. They compared striatal uptake of [11C]methylphenidate (labelling DAT, comparable to the way FP-CIT does) with F-DOPA in patients with PD in an early phase of the disease. They found that in mildly affected parkinsonian patients, striatal F-DOPA uptake is relatively higher than the uptake of methylphenidate in the caudate as well as in the putamen. It was suggested that this difference in tracer uptake could be the result of an upregulation of AADC and a downregulation of the plasma membrane DAT in the striatum of patients with early PD. No direct proof of this has been obtained from human studies, although it is in line with experimental animal studies, which suggest a compensation of the loss of dopaminergic neurones by increasing the relative synthesis and release of dopamine from the remaining dopaminergic neurones47, 48. Ito et al.19 concluded that in rats with early to advanced parkinsonism induced by 6-hydroxydopamine, [14C]L-DOPA levels underestimated the decrease in dopaminergic neurones and that [125I]β-CIT levels more precisely reflected the decrease, suggesting that DAT is a better indicator of dopaminergic neuron loss. According to our data, striatal uptake of both tracers was reduced in patients with early-stage PD compared to healthy controls and no significant difference was found in disease discrimination between the two tracers.We found that using either tracer, the striatal uptake in patients with unilateral PD (H&Y I) was significantly reduced bilaterally without overlap of putamen values between PD patients and healthy controls. This implies that both methods can possibly also be used to identify patients in a preclinical phase of the disease. This has been confirmed by others22, 30, 44. Values of healthy controls are needed to interpret correctly the results of functional

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neuroimaging. As different institutions may use different ROIs or different methods of analysis, each should obtain its own set of normal data. Our striatal uptake values of FP-CIT were higher than those of Innis et al.17 and Tissingh et al., who found a striatal SOR in healthy controls of 2.24 and 2.4344. Our caudate uptake values for FP-CIT were much higher than putaminal uptake values for FP-CIT (putamen:caudate ratio 0.83 for FP-CIT and 1.00 for F-DOPA) in healthy controls. These high caudate uptake values were consistent through the complete set of data and could not be explained by outliers. Tissingh et al. found similar high caudate:putamen ratios44. Other studies have shown lower caudate uptake values in healthy controls. Also our SORF-DOPA values were slightly higher than those of Ishikawa et al. who found uptake values of approximately 2.218.One of the problems in evaluating neuroimaging methods in patients with early-stage PD is the validity of the clinical diagnosis of PD in this group. As mentioned before, diagnosing PD correctly based on clinical symptoms can be difficult and misdiagnosis occurs in up to 25% of patients in general practice. We have tried to overcome this problem by making the selection of participating patients by movement disorder specialists and by evaluating the clinical diagnosis after a period of more than 1 year after the performance of the scans.We found a significant age-dependent decline in SORFP-CIT of 3.9% per decade in healthy controls, which is in line with the findings of others18, 25, and in a post-mortem study a decay of 4.7% per decade of pigmented neurones in the substantia nigra was demonstrated10. It has been speculated that this ageing effect on DAT binding can be explained by compensation of neuronal dopamine loss by decreasing DAT synthesis46. Our study shows that the ageing effect on striatal DAT binding is relatively small and adjusting uptake values for age did not influence its accuracy for diagnosing presynaptic dopaminergic deficit. Also no significant effect of age on the symptomatic threshold of disease in PD was reported by others4, 18.In our data, striatal F-DOPA uptake did not change with age, although the number of subjects may have been too small to detect any ageing effect. This is in agreement with others8, 18, 38, although some studies have shown an ageing effect with F-DOPA PET31, 45. This doubtful effect of ageing on F-DOPA uptake may perhaps be explained by a lack of AADC decline during ageing36, which is supported by post-mortem studies, indicating no decrease in AADC activity with age23. Another explanation may be that the remaining neurons produce less tyrosine hydroxylase (TH) per neuron resulting in a decrease in endogenous dopamine, while AADC and storage capacity for dopamine is maintained14.We found no significant effect of gender on striatal uptake values of either FP-CIT or F-DOPA in healthy controls. This is in agreement with some studies which also could not detect an effect of gender on FP-CIT uptake or FDOPA uptake20, 37. However, other studies have suggested that DAT tracer and F-DOPA uptake values are higher in women than in men13, 24, 25, 42. The lack of a significant effect of gender on striatal uptake values in our


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study may also be explained by the small number of participating healthy controls.In this study we examined the uptake of two tracers in the same patients with clinically strong evidence of having PD, also after a longer period of time. According to our study FP-CIT SPECT scans as well as F-DOPA PET scans are both able to detect early striatal dopaminergic defects and may therefore be of great help for the physician to diagnose PD correctly in an early phase of the disease. Patients with normal striatal uptake need to be reconsidered in terms of clinical condition. A person with clinical signs and symptoms which suggest parkinsonism but with normal striatal FP-CIT or F-DOPA uptake is not suffering from PD. However, it must be borne in mind that finding a striatal uptake reduction of either tracer does not provide by itself a clinical diagnosis of PD or other brain disease, but only demonstrates a striatal dopaminergic biochemical lesion.

ConclusionOur study demonstrates that both FP-CIT SPECT scans and F-DOPA PET scans are able to diagnose presynaptic dopaminergic dysfunction and to separate patients with PD from healthy controls with high values of sensitivity and specificity.

Acknowledgements:We thank GE Health for financial support.The experiments complied with the current laws of the Netherlands, including approval of the local medical ethics committee.

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21. Kaasinen V, Nurmi E, Bruck A, et al: Increased frontal [F-18]fluorodopa uptake in early Parkinson’s Disease: sex differences in the prefrontal cortex. Brain 124:1125-1130, 2001

22. Kaufman MJ, Madras BK: Severe depletion of cocaine recognition sites associated with the dopamine transporter in Parkinson’s-diseased striatum. Synapse 9:43-49, 1991


24. Kish SJ, Zhong XH, Hornykiewicz O, et al: Striatal 3,4-dihydroxyphenylalanine decarboxylase in aging: disparity between postmortem and positron emission tomography studies? Ann Neurol 38:260-264, 1995

25. Laakso A, Vilkman H, Bergman J, et al: Sex differences in striatal presynaptic dopamine synthesis capacity in healthy subjects. Biological Psychiatry 52:759-763, 2002

26. Lavalaye J, Booij J, Reneman L, et al: Effect of age and gender on dopamine transporter imaging with [123I]FP- CIT SPET in healthy volunteers. Eur J Nucl Med 27:867-869, 2000






32. Martin WRW, Palmer MR, Patlak CS, et al: Nigrostriatal Function in Humans Studied with Positron Emission Tomography. Annals of Neurology 26:535-542, 1989

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33. Meara J, Bhowmick BK, Hobson P: Accuracy of diagnosis in patients with presumed Parkinson’s disease. Age Ageing 28:99-102, 1999

34. Morrish PK, Sawle GV, Brooks DJ: Clinical and [18F] dopa PET findings in early Parkinson’s disease. J Neurol Neurosurg Psychiatry 59:597-600, 1995

35. Niznik HB, Fogel EF, Fassos FF, et al: The dopamine transporter is absent in parkinsonian putamen and reduced in the caudate nucleus. J Neurochem 56:192-198, 1991

36. Ortega Lozano SJ, Martinez Del Valle Torres MD, Jimenez-Hoyuela Garcia JM, et al: [Diagnostic accuracy of FP-CIT SPECT in patients with parkinsonism]. Rev Esp Med Nucl 26:277-285, 2007

37. Ota M, Yasuno F, Ito H, et al: Age-related decline of dopamine synthesis in the living human brain measured by positron emission tomography with L-[beta-11C]DOPA. Life Sci 79:730-736, 2006

38. Ryding E, Lindstrom M, Bradvik B, et al: A new model for separation between brain dopamine and serotonin transporters in I-123-beta-CIT SPECT measurements: normal values and sex and age dependence. European Journal of Nuclear Medicine and Molecular Imaging 31:1114-1118, 2004

39. Sawle GV, Colebatch JG, Shah A, et al: Striatal Function in Normal Aging - Implications for Parkinsons-Disease. Annals of Neurology 28:799-804, 1990

40. Sawle GV, Playford ED, Burn DJ, et al: Separating Parkinson’s disease from normality. Discriminant function analysis of fluorodopa F 18 positron emission tomography data. Arch Neurol 51:237-243, 1994



43. Staley JK, Krishnan-Sarin S, Zoghbi S, et al: Sex differences in [I-123]beta-CIT SPECT measures of dopamine and serotonin transporter availability in healthy smokers and nonsmokers. Synapse 41:275-284, 2001




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46. Vingerhoets FJG, Snow BJ, Schulzer M, et al: Reproducibility of Fluorine-18-6-Fluorodopa Positron Emission Tomography in Normal Human-Subjects. Journal of Nuclear Medicine 35:18-24, 1994

47. Volkow ND, Ding YS, Fowler JS, et al: Dopamine transporters decrease with age. J Nucl Med 37:554-559, 1996


49. Zigmond MJ, Berger TW, Grace AA, et al: Compensatory responses to nigrostriatal bundle injury. Studies with 6-hydroxydopamine in an animal model of parkinsonism. Mol Chem Neuropathol 10:185-200, 1989

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7Summary

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Summary

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Parkinson’s disease is a common neurodegenerative disorder, which mainly affects the elderly. To date only symptomatic therapies are available and no treatment exists yet to stop or even delay the underlying process. Neuroprotective therapies are interventions interfering with the basic pathogenetic mechanism of cell death, resulting in a slowing or even stopping of the progression of neuronal degeneration. Apoptosis seems to play a role in the pathogenetic mechanism of Parkinson’s disease. Inhibition of apoptosis may therefore have a neuroprotective effect in this disorder. A possible apoptosis-inhibitor with neuroprotective effects is TCH346.If neuroprotection would be available, this should be given in the beginning of the disease, as progression is fastest in this phase. However, diagnosing Parkinson’s disease correctly in the beginning can be difficult. Conventional imaging techniques of the brain, like CT or MRI scans, are not useful in this respect. However, functional imaging by means of PET and SPECT is able to visualize and measure the functioning of the presynaptic striatal dopaminergic system. This implies that SPECT and PET might be useful to diagnose Parkinson’s disease correctly, also at early stages of the disease. But diagnosing Parkinson’s disease correctly is not only important in a research setting. Also in daily clinical practice it can be difficult for the neurologist to make the right diagnosis in an early phase of the disease, as other (non-) neurodegenerative disorders may mimic Parkinson’s disease. The aim of this thesis is to evaluate if FP-CIT SPECT and F-DOPA PET can be useful in diagnosing Parkinson’s disease both in clinical practice and in research settings.

In chapter 2 an extensive overview of Parkinson’s disease is presented, in which epidemiology, symptoms (motor and non-motor), pathology findings, comparison with other movement disorders, subclassifications of Parkinson’s disease and brain metabolism in aging and Parkinson’s disease, are described. From this description, it becomes clear, that Parkinson’s disease is a common neurodegenerative disorder, mainly affecting the elderly. Motor symptoms according to the UK Parkinson’s Disease Society Brain Bank criteria, consist of the classic triad of resting tremor, rigidity and bradykinesia. Postural impairment has often been called the fourth major motor symptom of PD. Next to the motor symptoms, patients with Parkinson’s disease may also suffer from cognitive problems, depressive disorder, and hallucinations. Pathology is characterized by degeneration of neuromelanin-containing neurons in the pars compacta of the substantia nigra, mainly in the ventrolateral part. This pattern of cell loss is unique for Parkinson’s disease. Thereafter, other movement disorders are described. In some of these, the presynaptic dopaminergic system is not involved, like essential tremor, vascular parkinsonism, normal pressure hydrocephalus, drug-induced parkinsonism, and metabolic or endocrine disorders. But also movement disorders which may mimic idiopathic Parkinson’s disease and in which

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the presynaptic dopaminergic system is involved as well, like multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and cortico-basal ganglionic degeneration (CBD), are described. Finally, the metabolic topography of Parkinson’s disease seems to be characterized by relatively increased activity in striatum, thalamus, pons and cerebellum, whereas activity was decreased in several association areas. In normal aging, the metabolic pattern of the brain changes also.

In chapter 3 the correlation between F-DOPA uptake and motor behavior in monkeys is described. F-DOPA PET provides a means to assess the integrity of the nigrostriatal dopaminergic system, in humans and in animal models. The MPTP-lesioned monkey is a validated animal model of Parkinson’s disease. Although it has been reported that striatal F-DOPA uptake is decreased in MPTP-lesioned monkeys, the correlation between F-DOPA uptake and motor behavior has not been evaluated in a systematic manner yet. A total of eight monkeys received MPTP bilaterally in two phases. They underwent an F-DOPA PET scan and their behavior was being analyzed in the non-lesioned phase, after unilateral administration of MPTP and after a second administration of MPTP resulting in lesions bilaterally. After administration of MPTP, striatal F-DOPA uptake decreased ipsilaterally and parkinsonian symptoms score increased, while the amount of limb movements contralaterally decreased. A significant relationship was found between mean striatal F-DOPA uptake and mean parkinsonian scores. Also, F-DOPA uptake was correlated with the amount of limb movements contralaterally.

In chapter 4, the possible neuroprotective effect of the compound TCH346 in MPTP-lesioned primates is evaluated. Until so far, only symptomatic treatment is available for patients with Parkinson’s disease. There is no therapy available yet to delay or even stop the underlying processes. Apoptosis seems to play a role in the pathogenetic mechanism of Parkinson’s disease. By inhibiting apoptosis, the underlying process may be blocked and neuroprotection may thus be achieved. Eight monkeys received MPTP in a two-phase model and became parkinsonian. Four of them received TCH346 and four of them received saline. Behavior was analyzed and F-DOPA PET scans were performed in the naïve state, after administration of 2.5 mg MPTP into the left carotid artery and after a second administration of 1.25 mg MPTP into the right carotid artery. The first MPTP infusion, into the left carotid artery, caused mild parkinsonian symptoms, reduced amount of right limb movements and reduced F-DOPA uptake in the left striatum. In the saline-treated monkeys, the second MPTP treatment (into the right carotid artery), resulted in a worsening of the parkinsonian symptoms with a reduced amount of left limb movements and a decrease of the F-DOPA uptake in the right striatum. However, in the TCH346 treated monkeys, the second MPTP

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treatment did not further worsen parkinsonian symptoms, did not affect the amount of right limb movements and did not decrease the F-DOPA uptake in the right striatum. Thus, it can be concluded from this study, that TCH346 is able to prevent nigrostriatal damage and motor symptoms induced by MPTP in monkeys.

The aim of the study in chapter 5 is to compare FP-CIT SPECT and F-DOPA PET in patients with different stages of Parkinson’s disease and to assess their power in demonstrating striatal dopaminergic damage. Diagnosing Parkinson’s disease can be difficult in the early phases of the disease and PET and SPECT enable visualization and evaluation of the striatal dopaminergic functioning. F-DOPA PET is already being used since the eighties of the last century for quantification of striatal dopaminergic capacity. However, its use is restricted by the limited availability of F-DOPA. SPECT scans using FP-CIT are more widely available and could therefore form an attractive alternative. A total of 30 patients with Parkinson’s disease, consisting of 13 de novo patients and 17 patients with advanced stage of disease underwent both an FP-CIT SPECT scan and an F-DOPA PET scan. The patients were clinically examined in the ‘off-state’. Striatal uptake of F-DOPA and FP-CIT correlated well with each other and also putaminal uptake of both tracers correlated well with each other. Both imaging techniques correlated moderately with several clinical scores in a similar way. By means of both scanning methods the two patient groups could be well distinguished from each other. From this study it could be concluded that both techniques can be used to measure the presynaptic dopaminergic system in patients with different stages of Parkinson’s disease.

The study described in chapter 5 has been extended in chapter 6 in which FP-CIT SPECT scans and F-DOPA PET scans have been compared with each other in patients with Parkinson’s disease and in healthy volunteers. Ten healthy volunteers underwent an FP-CIT SPECT scan and ten healthy volunteers underwent an F-DOPA PET scan. These uptake values were compared with the uptake values derived from the study described in chapter 5. Uptake values of caudate, putamen and striatum of both FP-CIT and F-DOPA were significantly reduced in parkinsonian patients compared to the uptake values in healthy volunteers. By means of contralateral putaminal and striatal uptake values of FP-CIT SPECT as of F-DOPA PET, the group of healthy volunteers could be completely separated from the group with patients with Parkinson’s disease. It can therefore be concluded that FP-CIT SPECT and F-DOPA PET scans are both able to diagnose presynaptic dopaminergic damage, also in an early phase of the disease, with an excellent sensitivity and specificity.

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8Discussion and future perspectives

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Correlation between clinical parameters and striatal dopaminergic tracer uptake

Animal studiesThe studies described in this thesis investigate the relation between clinical parameters and striatal uptake measures of dopaminergic tracers in patients with PD and parkinsonian non-human primates. Also the relation between these parameters and pathological findings in monkeys is described.In monkeys we found a significant negative correlation between striatal F-DOPA uptake and parkinsonian symptom scores and a positive correlation between striatal F-DOPA uptake and the number of limb movements. This is in concordance with other studies. Uptake of β-CIT and fluoro-methyltyrosine (FMT) both correlate well with motor symptoms in MPTP-lesioned monkeys11-13, 26 and F-DOPA uptake is altered in MPTP-treated monkeys8-10. In our study striatal F-DOPA uptake and motor symptoms correlated with immunohistochemical findings. In monkeys with high parkinsonian scores and decreased striatal F-DOPA uptake, more gliosis in substantia nigra and striatum was observed compared to monkeys in which striatal F-DOPA uptake remained intact and only moderate parkinsonian scores were present. On the other hand, monkeys with normal F-DOPA uptake and moderate parkinsonian scores displayed more TH-positive neurons and fibres in substantia nigra and striatum than monkeys with a decrease in striatal F-DOPA uptake and high parkinsonian scores. These data are consistent with the results of Pate et al29 who show that a reduction of F-DOPA uptake is correlated with a reduced amount of dopamine nerve terminals in the striatum. Oiwa et al26 showed that in MPTP-treated monkeys different stages of parkinsonism correlated with different amounts of DA concentrations in the caudate nucleus and putamen of the contralateral hemisphere. FMT-PET uptake correlated well with these biochemical data and proved to be a good predictor of DA levels in the contralateral striatal regions. Despite those positive correlations between striatal F-DOPA uptake, motor behaviour and immunohistochemical findings, the exact quantitative relation between those parameters has not been explored in a systematic manner yet.

Human studiesAccording to the studies described in this thesis, a significant correlation was found between clinical examination in the ‘off’-state (UPDRS-III and H&Y) and striatal uptake of F-DOPA and FP-CIT. This is in agreement with other authors. Many studies have described a significant correlation between striatal uptake by means of F-DOPA PET or FP-CIT SPECT and clinical parameters. Correlation coefficients between striatal uptake and UPDRS-III scores ranged from 0.51 to 0.812, 4, 17, 31, 35, 39, 42, while the correlation coefficients in our studies are lower (0.38 and 0.45 for striatal uptake of respectively F-DOPA and FP-CIT). This discrepancy

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may be explained by methodological differences in data processing. Although patients were examined in the clinical ‘off’-state, long-lasting effects of antiparkinsonian therapy on motor signs cannot be excluded. Pirker et al showed that striatal uptake of [123I]β-CIT SPECT was significantly correlated with UPDRS-II and UPDRS-III, disease duration and Hoehn & Yahr stage. UPDRS motor sub scores for bradykinesia showed a good correlation, while rigidity showed a moderate, but significant correlation with striatal uptake. No significant correlation could be found between tremor scores and striatal uptake31. Bradykinesia appears to be directly related to the degree of nigral cell loss, striatal dopamine deficiency5 and to the decrease of homovanillic acid in cerebrospinal fluid36. However, no such correlation could be found for parkinsonian tremor5, 36.In patients with early PD striatal uptake values of FP-CIT are reduced to 70.9% (caudate), 46.8% (anterior putamen) and 24.0% (posterior putamen) of normal values42. This is in agreement with Morrish et al who found that symptom onset in Parkinson’s disease was estimated at a mean putaminal F-DOPA uptake of 75% of normal and a mean caudate F-DOPA uptake of 91% of normal22. They concluded that the mean preclinical period is less than seven years. Other studies estimated a preclinical period of 6.5 years25 and 3.1 years23. Estimation of the preclinical period may vary depending on the method of analysis and extrapolation. It is estimated that at least 30-50% of nigral cell loss has occurred at the onset of symptoms of PD19. At that time patients have lost approximately 80% of striatal dopamine content. Striatal reduction of F-DOPA uptake correlates with subsequent post-mortem dopaminergic cell density in the substantia nigra of post-mortem-confirmed patients with PD37.Nigral cell loss is just one of the processes involved in the pathogenetic mechanism of PD. Many mechanisms have been proposed to be involved in the initiation of the neurodegenerative process, like oxidative stress, glutamate excitotoxicity, free radical damage, mitochondrial (complex I) dysfunction and inflammatory processes or proteasomal dysfunction16, 19, 27. These processes will eventually result in nigrostriatal dysfunction and cell death. Braak et al have proposed an ascending pattern for the development of PD7. The olfactory system and brainstem nuclei are involved in the early stages, i.e. stage 1 and 2. Substantia nigra is involved in stage 3, while involvement of anteromedial temporal mesocortex is characteristic to stage 4. At stage 5 and 6 the cortical areas become involved7. F-DOPA PET and FP-CIT SPECT measure only a small piece of this complex process. Each of these imaging techniques measures an aspect of dopaminergic function. These imaging techniques however, do not measure the number of nigral dopaminergic neurons and results derived from those studies do not directly assess the causes of the measured biochemical abnormalities.


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Although in many cross-sectional studies in patients with PD reduction in striatal uptake correlated significantly with increasing severity of PD measured by UPDRS, this relationship remains unclear in longitudinal studies. In the CALM-PD and the REAL-PET study the possibility of dopamine agonists to modify disease progression in PD were tested1, 43. In both studies percentage loss of uptake from baseline correlated with change in UPDRS score from baseline. Both studies demonstrated a significantly lower loss in the rate of striatal tracer uptake in patients treated with dopamine agonists compared with L-DOPA. However the REAL-PET study showed that after 2 years of treatment motor function was superior in the patients treated with L-dopa compared with ropinirole43. In the CALM-PD study no significant differences in UPDRS scores between pramipexole treated patients and L-dopa treated patients could be detected after 22 months of treatment1. The discrepancy between a slower loss of striatal uptake and a worse clinical condition in dopamine-agonist treated patients compared to L-dopa may be explained by several factors. First, in the REAL-PET study patients were scored in the clinical ‘on’-condition, because of the inherent problems associated with true ‘off’- treatment assessments43. L-dopa has a greater symptomatic efficacy compared to ropinirole, which may explain the lower UPDRS-scores in the L-dopa treated group. Although patients in the CALM-PD group were clinically examined 12h after study drug and antiparkinsonian medications, long-lasting symptomatic effects confounding the UPDRS-scores cannot be ruled out1. Second, neuroimaging tracer dynamics may be influenced by the tested drugs. In the CALM-PD study, patients were scanned without stopping study drugs in advance1. Third, in early PD the temporal patterns for rate of loss of dopamine transporter and change in UPDRS-scores may not be congruent. It cannot be excluded that in early phases of PD striatal uptake decreases to a large degree, while the change in UPDRS is only minimal. This problem can be overcome by a long-term follow up1. Due to the mentioned problems faced with the interpretation of imaging studies for evaluation of therapies, some assume that imaging studies should not be used as surrogate endpoints in clinical trials of PD24.

Neuroprotective studies

According to our study described in this thesis, the compound TCH346 is able to prevent motor symptoms and nigrostriatal deficits induced by MPTP in primates. Therefore TCH346 seemed a promising agent for neuroprotective therapy in Parkinson’s disease. The animal study we performed was followed by a double-blind, randomised, controlled trial in humans with Parkinson’s disease28. In that study, no significant differences between any of the TCH346 doses administered and placebo were observed regarding time to the development of disability requiring dopaminergic treatment, and regarding changes in

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clinical rating scores. This is not the first time that a promising preclinical neuroprotective agent fails to show any neuroprotective effect in the clinical human situation. In 2003, the National Institutes of Health (NIH) committee proposed 59 possible agents to be tested as potential neuroprotective compounds34. However, until now no therapy has been proven to be able to stop or even slow the progression of Parkinson’s disease. This discrepancy between preclinical animal studies and clinical studies with parkinsonian patients may be explained by several factors28, 41, like inappropriate animal model or inappropriate study design. These factors have probably both played a role in the disagreement found between our preclinical and the clinical TCH346-studies. It is unlikely that the discrepancy in results can be explained by the use of a wrong tracer in our animal study: our animal study showed that administration of TCH346 to MPTP-treated primates prevents striatal dopaminergic defects as well as parkinsonian behaviour.

Inappropriate animal modelMany animal models use MPTP which induces acute damage, limited to dopaminergic neurons. This may not correctly reflect the underlying pathogenetic mechanisms of cell death in Parkinson’s disease3. Neuroprotective effects of a compound observed in a MPTP-treated animal model may therefore not be relevant for the human situation. However the exact mechanism of cell death in Parkinson’s disease is not known yet, making it difficult to design a perfect animal model.

Inappropriate study designDeveloping an appropriate study design to evaluate neuroprotective effects of a compound in Parkinson’s disease is difficult. While the evaluation of symptomatic treatment has obvious endpoints, like change in signs and clinical ratings scales, the measure of effects in neuroprotective treatment is less clear. An increase of symptoms may not be a direct reflection of the changes at a cellular or biochemical level. The Unified Parkinson Disease Rating Scale (UPDRS), which is often used in clinical trials as a measure for the severity of parkinsonian symptoms is relatively insensitive to small deteriorations in clinical state. This forms especially a problem for patients, enrolled in neuroprotective studies, as they are in the beginning of the disease when signs and symptoms can be mild and subtle. More specific and sensitive parameters need to be designed for accurate assessment of neuroprotective effects. Only patients with very short duration of signs and symptoms should be enrolled in neuroprotective studies. In the beginning of the disorder, progression is fastest and neuroprotection is most effective. However, it is not easy to enrol patients in the beginning of the disease, as signs and symptoms are subtle and easily misinterpreted both by patients and by physicians. This may lead to a delay in diagnosis. In this respect


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the possibility of performing PET or SPECT scans may be helpful in diagnosing Parkinson’s disease in an early phase of the disease. Many neuroprotective trials include an observation time of 1 to 2 years. This may be a too short period of time to evaluate a (small) long-term neuroprotective effect. Longer periods of follow-up and observation time may be needed to detect neuroprotective outcome. However, studies with a longer period of follow-up will be difficult to perform and the clinical relevance of small neuroprotective effects is doubtful.

FP-CIT SPECT and F-DOPA PET

In this thesis we evaluated the usefulness of both F-DOPA PET and FP-CIT SPECT in clinical practice and of F-DOPA PET in research settings (using animals).

The biological processesBoth tracers assess one or more aspects of the dopaminergic nerve terminal system. F-DOPA PET reflects the uptake and conversion of fluorodopa to fluorodopamine. This process contains several steps. After i.v. injection, F-DOPA crosses the blood-brain barrier and is taken up into cells. It is converted by aromatic acid decarboxylase (AADC) to fluorodopamine. PET scans with F-DOPA allow quantification of striatal dopa decarboxylase activity and storage capacity of F-dopamine.FP-CIT belongs to a group of compounds derived from cocaine, which has a high affinity for the dopamine transporter (DAT). Removal of dopamine from the synaptic cleft is mainly performed by reuptake through the DAT. DAT levels correlate with the density of DA nerve terminals, but are also regulated by striatal DA concentration and is regulated at several other levels40.Both imaging techniques do not calculate the number or density of nigral dopaminergic neurons. Results from tracer uptake studies do not directly quantify the biologic processes as in vitro studies. Therefore, the correspondence between tracer uptake studies and biological parameters will always be indirect33 and will always need to be validated, to obtain a measure of specific uptake. The pattern of F-DOPA or FP-CIT uptake in patients with PD is practically identical. Uptake in caudate remains normal in the early phases, while putaminal uptake is reduced, mainly the dorsal part of the putamen18. Although both putamina often show reduced uptake of tracer, the putamen contralateral to the clinical signs and symptoms will be most affected.

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Comparison FP-CIT and F-DOPAFrom the studies described in this thesis it can be concluded that using either FP-CIT SPECT scans or F-DOPA PET scans patients with de novo – clinically typical- Parkinson’s disease can be separated completely from healthy volunteers. This is in agreement with Ishikawa et al. F-DOPA PET and FP-CIT SPECT significantly discriminated patients with PD and healthy volunteers with comparable accuracy17. Lee et al examined the striatal uptake of different dopaminergic tracers in patients with PD20. DAT labelling by means of methylphenidate (MP) was compared with F-DOPA uptake in patients with PD at an early stage of the disease. In mildly affected parkinsonian patients, striatal F-DOPA uptake was relatively higher than the uptake of MP in caudate and striatum. This pattern suggests up regulation of AADC activity and down regulation of DAT. Although no direct proof has been given of this assumption in human studies, it is in line with experimental animal studies, which suggests a compensation of the loss of dopaminergic neurones by increasing the relative synthesis and release of dopamine from the remaining dopaminergic neurons46, 47.According to the studies in this thesis, no significant difference could be detected between FP-CIT SPECT and F-DOPA PET in the detection of presynaptic dopaminergic deficits. This may possibly be due to the small sample size. Until so far, there is no convincing evidence for superiority of one of those tracers in assessment of presynaptic dopaminergic deficits33.

F-DOPA PET and FP-CIT SPECT in clinical settingWe have compared both techniques in patients with clinically strong evidence of Parkinson’s disease, which are not difficult to diagnose correctly for neurologists. It is more tempting and clinically useful to compare both scanning methods in patients with only subtle parkinsonian signs and symptoms, which cause problems to be diagnosed correctly in clinical practice. It remains to be investigated, whether those patients with recent or subtle parkinsonism can also be discriminated from patients with suspicious symptoms but without Parkinson’s disease by means of these imaging techniques. Which tracer should be used in clinical practice depends on several factors. FP-CIT SPECT scans are widely available, but have a lower spatial resolution. F-DOPA PET scans have a better spatial resolution, but restricted availability of PET instruments and the difficult production of F-DOPA limit its use. Patient preparation necessary for F-DOPA PET and FP-CIT SPECT scans is different. For F-DOPA PET it is recommended that patients do not consume large amounts of protein before the start of the scan21. Patients are allowed to take their (antiparkinsonian) medication. Only COMT-inhibitors should be taken into account before performing a F-DOPA PET scan32. In contrast, drugs interfering with the dopamine receptor like cocaine, amphetamine-like drugs (including methylphenidate), benzatropine, bupropion, mazindol, phenteramine, phentanyl and sertraline, have to be stopped 5 half-times before undergoing a FP-CIT


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SPECT scan38. Also other selective serotonin reuptake inhibitors (SSRI’s), like citalopram, escitalopram and paroxetine should be stopped6, 30. Antiparkinsonian medication is allowed to be continued for FP-CIT SPECT scans. Acquisition time of F-DOPA PET differs from FP-CIT SPECT. Acquisition time for FP-CIT SPECT scans is about 45 minutes. For F-DOPA PET scans one static 3D acquisition of 6 minutes is performed. This short scanning time may be advantageous for patients with Parkinson’s disease. All the above mentioned factors may influence the final decision which scanning technique to prefer as sensitivity and specificity of FP-CIT SPECT and F-DOPA PET are almost equal.

Future perspectives

In the recent past, many centers acquired a PET camera. About 10 years ago, only 3 centers in the Netherlands possessed one or more PET cameras, while nowadays circa 30 – 40 PET cameras are available in the Netherlands. PET cameras are mostly used as a diagnostic tool in oncology using FDG as radiotracer. FDG can be transported easily and is commercially available. This makes it unnecessary for a hospital to produce FDG by itself and therefore to possess an on-site cyclotron. Until now, F-DOPA is not commercially available. However, if neurologists discover the possibility of performing PET scans in their own center, it may be expected that the demand for F-DOPA PET scans will increase and that F-DOPA will become commercially available. SPECT is often used to study the dopamine transporter (DAT) system. As PET provides higher resolution than SPECT, new dopamine transporter radiotracers for PET, labelled with 11C or 18F, have been developed14, 15, 44, 45. It may be expected that one or more of the 18F-labeled DAT radiotracers will become commercially available in the future.The development of new PET cameras with higher spatial resolution results in higher quality of PET images and the ability to visualize small cerebral regions. The combination of the development of commercially available presynaptic dopaminergic radiotracers for PET and the higher resolution of PET cameras will probably result in high-quality diagnostic tools for detecting dopaminergic striatal deficits in daily clinical practice as well as in experimental settings for the assessment of possible neuroprotective effects of new therapies.

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25. Nurmi E, Ruottinen HM, Bergman J, et al: Rate of progression in Parkinson’s disease: a 6-[18F]fluoro-L-dopa PET study. Mov Disord 16:608-615, 2001

26. Oiwa Y, Eberling JL, Nagy D, et al: Overlesioned hemiparkinsonian non human primate model: correlation between clinical, neurochemical and histochemical changes. Front Biosci 8:a155-a166, 2003

27. Olanow CW: The pathogenesis of cell death in Parkinson’s disease--2007. Mov Disord 22 Suppl 17:S335-S342, 2007

28. Olanow CW, Schapira AH, LeWitt PA, et al: TCH346 as a neuroprotective drug in Parkinson’s disease: a double-blind, randomised, controlled trial. Lancet Neurol 5:1013-1020, 2006


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30. Peremans K, Goethals I, De Vos F, et al: Serotonin transporter and dopamine transporter imaging in the canine brain. Nucl Med Biol 33:907-913, 2006

31. Pirker W: Correlation of dopamine transporter imaging with parkinsonian motor handicap: how close is it? Mov Disord 18 Suppl 7:S43-S51, 2003

32. Psylla M, Gunther I, Antonini A, et al: Cerebral 6-[18F]fluoro-L-DOPA uptake in rhesus monkey: pharmacological influence of aromatic amino acid decarboxylase (AAAD) and catechol-O-methyltransferase (COMT) inhibition. Brain Res 767:45-54, 1997

33. Ravina B, Eidelberg D, Ahlskog JE, et al: The role of radiotracer imaging in Parkinson disease. Neurology 64:208-215, 2005

34. Ravina BM, Fagan SC, Hart RG, et al: Neuroprotective agents for clinical trials in Parkinson’s disease: a systematic assessment. Neurology 60:1234-1240, 2003

35. Ribeiro MJ, Vidailhet M, Loc’h C, et al: Dopaminergic function and dopamine transporter binding assessed with positron emission tomography in Parkinson disease. Arch Neurol 59:580-586, 2002

36. Rinne UK, Sonninen V: Brain catecholamines and their metabolites in Parkinsonian patients. Treatment with levodopa alone or combined with a decarboxylase inhibitor. Arch Neurol 28:107-110, 1973


38. Tatsch K, Asenbaum S, Bartenstein P, et al: European Association of Nuclear Medicine procedure guidelines for brain neurotransmission SPET using (123)I-labelled dopamine D(2) transporter ligands. Eur J Nucl Med Mol Imaging 29:BP30-BP35, 2002


40. Uhl GR, Walther D, Mash D, et al: Dopamine transporter messenger RNA in Parkinson’s disease and control substantia nigra neurons. Ann Neurol 35:494-498, 1994

41. Waldmeier P, Bozyczko-Coyne D, Williams M, et al: Recent clinical failures in Parkinson’s disease with apoptosis inhibitors underline the need for a paradigm shift in drug discovery for neurodegenerative diseases. Biochem Pharmacol 72:1197-1206, 2006

42. Wang J, Zuo CT, Jiang YP, et al: 18F-FP-CIT PET imaging and SPM analysis of dopamine transporters in Parkinson’s disease in various Hoehn & Yahr stages. J Neurol 254:185-190, 2007

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44. Wuest F, Berndt M, Strobel K, et al: Synthesis and radiopharmacological characterization of 2beta-carbo-2’-[18F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane ([18F]MCL-322) as a PET radiotracer for imaging the dopamine transporter (DAT). Bioorg Med Chem 15:4511-4519, 2007

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47. Zigmond MJ, Berger TW, Grace AA, et al: Compensatory responses to nigrostriatal bundle injury. Studies with 6-hydroxydopamine in an animal model of parkinsonism. Mol Chem Neuropathol 10:185-200, 1989

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9Samenvatting

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De ziekte van Parkinson is een veel voorkomende neurodegeneratieve aandoening, die vooral de oudere bevolking treft. Behandeling bestaat tot nu toe uit symptomatische behandeling. Therapieën waarbij wordt getracht het degeneratieve proces te vertragen dan wel stop te zetten, zijn nog niet beschikbaar. Neuroprotectieve therapieën zijn interventies die interfereren met het onderliggende pathogenetische mechanisme van celdood, waardoor de progressie van neuronale degeneratie wordt vertraagd of zelfs gestopt. Apoptose lijkt een belangrijke rol te spelen in de pathogenese van de ziekte van Parkinson en het afremmen van apoptose kan derhalve resulteren in neuroprotectie. Een mogelijke apoptose-remmer met neuroprotectieve effecten is TCH346. Als neuroprotectie beschikbaar zou zijn, dan zou dit in het begin van de ziekte moeten worden gegeven, aangezien achteruitgang het snelst verloopt in de vroege fasen. Het kan echter lastig zijn om in de vroege fase de diagnose ziekte van Parkinson correct te stellen. Conventionele beeldvormingtechnieken van de hersenen, zoals CT- of MRI scans zijn hiervoor minder goed bruikbaar. Met behulp van functionele beeldvorming door middel van PET en/of SPECT kan het presynaptische striatale dopaminerge systeem worden gevisualiseerd en gemeten. Dit houdt in dat SPECT en PET nuttig kunnen zijn om de diagnose ziekte van Parkinson correct te stellen, ook in de vroege fasen van de ziekte. Het stellen van de juiste diagnose van de ziekte van Parkinson is niet alleen van belang voor onderzoeksdoeleinden, maar ook in de dagelijkse klinische praktijk kan het voor de neuroloog moeilijk zijn om de juiste diagnose te stellen. Andere (niet)-neurodegeneratieve aandoeningen kunnen lijken op de ziekte van Parkinson en hiermee worden verward. Het doel van dit proefschrift is om te evalueren of FP-CIT SPECT en F-DOPA PET bruikbaar kunnen zijn zowel in de klinische praktijk voor het stellen van de diagnose ziekte van Parkinson, als bij wetenschappelijk onderzoek.

In hoofdstuk 2 wordt een uitgebreid overzicht van de ziekte van Parkinson gegeven, waarin epidemiologie, symptomen (motorische en niet-motorische), post-mortem bevindingen, vergelijkingen met andere bewegingsstoornissen, subclassificaties van de ziekte van Parkinson en het hersenmetabolisme van de ouderdom en de ziekte van Parkinson worden beschreven. Uit deze beschrijving volgt dat de ziekte van Parkinson een veel voorkomende neurodegeneratieve aandoening is, die vooral de oudere bevolking treft. Motorische symptomen bestaan volgens de UK Parkinson’s Disease Society Brain Bank Criteria, uit de klassieke trias van rusttremor, rigiditeit en bradykinesie. Houdingsinstabiliteit wordt vaak het vierde belangrijke symptoom van de ziekte van Parkinson genoemd. Naast de motorische symptomen kunnen Parkinsonpatiënten ook last hebben van cognitieve problemen, depressiviteit en hallucinaties. De ziekte wordt gekenmerkt door degeneratie van neuromelanine-bevattende neuronen in de pars compacta van de substantia nigra,

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vooral in het ventrolaterale deel. Dit patroon van celverlies is uniek voor de ziekte van Parkinson.Daarna worden andere bewegingsstoornissen beschreven. Bij enkele van deze aandoeningen is het presynaptische dopaminerge systeem niet betrokken, zoals essentiële tremor, vasculair parkinsonisme of endocriene aandoeningen. Maar ook andere bewegingsstoornissen worden beschreven waarbij het presynaptische dopaminerge systeem wel betrokken is, zoals multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and cortico-basal ganglionic degeneneration (CBGD).Tot slot wordt de metabole topografie van de ziekte van Parkinson gekenmerkt door relatief toegenomen metabolisme in het striatum, de thalamus, pons en het cerebellum, terwijl het metabolisme is afgenomen in meerdere associatiegebieden. Het metabole patroon van de hersenen verandert ook met veroudering.

Hoofdstuk 3 beschrijft de correlatie tussen striatale F-DOPA opname en motorisch gedrag in apen. F-DOPA PET is een manier om de integriteit van het nigrostriatale dopaminerge systeem te beoordelen, zowel in mensen als in diermodellen. Apen, waaraan MPTP is toegediend, zijn een gevalideerd diermodel voor de ziekte van Parkinson. Hoewel beschreven is dat striatale F-DOPA opname afgenomen is in MPTP-gelaedeerde apen, is de correlatie tussen F-DOPA opname en motorisch gedrag nog niet systematisch geëvalueerd. Acht apen kregen in 2 fases MPTP beiderzijds toegediend. Ze ondergingen een F-DOPA PET scan en hun gedrag werd geanalyseerd in de uitgangsfase, na eenzijdige toediening van MPTP en na een tweede toediening van MPTP contralateraal aan de eerste toediening van MPTP waardoor beiderzijds laesies ontstonden. Na toediening van MPTP nam ipsilateraal de striatale F-DOPA opname af, evenals de hoeveelheid doelgerichte bewegingen van de contralaterale ledematen en steeg de score voor parkinsonsymptomen. Striatale F-DOPA opname correleerde significant met de scores voor parkinsonsymptomen. Ook correleerde de F-DOPA opname met de hoeveelheid doelgerichte bewegingen van de contralaterale ledematen.

In hoofdstuk 4 wordt het mogelijk neuroprotectieve effect van de stof TCH346 in MPTP-gelaedeerde apen beoordeeld. Tot nu toe bestaat alleen symptomatische therapie voor patiënten met de ziekte van Parkinson. Er is nog geen therapie beschikbaar om het onderliggende proces te vertragen of zelfs te stoppen. Apoptose lijkt een belangrijke rol te spelen in de pathogenese van de ziekte van Parkinson. Door apoptose te remmen, zou het onderliggende proces kunnen worden geblokkeerd, resulterend in neuroprotectie. Acht apen kregen MPTP toegediend in een 2-fase model en werden parkinsonistisch. Vier van deze apen kregen TCH346 toegediend en vier van hen placebo. Gedragsanalyse en F-DOPA PET scans werden verricht in de uitgangfase, na toediening van 2,5 mg

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MPTP in de linker arteria carotis en na een tweede toediening van 1.25 mg MPTP in de rechter arteria carotis. De eerste toediening van MPTP, in de linker arteria carotis, veroorzaakte parkinsonsymptomen en verlaagde het aantal doelgerichte bewegingen van de rechter ledematen. De F-DOPA opname in het linker striatum nam af. In the apen, die een zoutoplossing kregen, leidde de tweede toediening van MPTP ( in de rechter arteria carotis), tot een verergering van de parkinsonissymptomen, waarbij de hoeveelheid doelgerichte bewegingen van de linker ledematen sterk afnam. De F-DOPA opname in het rechter striatum was bij deze apen duidelijk verminderd. De dieren die TCH346 hadden gekregen, ontwikkelden echter geen klachten aan de linkerkant en bleven motoor gelijk. De opname van F-DOPA in het rechter striatum bleef bij deze groep dieren eveneens gelijk. Hieruit kan worden geconcludeerd, dat TCH346 in staat is om de gevolgen van MPTP, zowel de nigrostriatale schade als de motore parkinsonsymptomen, te voorkomen.

In hoofdstuk 5 worden FP-CIT SPECT scans en F-DOPA PET scans met elkaar vergeleken in Parkinsonpatiënten in verschillende ziektestadia. Tevens wordt het vermogen om striatale dopaminerge defecten aan te tonen van beide afbeeldingstechnieken beoordeeld. Het kan moeilijk zijn om de ziekte van Parkinson vast te stellen in het beginstadium. PET- en SPECT-scans kunnen behulpzaam zijn in het beoordelen van striatale dopaminerge functie. F-DOPA PET wordt al sinds de jaren tachtig van de vorige eeuw gebruikt voor kwantificatie van de striatale dopaminerge capaciteit. Het gebruik van F-DOPA PET scans is echter beperkt doordat F-DOPA niet makkelijk beschikbaar is. FP-CIT SPECT scans zijn ruimer voorhanden en kunnen derhalve een aantrekkelijk alternatief vormen. In totaal ondergingen 30 Parkinsonpatienten, waarvan 13 de novo en 17 in een gevorderd ziektestadium, zowel een FP-CIT SPECT scans als een F-DOPA PET scan. De patiënten werden lichamelijk onderzocht zonder dopaminerge medicatie. Striatale F-DOPA opname correleerde goed met striatale FP-CIT opname. Dit gold ook voor de correlatie tussen beide tracers met betrekking tot de opname ter plaatse van het putamen. Beide technieken correleerden matig met meerdere klinische parameters op een soortgelijke manier. Door middel van (één van) beide scanmethoden kunnen de twee patiëntengroepen goed van elkaar worden onderscheiden. Deze studie toont daarom aan dat beide technieken kunnen worden gebruikt om het presynaptische dopaminerge systeem te beoordelen in parkinsonpatiënten met verschillende ziektestadia.

De studie uit hoofdstuk 5 is verder uitgebreid in hoofdstuk 6, waarin FP-CIT SPECT scans en F-DOPA PET scans met elkaar zijn vergeleken in patiënten met de ziekte van Parkinson en gezonde vrijwilligers. Tien gezonde vrijwilligers kregen een FP-CIT SPECT scan en 10 gezonde vrijwilligers ondergingen een F-DOPA PET scan. Deze opnamewaarden werden vergeleken met de opnamewaarden verkregen uit de studie, die beschreven is in hoofdstuk

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5. Opname in de nucleus caudatus, het putamen en het striatum van zowel F-DOPA als FP-CIT was significant lager in parkinsonpatiënten vergeleken met de waarden van gezonde vrijwilligers. Op basis van opname in het contralaterale putamen of het contralaterale striatum van zowel FP-CIT als F-DOPA kunnen de patiënten volledig worden onderscheiden van de groep gezonde controlepersonen. Daarom kan worden geconcludeerd dat zowel FP-CIT SPECT scans als F-DOPA PET scans in staat zijn om presynaptisch dopaminerge defecten aan te tonen, ook in de beginstadia van de ziekte van Parkinson, met een uitstekende sensitiviteit en specificiteit.

10List of abbreviations

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AADC Aromatic Acid DecarboxylaseAD Alzheimer’s DiseaseADL Activities of Daily LivingANOVA Analysis Of VarianceBAX Bcl-2–Associated X proteinCBD Cortico Basal DegenerationCGP 3466B dibenzo[b,f]oxepin-10-ylmethyl-methyl-prop-2-ynyl-amineCFT 2beta-Carbomethoxy-3beta-(4-Fluorophenyl)TropaneCOV Coefficient Of VariationCSF Cerebro Spinal FluidCT Computed TomographyCOMT Catechol-O-Methyl-Transferase DA DopamineDAT Dopamine TransporterDATATOP Deprenyl And Tocopherol Antioxidative Therapy Of ParkinsonismDNA Deoxyribonucleic AcidDTBZ DihydrotetrabenazineEMG ElectromyographyET Essential TremorFDG FluorodeoxyglucoseF-DOPA 6-[18F]-fluoro-L-3,4-dihydroxyphenylalanineFMT Fluoro-MethyltyrosineFP-CIT N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) nortropaneFWHM Full Width Half MaximumGAPDH Glyceraldehyde-3-phosphate DehydrogenaseH &Y Hoehn & YahrIBZM IodobenzamideIM IntramuscularkeV Kilo electron VoltMAO Mono-amino OxidaseMBq Mega BecquerelMP MethylphenidateMPTP 1,2,3,6-methyl-phenyl-tetrahydroyridineMRI Magnetic Resonance ImagingMSA Multi System Atrophy NIH National Institutes of Health NMSP N-methylspiperoneNPH Normal Pressure Hydrocephalus

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OHDA 6-HydroxydopaminePD Parkinson’s DiseasePDQ Parkinson’s Disease QuestionnairePET Positron Emission TomographyPIGD Postural Instability / Gait Disorder dominant Parkinson’s DiseasePSP Progressive Supranuclear Palsy ROI Region Of InterestRNA Ribonucleic AcidROC Receiver Operating CharacteristicSD Standard DeviationSN Substantia NigraSOR Striato-occipital RatioSPECT Single Photon Emission Computed TomographySPM Statistical Parametric MappingSRI Striato-reference IndexSSRI Selective Serotonin Reuptake InhibitorTCH346 dibenzo[b,f]oxepin-10-ylmethyl-methyl-prop-2-ynyl-amineTH Tyrosine HydroxylaseUPDRS Unified Parkinson’s Disease Rating Scale

11Dankwoord

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Promoveren hoef je gelukkig niet in je eentje te doen. Ik ben dan ook erg blij dat heel veel mensen op verschillende fronten me de afgelopen jaren hebben gesteund en geholpen. Daarvoor wil ik hen allemaal heel erg bedanken. Bovenal wil ik alle deelnemers aan de verschillende onderzoeken bedanken, want zonder hun inzet was dit niet mogelijk geweest. Verder realiseer ik me, dat ik waarschijnlijk mensen vergeet.

Allereerst wil ik mijn promotores, Prof. Dr. K.L. Leenders en Prof. Dr. R.A. Dierckx en co-promotor Dr. P.L. Jager bedanken. Nico, bedankt voor je vertrouwen in mij gedurende al die jaren en de ruimte die je me hebt gegeven. De weg was niet altijd even gemakkelijk, maar gelukkig is de eindstreep in zicht. Rudi, je raakte pas in een later stadium bij dit onderzoek betrokken, maar ik ben blij met je bijdrage aan dit proefschrift. Jouw kritische blik en steun had ik niet willen missen. Piet, jouw optimisme, enthousiasme, relativeringsvermogen en laagdrempeligheid hebben me meerdere keren op weg geholpen en gehouden; bedankt hiervoor.

De leden van de leescommissie, Prof. Dr. B.R. Bloem, Prof. Dr. A.A. Lammertsma, en Prof. Dr. J.H.A. De Keyser, wil ik bedanken voor hun tijd en inspanning om mijn proefschrift kritisch door te lezen. Dan wil ik Paul Maguire bedanken. Paul, zonder jouw expertise en kennis op het gebied van fysica, methodologie en farmacokinetiek had ik het nooit gered. Ook de momenten van reflectie over wetenschap had ik niet willen missen.

Iedereen die betrokken is geweest bij de werkgroep bewegingsstoornissen wil ik bedanken voor de jarenlange fijne samenwerking. Ik heb met een aantal van jullie nog steeds contact en dat koester ik. Bedankt dat dat mogelijk is.Cobi Bolwijn, jij was mijn steun en toeverlaat bij alle trials die we samen hebben gedaan. Het was altijd ontzettend gezellig met jou en ik ben blij dat je weer je plekje gevonden hebt.Martje Drent, onze Parkinsonverpleegkundige, jouw hulpvaardigheid, kunde en charme zijn onmisbaar geweest tijdens onze patiëntencontacten. Lammy Veenma, samen hebben we heel wat analyses van PET-studies mogen doen. Het was heerlijk om met jou samen te mogen werken.En uiteraard wil ik ook Renee Staal bedanken. Jij bent al jarenlang de spil van de ‘bewegingsstoornisclub’. Jij hebt altijd alles wonderbaarlijk goed piekfijn geregeld. Jouw gezelligheid, humor en medeleven zorgden voor een goede sfeer.

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En dan al mijn kamergenoten op de GNIP-kamer. Het zijn er heel wat geweest: Joost van Oostrom, Esther Gieteling, Anna Bartels, Joyce Oen, Jolanda Lutke Farwick, Jeroen de Vries, en alle anderen die gedurende korte of langere tijd op de GNIP-kamer werkten. Dankzij jullie allemaal was er altijd een geweldige sfeer. Bedankt voor al die jaren samenwerking en gezelligheid. In het bijzonder wil ik Axel Portman en Marije van Beilen noemen. Axel, wij zijn samen echt letterlijk met niets begonnen. Jij hebt mij wegwijs gemaakt in de wondere wereld der neurologie en de even zo wondere wereld van echte Groningers. Bedankt voor de leuke tijd. Marije, als enige niet-medicus ben je anders dan alle andere kamergenoten. Jij hebt altijd een verfrissende blik op de zaken en je hebt me met al je adviezen en raadgevingen op verschillende vlakken heel veel geholpen.

Verder wil ik alle andere assistenten neurologie bedanken. Ik heb met jullie een geweldig leuke tijd gehad, zowel professioneel als ook buiten werktijd; bedankt hiervoor. Ook de stafleden neurologie, evenals het secretariaat neurologie wil ik bedanken voor hun prettige samenwerking. Vooral Bauke de Jong en Teus van Laar wil ik bedanken voor hun enthousiaste begeleiding en samenwerking.

Ook wil ik Gerda Andringa bedanken. Gerda, toen ik nog helemaal aan het begin van mijn promotietraject stond, was jij al bijna klaar. Ik ben blij dat ik de kunst bij jou mocht afkijken. Bedankt voor de fijne samenwerking. Ook de overige Nijmegenaren, M. Faassen, T. Peters en A. Hanssen wil ik bedanken voor hun professionele dierverzorging.

Veel dank ben ik verschuldigd aan Sharon Jonkman. Sharon, altijd enthousiast en accuraat, jij hebt enorm veel energie en tijd gestoken in de uitwerking van de FP-CIT SPECT scans, waarvoor dank.

Ook de overige MNW-ers van de afdeling NGMB wil ik allemaal bedanken voor hun inzet en samenwerking.

Speciale dank voor de secretaresses van de SPECT- en de PET kant die al die aanvragen voor F-DOPA PET en FP-CIT SPECT scans verwerkten en de afspraken regelden.

Bovendien wil ik Ruud Kortekaas bedanken. Ruud, jij hebt me met jouw enthousiasme en kennis van proefdieren en statistiek meerdere malen geholpen, zelfs als je daarvoor naar mijn huis moest komen. Bedankt hiervoor.

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Natuurlijk wil ik ook de stafleden NGMB bedanken. Adrienne Brouwers, Andor Glaudemans, Jan Pruim, Riemer Slart, bedankt voor de mogelijkheid om tijdens mijn opleiding nog aan mijn proefschrift te mogen werken.

Verder wil ik ook de assistenten NGMB bedanken voor hun samenwerking en hulp. KP Koopmans, je bent me al voorgegaan in promotieland en wist me altijd op te beuren als ik het weer eens niet zag zitten. Niels Veltman, Olga Mirankova, Leo Weijs, Ronald van Rheenen; sommige van jullie gaan ook het promotietraject in: veel succes hiermee. Heel erg bedankt voor jullie begrip, als ik weer eens een wetenschapsdag had.

Als moeder lukt het niet te promoveren, als het thuisfront niet goed geregeld is. Daarvoor ben ik heel veel mensen dank verschuldigd die volledig belangeloos voor me klaar stonden om onze kinderen op te vangen: Marcel de Jong, Henriet Kampman, Emma en Margreet Zorgdrager, maar vooral Saskia Popken. Zonder jullie hulp was dit niet mogelijk geweest.

Mijn paranimfen, Esther Zeinstra en Emma Zorgdrager, wil ik bedanken voor al hun hulp en steun. Ik beschouw het als een eer dat jullie beiden mijn paranimf willen zijn. Bedankt dat jullie mij letterlijk terzijde willen staan bij mijn verdediging.

Dank aan mijn (pleeg)familie. Zonder familie ben je nergens. Jullie hebben me opgevangen en geaccepteerd, toen ik het nodig had en ik kan niet verwoorden, hoe zeer ik dat altijd heb gewaardeerd. Leave heit en mem. Spitigernôch kinne jimme dit net mear meimeitsje, sa as jimme al sa folle mist hawwe. Jimme hawwe my it libben jûn en fan jimme ha ik leard troch te setten ek as it net sa noflik rint! Tige tank!

En dan wil ik mijn gezin bedanken, te beginnen met mijn schatjes, mijn boefjes: Tjeart, Aafke en Riksta. Via jullie heb ik de wonderen van het leven opnieuw leren ontdekken. Het is een feest om na het werk jullie armpjes om me heen te voelen en me mee te laten slepen in jullie (fantasie)wereld. Gelukkig heeft mem het boekje nu af, zodat we weer meer tijd samen kunnen doorbrengen.En tot slot wil ik Vincent bedanken. Jij kent het promotietraject uit eigen ervaring en hebt me altijd geholpen door te zetten. Jij was degene die ervan overtuigd was dat het me zou lukken, ook als ik het zelf niet meer zag zitten. Je hebt me de ruimte gegeven om ook thuis te kunnen werken en nam dan de zorg voor onze kinderen op je. Bedankt voor jouw rotsvaste vertrouwen in mij en al je steun, begrip, geduld en liefde tijdens dit project!

tch346 prevents motor symptoms and loss of striatal fdopa uptake in bilaterally mptp-treated...

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