lung imaging using oxygen-enhanced mri in small animals · lung imaging using oxygen-enhanced mri...

Lung imaging using oxygen-enhanced MRI in small animalsMaster of Science Thesis in Medical Radiation Physics

Daniel AlamSupervisor: PAu idi rof., Lars E. Olsson gust 2010

Imaging centre AstraZeneca R&D Mölndal, Sweden

[email protected] epartment of Radiatioahlgrenska Academy n Physics

Lung imaging using oxygen-enhanced MRI in small animals Daniel Alamidi

Abstract BACKGROUND Oxygen enhanced magnetic resonance imaging (OE-MRI) is a novel method of assessing regional ventilation and oxygen diffusion from the alveoli into the capillaries of the lung. The paramagnetic nature of oxygen and deoxyhemoglobin in blood shortens the T1 of the oxygenated tissues. MRI can visualize the effect by a signal increase on T1 weighted images acquired with subjects during breathing of increased oxygen concentration compared

ata on the utility of this technique in rodents are limited. to room air. D S To develop a protocol for OE-MRI on freely breathing live rodents. OBJECTIVE METHODS A T1-measurement protocol for the lung was established. Measurements on both Nickel-doped agarose gel phantoms and living rat lungs for optimization with respect to repetition time/inversion time/echo train length at a 4.7 T MRI scanner were performed. An optimized cardiac triggered inversion recovery RARE imaging sequence was developed. In order to achieve adequate signal from the lung parenchyma, maintaining practical acquisition

ompensating for rapid physiological motion. times, and c RESULTS The optimized parameters from the phantom studies were: repetition time of 6000 ms, 6 inversion time values and an echo train length of 6. The mean T1 was measured with ardiac triggering to 1682 ± 203 ms (12 %) and 1769 ± 188 ms (11 %) in the right and left

y. clung, respectivel CONCLUSIONS An optimized T1-measurement protocol was established for OE-MRI in rodents. Due to hardware problems that affected the images with ghosting artifacts no conclusions can be drawn about the oxygen-induced changes.

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Summary in Swedish (Sammanfattning på svenska) MR-bildtagning av lungor i smådjur med förhöjd syrenivå Kroniskt obstruktiv lungsjukdom (KOL) är en folksjukdom som idag enklast diagnostiseras med hjälp av spirometri. Med en spirometer kan man mäta lungfunktionen men dessvärre inte erhålla någon detaljerad information om sjukdomens utbredning i lungorna. För att vidare orientera sig i sjukdomens distribution gör man en lungröntgen eller datortomografi, vilket utsätter kroppen för joniserande strålning. Under senare år har Magnet Resonans (MR) kameror gjort stora framgångar vid bildtagning av lungor. Genom att använda sig av syre som kontrastmedel har en ny lungfunktionsmetod växt fram, syreförstärkt MR bildtagning. Det fria syret som objektet andas in har en paramagnetisk egenskap vilket kommer att åstadkomma en signalskillnad i lungvävnaden. Signaländringen kommer att reflektera lungfunktionen och dess förmåga att överföra syre från alveolerna i lungan till blodet. Denna bildtagningsmetod har under det senaste decenniet börjat tillämpas i den kliniska verksamheten med goda resultat. Syftet med detta arbete var att utarbeta ett MR protokoll för mätning av den signaländring som uppkommer vid inandning av syre på smådjur. Till en början optimerades protokollet med simuleringar och mätningar på MR fantom (geler). Därefter gjordes mätningar på fritt respirerande råttor.

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Abbreviations, acronyms and symbols 129Xe Xenon-129 3He Helium-3 81mKr Krypton-81m 99mTc Technetium-99m 3D Three-dimensional B0 External magnetic field BOLD Blood Oxygenation Level Dependent effect used in functional MRI BPM Beats per minute CO2 Carbon dioxide COPD Chronic Obstructive Pulmonary Disease CT Computed Tomography CV Coefficient of variation, standard deviation as a % of the mean Diffusion Process by which molecules spread from areas of high concentration, to areas of low

concentration DTPA Diethylenetriaminepentaacetic acid ETL Echo train length FID Free induction decay FOV Field of view GRE Gradient-echo IDL Interactive data language, programming software for data analysis IR Inversion recovery M0 Net longitudinal magnetization at equilibrium MR Magnetic resonance MRI Magnetic resonance imaging Mxy Net transverse spin magnetization Mz Net longitudinal spin magnetization NEX Number of excitations O2 Molecular oxygen OE-MRI Oxygen-enhanced magnetic resonance imaging Perfusion Flow of blood to reach an organ or tissue PFT Pulmonary function test RARE Rapid Acquisition with Relaxation Enhancement pulse sequence RF Radiofrequency ROI Region of interest SD Standard deviation SE Spin-echo SNR Signal to noise ratio SI Signal intensity T1 Time-constant for longitudinal relaxation T2 Time-constant for transversal relaxation due to spin interactions T2* Time-constant for transversal relaxation due to a combination of magnetic field

inhomogeneities and spin interactions TD Delay time TE Echo time of pulse sequence; time between slice excitation and measurement of

signal TEeff Effective echo time, the echo time that contributes the central segment of k-space TI Inversion time TR Repetition time of pulse sequence; time between two consecutive excitations of the

same slice

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Ventilation Exchange of air between the lungs and the atmosphere

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Contents 1. Introduction and aims ...................................................................................................... 7

1.1. Introduction ................................................................................................................ 7

1.2. Aims ........................................................................................................................... 7

2. Theory ................................................................................................................................ 9

2.1. Respiratory system ..................................................................................................... 9

2.1.1. Anatomy and physiology of the human respiratory system ............................... 9

2.1.2. Respiratory function ......................................................................................... 10

2.2. The Lung .................................................................................................................. 11

2.2.1. Lung diseases ................................................................................................... 11

2.2.2. Lung function measurements ........................................................................... 11

2.2.3. Rat lung anatomy data ..................................................................................... 11

2.3. Challenges with MRI of the lung ............................................................................. 12

2.4. OE-MRI of the lung ................................................................................................. 13

2.4.1. Principles.......................................................................................................... 13

2.4.2. Background ...................................................................................................... 14

2.5. Inversion recovery RARE pulse sequence ............................................................... 14

2.6. T1 - relaxation .......................................................................................................... 15

2.6.1. T1 - calculation................................................................................................. 16

3. Material and methods .................................................................................................... 18

3.1. Hardware and software ............................................................................................ 18

3.2. Scan parameters ....................................................................................................... 18

3.3. Polarity restoration ................................................................................................... 18

3.1. Phantom studies ....................................................................................................... 19

3.1.1. General ............................................................................................................. 19

3.1.2. T1-calculation with Solver and IDL ................................................................ 20

3.1.3. SNR calculation and reduction ........................................................................ 20

3.1.4. TR evaluation ................................................................................................... 20

3.1.5. TI evaluation .................................................................................................... 21

3.2. In vivo studies .......................................................................................................... 21

3.2.1. General ............................................................................................................. 21

3.2.2. Animal preparation .......................................................................................... 21

3.2.3. T2 measurements ............................................................................................. 22

3.2.4. T1 measurements ............................................................................................. 22

3.2.5. T1 measurements with cardiac triggering ........................................................ 22

3.2.6. T1 measurements with combined cardiac and respiratory triggering .............. 22

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3.2.7. Oxygen-enhanced T1 measurements ............................................................... 23

4. Results .............................................................................................................................. 24

4.1. Phantom studies ....................................................................................................... 24

4.1.1. T1-calculation with Solver and IDL ................................................................ 24

4.1.2. SNR reduction .................................................................................................. 25

4.1.3. TR evaluation ................................................................................................... 25

4.1.4. TI evaluation .................................................................................................... 26

4.2. In vivo studies .......................................................................................................... 27

4.2.1. T2 measurements ............................................................................................. 27

4.2.2. T1 measurements ............................................................................................. 27


4.2.4. T1 measurements with combined cardiac and respiratory triggering .............. 28

4.2.5. Oxygen-enhanced T1 measurements ............................................................... 28

5. Discussion ........................................................................................................................ 29

5.1. Polarity restoration ................................................................................................... 29

5.2. Scan parameters ....................................................................................................... 29

5.3. Phantom studies ....................................................................................................... 30

5.3.1. SNR reduction .................................................................................................. 30

5.3.2. TR evaluation ................................................................................................... 30

5.3.3. TI evaluation .................................................................................................... 30

5.4. In vivo studies .......................................................................................................... 31

5.4.1. T2 measurements ............................................................................................. 31


5.4.1. Oxygen-enhanced T1 measurements with combined triggering ..................... 31

Acknowledgements .................................................................................................................. 32

References ................................................................................................................................ 33


1. Introduction and aims 1.1. Introduction

One of the most common lung diseases is chronic obstructive pulmonary disease (COPD). COPD is characterized by limitation of airflow during expiration due to emphysema, chronic bronchitis or both and is in a decade expected to be the third-leading cause of death (Devereux G 2006). This reduction in outflow of air during expiration can be measured by pulmonary function tests. Spirometry (measuring of respiration) is the most straightforward method of measuring lung function and investigating diseases such as COPD. Nevertheless, this technique is incapable of identifying regional distributions and the location of pulmonary disorders. Hence, a three-dimensional (3D) method is required, i.e. imaging. Inhalation of radioactive gases is a method for lung imaging but with some significant limitations; the radiation dose from the radioactive substances and poor spatial resolution. The most common way of detecting lung abnormalities is with a standard chest x-ray. A better insight of pulmonary diseases can be achieved by computed tomography (CT) imaging. It is valuable for the evaluation of morphological changes and regional pulmonary functional tests. However, CT-imaging does not supply any functional information of the lungs and it exposes the body to ionizing radiation. MRI of the lung is challenging since the lung has low proton density and therefore creating a low signal. It furthermore contains air-tissue interfaces generating susceptibility artifacts. Additionally, the influence of respiratory and cardiac motion has to be controlled to avoid motion artifacts. Several methods have been proposed in order to overcome these difficulties. They consist of breath hold imaging, respiratory and cardiac triggering procedures and use of pulse sequences with very short echo times (TEs). In addition, MRI with hyperpolarized noble gases such as Helium (3He) and Xenon (129Xe) render direct MR visualization of gaseous possible. Excellent results have been achieved for lung imaging, but the costs for additional equipment and production of noble gases currently limit its broad application. A new technique in development devoted to lung functional imaging is oxygen-enhanced MRI (OE-MRI), inhaling of molecular oxygen as a contrast agent, which enhances the signal of the protons in the pulmonary capillaries. This method provides means to directly study oxygen uptake of the lungs and is useful for the diagnostic of respiration related diseases.

1.2. Aims The overall aim of this project was to perform oxygen-enhanced MR imaging on living rodents during free breathing. Throughout the task, a T1-measurement protocol for the lung was established. To achieve these goals, the project in developing this new technique for rodents was divided into three main aspects: Initially gel phantoms were used to optimize the parameters of a pulse sequence made for T1-relaxation time measurements. The signal to noise ratio (SNR) in vivo in lung is lower than in phantoms due to the low proton density. The SNR was thus virtually reduced in the phantoms

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by adding noise to the images with help of simulations to investigate the results in a realistic environment. In the second part of the project the optimized sequence was verified in vivo in lung tissue of rats. The accuracy and precision of T1 was improved with cardiac triggering and a respiratory technique that blocked the acquisition of the signal during the inspiration of the animal. The final part of the project considered the examination of oxygen induced changes in T1 relaxation time of lung parenchyma in rodents.

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2. Theory 2.1. Respiratory system 2.1.1. Anatomy and physiology of the human respiratory system

The human body is dependent on a constant supply of oxygen (O2) to every single cell of the organism. Respiration is the physiological process by which organisms supply O2 to their cells and the cells use that O2 to produce high energy molecules. A respiratory system consists of conducting airways: trachea, bronchi, bronchioles and terminal bronchioles. The acinar airways, where actual gas exchange occurs, includes transitional bronchioles, respiratory bronchioles, alveolar ducts and alveolar sacs (Faller A et al. 2004) (Figure 1).

Figure 1. Detailed illustration of the lung and the respiratory system with conducting and acinar airways (Mayo M L 2009).

The alveolar sacs are closely packed air sacs, like individual grapes within a bunch (Figure 2). The lung can be regarded as a collection of these small 500 million bubbles. The individual alveoli are tightly wrapped in blood vessels, allowing gases in the alveoli to easily diffuse into the blood.

Figure 2. A schematic view of alveoli (Faller A et al. 2004).

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Fresh air is taken in via the upper airways (the nasal/oral cavities, pharynx and larynx) through the lower airways (trachea, primary bronchi and bronchial tree) and into the small bronchioles and alveoli within the lung tissue. The upper airways are the means of transportation of the inspired and expired air, and they warm it, humidify it, purify it and regulate it (sense of smell). The trachea is divided into two equally sized bronchi, which in turn diverge into two daughter branches (Hlastala M P et al. 2001). Additionally, the lung filters unwanted materials from the circulation and acts like a reservoir for blood. The lung structure has an intricate structure; it is elastic and remarkably durable (Schwartzstein R M et al. 2005, West J B 2008).

2.1.2. Respiratory function The breathing mechanism is driven by the diaphragm; the thin, dome-shaped muscle at the base of the thoracic cavity. During inspiration, the volume of the thoracic cavity increases and air is drawn into the lung. The volume increase causes the internal pressure of the chest to become lower than atmospheric pressure, resulting in a flow of air into the airways. The driving forces for gas exchange between the lung and the environment are the pressure differences. The processes of internal respiration concern the exchange of O2 and carbon dioxide (CO2) between blood and cells in different tissues. On the contrary, external respiration is the process by which outer air is drawn into the body in order to supply lungs with O2, and “used” air is expelled from the lungs in order to remove the CO2 from the body. The oxygen transport can be divided into three steps: (1) Ventilation, or breathing, involves the physical movement of air in and out of the lungs; (2) Gas diffusion, exchange of gases between the alveoli and the pulmonary capillaries (Figure 3); (3) Perfusion, circulation of blood between the lungs and organs.

Figure 3. Gas exchange in the lung. The exchange of gases in the lung transforms deoxygenated (poor in oxygen) venous blood that is rich in CO2 into oxygenated (rich in O2) arterial blood with low CO2 content (Faller A et al. 2004).

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2.2. The Lung 2.2.1. Lung diseases

Many of the properties and mechanics governing lung function are impaired in case of a disease. One of the most common lung diseases is COPD. It is currently the fourth leading cause of mortality in the western world. With an increase in smoking in developing nations; COPD is expected to be the third-leading cause of death worldwide within ten years. The disease is associated with several risk factors where cigarette smoking clearly is the most significant (Devereux G 2006). COPD is characterized by limitation of airflow during expiration due to either emphysema, chronic bronchitis or both. Emphysema is a condition of the lung that destroys the alveolar walls, resulting in loss of elasticity of lung tissue. Chronic bronchitis is the inflammation of the bronchi in the lungs and destruction of structures supporting the alveoli. These symptoms lead to a reduction in the outflow of air during expiration that can be measured by pulmonary function tests. In pulmonary diseases, like COPD, alterations in the ventilation and/or perfusion progresses and impaired oxygen diffusion from the alveoli into the capillaries are the main cause of respiratory disorders. These abnormalities can be detected by pulmonary function test measurements (Devereux G 2006).

2.2.2. Lung function measurements In case of a disease in the lung, pulmonary function tests (PFTs) can be vital for measuring lung function. Spirometry is the most common way of measuring lung function and investigating diseases such as these. The volume and/or flow of air that can be inhaled and exhaled is examined. However, this method only provides information on a global scale, when disorders of the lung have reached more advanced stages. PFT is a relative insensitive method. Lung ventilation scintigraphy is an imaging method that uses inhalation of radioactive gases such as 133Xe, 81mKr or 99mTc- labeled diethylenetriaminepentaacetic acid (DTPA). The pulmonary ventilation function is evaluated with a gamma camera but the radioactive dose and poor spatial resolution limits the use of this method. Another common image modality of choice for detecting lung abnormalities is a chest x-ray. A better insight of the lung disease is attained with CT-imaging as it rapidly provides more detailed information. Nonetheless, CT-imaging does not provide any functional information of the lungs and there are restrictions related to CT, as the body is exposed to ionizing radiation with each imaging session (Mills et al. 2003). MRI of the lung has recently provided excellent results for evaluation of ventilation by utilization of hyperpolarized noble gases (Ohno Y et al. 2007). An alternative approach to hyperpolarized gas MRI is a new technique devoted for functional lung imaging, OE-MRI.

2.2.3. Rat lung anatomy data The respiratory system of rats is similar to that of humans, but with smaller dimensions and higher breathing frequency. Rodents have high respiratory rates, 71-146 breaths per minute, and high cardiac rates, 320-480 beats per minute (BPM), compared to human beings with 12-

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70 breaths per minute and 70 BPM (Grant K 2009, Pass D et al. 1993). The total lung capacity (TLC) of the rat is about 10 ml compared to 6 l of a human. There are 4 lobes in the ight rat lung (3 in human right lung) and only a single lobe in the left lung (2 lobes in human eft lung).

rl Further, the parenchyma of the rat lung occupies double as large fraction of the total lung than the human (rat: 24%, human: 12% lung volume). The blood-gas barrier thickness, diffusion distance in the rats (0.40 μm) is somewhat smaller than that of the human (0.62 μm). The thickness might have important consequences for both gas exchange and lung mechanics. In case of an inflammation of the lung tissue, plasma leakage into the alveoli increases the diffusion distance and impairs alveolar gas exchange. Lastly, rat lungs have fewer respiratory bronchioles and airway generations than human lungs do. Lung anatomy comparison between rodents and humans are presented in Table 1 (Grant K 2009, Irvin C G et al. 2003, Lindstedt S L et al. 2002, Sahebjami H 1992).

Table 1. Lung anatomy comparison between rodents and humans.

Rodent HumanRespiratory rate 71-146 12-70Cardiac rate 320-480 70Total lung capacity (TLC) 10 ml 6,000 mlRight lobes in lung 4 3Left lobes in lung 1 2Fraction of parenchyma of total lung 24 % 12 %Blood-gas barrier thickness 0.40 μm 0.62 μmAirway generations Single generation Several generations

2.3. Challenges with MRI of the lung MR Lung imaging is facing many difficulties because of the morphology, physiology and composition of the lung. Due to the air within the lung, it has inherently low average proton density (approximately 20-30 % of the soft tissue) generating weak signal, resulting in low SNR (Dietrich O 2009). Additionally, the signal is hampered by multiple air-tissue interfaces within the alveoli in the lung. The heterogeneous microstructure of lung parenchyma generates large local variations of susceptibility within small spatial scales between paramagnetic oxygen in air and diamagnetic tissue. These susceptibility variations influence the magnetic fields within the lung that rapidly dephase the already low MR signal, resulting in a very short T2*. Therefore sequences other than conventional Gradient-Echo (GRE) should be used for lung MRI, for example a Turbo Spin-Echo (TSE) sequence with a short echo spacing (Kauczor H-U et al. 1999). Moreover, signal distortions due to cardiac pulsation and respiration motion are a major problem in lung MRI. This is particularly apparent in small rodents, because of their higher cardiac and respiratory rates. To overcome these difficulties many strategies have been used. They consist of breath hold imaging, respiratory and cardiac triggering procedures and use of pulse sequences with extremely short TE (Beckmann N et al. 2007).

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2.4. OE-MRI of the lung 2.4.1. Principles

OE-MRI was first proposed in 1996 for evaluation of regional ventilation using molecular O2 as a contrast agent (Edelman R R et al. 1996). When compared with hyperpolarized gas imaging (see theory section 2.2.2), OE-MRI offers several benefits: the oxygen ventilation technique provides ways to directly study oxygen uptake from the air space to the pulmonary blood system; oxygen is easily reached as a part of emergency equipment in most MR suites; OE-MRI is very cost-effective since it does not require any supplementary expensive equipment (Mai V M et al. 2005). The underlying principle of OE-MRI is the weakly paramagnetic property of molecular oxygen caused by the presence of two unpaired electrons in their outer shells. The contrast mechanism works similar like a gadolinium-based contrast agent, but with a smaller magnitude: i.e., the T1 of the protons in blood is shortened depending on the O2 concentration (Edelman R R et al. 1996). Accordingly, oxygen-enhanced lung imaging can be regarded as imaging of lung function, as it provides information about three physiological parameters: The inhaled oxygen must be transferred to the lung region; subsequently adequate ventilation of the area is an essential condition for oxygen-induced reduction of T1 relaxation. The intermediate step regards the exchange of oxygen between the alveoli and the pulmonary capillaries, i.e. diffusion, is required for signal enhancement. As a final point, fresh capillary blood must be supplied in which the oxygen can be solved; thus, lung perfusion is for that reason a third constraint for the observation of reduced T1 values (Edelman R R et al. 1996, Loffler R et al. 2000). MRI with hyperpolarized gases and perfusion measurements with Gadolinium-DTPA are performed to provide information about ventilation and perfusion, respectively. OE-MRI has the potential to bring out information of these two physiological parameters as well as providing diffusion information of the gas exchange in lung. Oxygen-induced relative T1 reductions between 7 % and 14 % have been observed after inhalation of pure oxygen. The signal within each single voxel is averaged over various kinds of tissue such as blood, vessels, alveolar cells and surrounding tissue (Nakagawa T et al. 2001, Dietrich O et al. 2009). Subsequently, during gas exchange oxygen diffuses across the alveolar membrane into the pulmonary capillary blood and initially dissolves into blood plasma as molecular oxygen. Oxyhemoglobin is generated when the oxygen molecules couples with hemoglobin. It is known that oxyhemoglobin is diamagnetic and that deoxyhemoglobin is paramagnetic. This means that deoxygenated blood has a shorter T2* and hence lower MR signal than fully oxygenated blood in gradient-echo (GRE) sequences with its strong T2* weighting. Blood oxygenation level dependent (BOLD) contrast is based on this effect of deoxyhemoglobin and is frequently used in functional MRI (McRobbie D W et al. 2003). After pure oxygen inhalation the molecular oxygen will dissolve and the hemoglobin will be saturated with oxygen. Consequently the concentration dissolved oxygen in the blood increases approximately fivefold. Hence the most important component for the T1 effect is caused by the increased concentration of dissolved oxygen in the capillary blood of the lung.

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This effect can be detected by T1-weighted MRI sequences as regions of increased signal intensity (Tadamura E et al. 1997, Watt K N et al. 2008). In addition, an increase in oxygen concentration within the blood results in a prolongation of T2* when T1 is measured in lung. However, this has only a minor impact on the signal intensity in T1 weighted sequences (Ohno Y et al. 2007).

2.4.2. Background Throughout the last decade several researchers have declared the potential of using OE-MRI in clinical applications for evaluation of lung function. Oxygen induced MRI for patients with pulmonary diseases has been successfully established which demonstrates the significance of oxygen enhancement. This includes ventilation abnormalities and emphysema, as well as assessing parameters of pulmonary function measurements for patients with lung cancer (Loffler R et al. 2000, Edelman R R et al. 1996, Chen Q et al. 1998). Moreover, OE-MRI was found to be as effective as quantitative CT for smoking-related lung functional loss assessment and stage classification concerning patients with smoking-related COPD (Ohno Y et al. 2008). In contrast, regarding preclinical animal studies the publications are very limited and further experimental and theoretical work is required. In mice only one study can be found (Watt K N et al. 2008), and not a single work on rats. Furthermore, external magnetic fields (B0) over 3 T are preferred to improve the SNR when small animals are studied. This induces problems for lung studies since the susceptibility variations are proportional to B0. In addition, small animals have high breathing and cardiac frequencies, which leads to signal distortions.

2.5. Inversion recovery RARE pulse sequence A basic Spin-Echo (SE) sequence has a long acquisition time since only one line of k-space data is collected after an excitation. However a SE image can be acquired with dramatic time saving by utilization of a Turbo SE (TSE) sequence. TSE is a commercial version of Rapid Acquisition with Relaxation Enhancement (RARE) sequence, which collects more than one line of data for each excitation by producing a train of echoes. The echo train is formed by multiple refocusing, 180˚, radiofrequency (RF) pulses (Figure 4). The acquisition time is proportional to the echo train length (ETL), i.e. the number of echoes acquired. For instance if 10 echoes are individually phase-encoded in RARE, the total acquisition time is decreased by a factor of 10. This echo train formation can be continued as long as sufficient transverse magnetization remains to form an echo, i.e. as long the T2 relaxation permits (McRobbie D W et al. 2003, Vlaardingerbroek M T et al. 1996). For enhancement of T1 contrast and T1 relaxation measurements an inversion pulse is implemented to the RARE sequence (Vaninbroukx J et al. 2003).

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Figure 4. Pulse sequence diagram of a RARE sequence with a train of echoes formed by multiple refocusing pulses. The top line, RF, shows the applied radiofrequency pulses, GS the slice-selective gradient, GR the readout gradient (frequency-encoding). Centrically reordered phase encoding is used to produce the highest SNR (Stock W K et al. 1999).

2.6. T1 - relaxation T1 relaxation (also known as thermal, longitudinal or spin-lattice relaxation) describes the recovery of the longitudinal magnetization to its initial value at thermal equilibrium (M0) along B0, just after applying an RF excitation pulse. It results from interactions amongst the water protons and protons attached to the surrounding molecules (the lattice) with fluctuating magnetic dipole moments at the larmor frequency. There are two energy states the hydrogen nucleus can occupy in the presence of B0; up, the lower energy state where the magnetic dipole moment points along B0, or down, the higher energy state where the magnetic dipole moment points opposite B0. At M0, thermal equilibrium, a slightly increase of dipoles is observed in the lower energy state along B0. In the excitation process, after a 90° pulse, there is an equal population of hydrogen dipoles in the lower and higher energy states. To give away energy and return to the lower energy state, the protons interact with the lattice, which can absorb the energy. In order to enable this energy transfer, the magnetic dipole moments of the neighboring protons or other nuclei or molecules has to fluctuate at the larmor frequency and thereby satisfy the resonance condition. Due to the fluctuating fields the spins can change from high to low energy states through interaction with the lattice, and contribute to a relaxation in magnetization. T1 is defined as the time it takes for Mz to recover to a value about 63 % of M0 after a 90˚ excitation of the longitudinal magnetization. Accordingly, longitudinal relaxation can occur only when a proton encounters another magnetic field fluctuating near the Larmor frequency and therefore, different amounts of spins will contribute to T1 relaxation in different types of tissue. Information about the mobility of molecules, particularly water molecules, and hence the binding of water molecules, for example macromolecules can be generated from T1. In addition there are several factors affecting T1: B0; free water content; lipid and macromolecule content; molecular motion; viscosity; temperature. For instance, the T1 relaxation time is prolonged as B0 increases.

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2.6.1. T1 - calculation The gold standard to measure T1 is by a series of inversion recovery (IR) acquisitions with varying inversion times (TI). The IR method may be describes as:

Prepare (180˚) - TI - 90˚ (detection) - TD In its simplest form the first part within an IR sequence involves inverting the initial magnetization (which is initially in its equilibrium state) with an 180˚ preparation pulse. After this pulse the longitudinal magnetization will recover exponentially during TI at a rate described by T1. Subsequently after TI, acquisition of the signal is performed during a readout time, d. The readout sequence is typically a SE such as RARE (Figure 4), which is used in this study. Hence a 90˚ RF pulse is applied, which tips the current longitudinal magnetization into the transverse plane, where it after the refocusing pulses (180˚ pulses) gives rise to an echo and a free induction decay (FID). The sequence is then repeated after a delay time TD (Figure 5).

Figure 5. Pulse sequence scheme of the inversion recovery RARE sequence where d, is the readout time during the readout and Td, is the delay time after the acquisition before the next IR pulse.

The sequence is repeated several times with different TIs. In this way the recovery curve can be sampled where the signal intensity (SI) is plotted versus the TI (Figure 6). The time dependence of th I n l d ) bed as: e S a d ongitu inal magnetization (Mz is descri𝑆𝐼 ∝ 𝑀 = 𝑀 [1 − (1 − 𝑀 /𝑀 ) exp (− 𝐼/𝑇 )] 𝑇where 𝑀 is the magnetization at the beginning of the 𝑇𝐼 period. This equation can be developed and simplified c s

[2.1]

if TR is held on tant: 𝑆𝐼(𝑇𝐼) ∝ 𝑀 = 𝐴 − 𝐵𝑒 / where 𝑆𝐼(𝑇𝐼) is the signal intensity (~𝑀 ) at time 𝑇𝐼, 𝐴 is a constant for the offset (𝑀 ) and 𝐵 is a constant for the proton density and 𝑀 (Kingsley P B 1999, Gupta R K 1980). The three constants 𝐴, 𝐵 and 𝑇 are obtained by minimizing the difference between the observed data and the calculated values, i.e., a three-parameter nonlinear least square method fit generates the values of the three constants. For perfect pulses only a two-parameter fit is needed to fit the data because 𝑀 /𝑀 = −1 for fully relaxed IR (Levy G C et al. 1975).

[2.2]

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2000 4000 6000

-100000

-50000

0

50000

100000

TI [ms]

SI [

a.u

]

Figure 6. Inversion recovery plot of SI within a circular region of interest (ROI) at a gel phantom with a T1 of 1500 ms. 8 TI points (of 100, 300, 400, 500, 1200, 2000, 3500 and 5000 ms) were used to sample the curve. The standard deviations (SDs) are smaller than symbols.

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3. Material and methods 3.1. Hardware and software

All the experiments were performed on a 4.7 T Bruker MRI system (Bruker Biospin 47/40, Ettlingen, Germany) and operated from Linux computers running Paravision 5.0 (Bruker, Ettlingen, Germany). The gel-containing vials and living rodents were scanned using a 72 mm quadrature coil (Model no: 1P TP9455, Serial no: S 0027).

3.2. Scan parameters An inversion recovery RARE pulse sequence (section 2.5) was used for the phantom and in vivo studies with following parameters: TE = 3.52 ms; bandwidth = 200 kHz; field of view (FOV) = 7 x 6.5 cm; matrix size = 196 x 256; number of excitations (NEX) = 2; and slice thickness = 5 mm. The frequency and phase encoded pixel sizes were 0.36 mm/pixel and 0.25 mm/pixel respectively. Initially, an ETL of 4 was used and a non-selective inversion pulse was applied. Centrically reordered phase encoding was utilized that collects the center of the k-space during the first part of the readout interval, resulting in a short effective echo time (TEeff) (Figure 4). The lines around the center of the k-space determine the contrast of the acquired image, while the outer part of the raw data matrix provides information on details of the image (Zhou X J 2004a). A short TEeff is desired to avoid T2 weighting. Centrically reordered phase encoding provides higher signal intensity, a shorter TEeff and a higher SNR.

3.3. Polarity restoration The phase information of the transverse magnetization is ignored when magnitude MR images are used. As a result the sign is lost from the inversion recovery curve (Figure 7). It is preferable to restore the polarity of the inversion recovery data, as this will reduce the variance in the estimation of T1 (Gowland P A et al. 2003, Zhou X J 2004b). In this project magnitude images were sampled, hence a straightforward polarity restoration technique was implemented for the T1 calculations. To begin with, the minimum point at a magnitude curve was obtained, the lowest signal value A in Figure 7. The polarization of the TIs less than or equal to this minimum point was switched, resulting in a polarity-restored IR curve, hence point A was moved to point B in Figure 7. This procedure was also performed for the points ± 1 steps from the minimum point, the values within the circle in Figure 7. Accordingly three different inversion recovery plots were acquired. The sum of squared error at the three recovery curves was calculated, and the curve with smallest amount of inaccuracy was selected. Data analysis with standard interactive data language (IDL) (version 6.4, ITT Visual Information Solutions, Boulder, CO, USA) software was performed. ROIs were positioned on a set of IR magnitude images acquired with different inversion times (Figure 8) and polarity-restored IR plots were performed.

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0 2 000 4 000 6 000-100000

-50000

0

50000

100000Polarity-restored data

Magnitude data

TI [ms]

Sign

al [

a.u

]

A

B

Figure 7. Polarity-restored and magnitude IR curves for a gel phantom. The arrow points at the lowest signal value (A) on the magnitude curve. TIs less than or equal to the minimum point (A) were polarity-restored; the sign of the minimum point A was switched, resulting in a new position B. The points within the circles, ± 1 steps from the minimum point were also polarity restored, to decide the optimal IR curve for the T1 assessment. The SD is less than 1 % and is therefore too small to present graphically.

3.1. Phantom studies 3.1.1. General

Phantom studies were performed to optimize and establish timing constrains for a pulse sequence scheme. The sequence design was subsequently used for the in vivo experiments. The particular aim was to keep the scan time as low as possible and acquiring T1 values with good accuracy and precision. The main parameters optimized in the sequence were the length of the repetition time (TR) and the number of TI values used for the T1 assessment.

Figure 8. Circular ROIs placed at a 5 mm single slice axial MRI image of 8 gel phantoms with T1 values of 1000 – 1800 ms. The rectangular ROI in the right corner represent the noise area for the SNR calculation.

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Eight nickel-doped agarose gel phantoms in vials with T1 values of 1000 – 1800 ms were used (Alamidi D et al. 2009). Earlier experiments in rat lung confirmed this range of T1-values. The vials were scanned in an axial projection with a slice thickness of 5 mm (Figure 8).

3.1.2. T1-calculation with Solver and IDL To assure that the estimation in IDL was acceptable, T1 calculations at the eight gel phantoms with the Solver tool in Microsoft Excel software were done. The function of the Solver is to adjust parameters with the least square method, similar to the IDL software. The Solver tool use modulus data, hence the absolute value of equation [2.2] is applied. Mean signal intensity values were obtained from circular ROIs placed on the gel phantom images (Figure 8). Estimation of T1 over the eight vials with T1 values of 1000 – 1800 ms was also done with the IDL software for evaluation between the two methods. T1 maps were generated with the polarity-restored data in the IDL software. A pixel-by-pixel three-parameter nonlinear least square T1 method fit was applied (section 2.6.1), i.e., T1 was calculated for each pixel. Circular ROIs were drawn on the T1 map to obtain the mean T1 values of the pixels. The inversion recovery RARE sequence with a constant TR was used and the TI values were 100, 300, 400, 500, 1200, 2000, 3500 and 5000 ms for the both methods.

3.1.3. SNR calculation and reduction For the T1 assessments, a set of TI values were used. The image with longest TI, i.e. with highest signal, was selected for the SNR calculation. The noise was determined by placing a rectangular ROI in the noise area of the image (Figure 8). A single circular ROI to obtain the signal intensity was placed in the phantom with a T1 value of 1500 ms (earlier results have shown that T1 in rat lung is around 1500 ms). The SNR was calculated by dividing the mean signal in the circular ROI with the mean signal in the noise. The SNR in vivo is lower than in phantoms because of the low proton density and signal in lung. To investigate the results in a realistic situation SNR was reduced in the phantom images using simulations with standard IDL software. The purpose of the simulations was to optimize the sequence for in vivo studies, and to keep the scan time as low as possible. Stochastic noise that decreased the existing SNR to a ratio of 40 and 20 was added to the images. The noise was defined as a Gaussian distribution of random numbers, with a mean of zero and a SD of one. T1 was estimated by a three-parameter nonlinear least square method fit (section 2.6.1) in the IDL software.

3.1.4. TR evaluation T1 measurements with different TRs of 3000, 4000, 6000 and 8000 ms were performed to obtain the relationship of TR and the precision of T1. The TIs were adjusted with a range from 100 ms up to the respective TR to receive a full relaxation curve (Table 2). Setting TR significantly longer than the longest TI increases the measurement time without improving accuracy or precision (Kingsley P B et al. 2001). Accordingly, TR was set just slightly longer than the longest TI.

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Table 2. TR and TI values for the T1 phantoms measurements.

TR [ms] TI [ms] ---> 8000 7000 6000 5000 3500 2000 1200 500 400 300 1006000 5000 3500 2000 1200 500 400 300 1004000 3500 2000 1200 500 400 300 1003000 2000 1200 500 400 300 100

3.1.5. TI evaluation

The correlation of the number of TI values with the T1 precision was studied when the TR parameter was established. With fewer TI points the scan time is improved. The experiment started with an acquisition of 8 TI points and was consecutively reduced to 3 TI points. A constant TR of 6000 ms was used for all measurements. After the acquisitions, simulations and T1 calculations in IDL were performed.

3.2. In vivo studies 3.2.1. General

The optimized inversion recovery RARE pulse sequence design was verified in vivo in rat lungs to establish the timing constrains for the sequence. The in vivo studies were performed with the same geometry and parameters as the phantom studies, i.e. in an axial projection with a slice thickness of 5 mm. The slice was carefully positioned slightly over the diaphragm to include as much lung parenchyma as possible. T1 maps were calculated in the same way as for the phantom studies. Mean T1 values from ROIs placed on the relaxation maps in lung were obtained. The position and shape of the ROIs in which the maps were determined were carefully chosen to avoid contributions from large vessels. The relation between ETL and SNR was examined with the anticipation to decrease the scan time. To improve the accuracy and precision of the T1 calculations cardiac triggering was performed.

3.2.2. Animal preparation

Wistar rats (280 ± 35 g, mean ± SD) were prepared in accordance with the guidelines established by the Animal Ethics Committee of Göteborg’s University, with the ethical approval number 400-2008. Rats were lightly anaesthetized with isoflurane (5 %, Abbot Scandinavia, Solna, Sweden) for 1-2 min and immediately injected with anesthetic solutions Zoletil (40 mg/kg, VIRBAC S.A. Titulaire de I’AMM, 06516 Carros, France) and Domitor (0.4 mg/kg, Orion Pharma, Espoo, Finland). Next the animals were transferred to a dedicated rat holder (Bruker, Ettlingen, Germany) in supine position, head up, and allowed to breathe room air or pure oxygen spontaneously. The bed was inserted to the center of the scanner with the lungs in the center of the coil.

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Cardiac and respiratory monitoring systems (SA instrument, Inc) were used throughout the imaging. A pneumatic pillow was taped on the rats’ abdomen for respiratory monitoring. The core temperature was measured with a rectal thermo sensor and was maintained at 37˚C ± 1˚C by means of heated water tubes. The bed was equipped with a fluid based (fluorocarbon) heating system, which prevented the animals from cooling down during imaging. By running a set of conventional proton-localizers it was ensured that the lungs were positioned in the center of the coil and magnet. All measurements were performed with spontaneously breathing animals.

3.2.3. T2 measurements A long ETL without letting the signal totally decay due to T2 decay is desirable to minimize the scan time. Therefore T2-measurements in vivo in rat lung were made to examine the ETL relation to the SNR. The same sequence and scan parameters as for the T1 studies was used (section 3.2), but with 8 echoes (3.52, 7.04, 10.56, 14.05, 17.6, 21.12, 24.64, 28.16 ms). A constant TR of 2000 ms and no IR pulse was used. The filling of k-space was also changed to multi echo sampling, each echo filling a different k-space. The measured signal versus TE was fitted to an exponential decay Mxy = M0exp(-TE/T2). For the evaluation of the T2 values, T2 maps were generated with the IDL software. A T2 method fit to the exponential decay (see above) was applied for the corresponding pixels. Mean T2 values of the pixels from ROIs placed on the T2 maps in lung were obtained.

3.2.4. T1 measurements T1 measurements were performed on 4 rats breathing air with an ETL of 4 and 6 with a constant TR of 6000 ms and six TI values of 100, 500, 1200, 2000, 3500 and 5000 ms. The same inversion recovery RARE sequence and scan parameters was used as for the phantom studies (section 3.2).

3.2.5. T1 measurements with cardiac triggering To increase the accuracy and precision of T1, motion artefacts were reduced using cardiac triggering, where no respiratory triggering was used. T1 measurements were performed on 4 new rats breathing air with the IR RARE sequence and scan parameters as for the phantom studies (section 3.2). The TR was 6000 ms and six TI values of 100, 500, 1200, 2000, 3500 and 5000 ms were acquired with an ETL of 6.

3.2.6. T1 measurements with combined cardiac and respiratory triggering

In order to keep TR constant and increase the accuracy and precision of T1, combined cardiac and respiratory triggering was used. Cardiac triggering was on together with the respiratory triggering technique where the acquisition of the signal was blocked during the inspiration of the animal. The combined triggering method with a constant TR of 6000 ms was tested on four new animals and six TI values of 100, 500, 1200, 2000, 3500 and 5000 ms were acquired with an ETL of 6. The scan parameters were the same as for the phantom studies (section 3.2).

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3.2.7. Oxygen-enhanced T1 measurements Two T1 measurements on four rats were performed with the combined cardiac and respiratory triggering technique. The animals were breathing air during the first acquisition and inhaling pure oxygen on the second. To ensure high oxygen concentration in the lung, a delay time of minimum 2 minutes was allowed between air and oxygen acquisitions.

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4. Results 4.1. Phantom studies 4.1.1. T1-calculation with Solver and IDL

T1-calculations on eight gel phantoms in the Solver and IDL software were done where an excellent correlation (r = 1, p < 0.0001) between the two methods is observed (Table 3). The estimated T1 values are practically the same for the two methods. ROIs are the mean signal within the ROI for the Solver and averaged T1s for pixels within the ROI for the IDL software. An obtained T1 map used for the T1 calculation in the IDL software is shown in Figure 9.

Table 3. T1 relaxation times [ms] on eight gel phantoms calculated with the Solver tool and IDL software.

Solver IDL

Nr Estimated T1 Average T1 ± SD

1 974 973 ± 11

2 1076 1076 ± 15

3 1168 1169 ± 13

4 1246 1247 ± 15

5 1354 1356 ± 19

6 1434 1437 ± 20

7 1546 1550 ± 20

8 1784 1790 ± 32

Figure 9. T1 map generated with the polarity-restored data in the IDL software on 8 gel phantoms with T1 values of 1000 – 1800 ms.

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4.1.2. SNR reduction

The stochastic noise that decreased the existing SNR to a ratio of 40 ± 1 and 20 ± 1 was added to the original phantom images. The SNR reduced images are shown in Figure 10.

Figure 10. Axial MRI images on a 5 mm single slice of 8 gel phantoms with T1 values of 1000 – 1800 ms. Image A has the original SNR of 114, for B and C is the SNR decreased to a ratio of 40 ± 1 and 20 ± 1, respectively.

4.1.3. TR evaluation The results from the TR evaluation are presented in Figure 11 where the coefficient of variation (SD/mean) is plotted versus mean T1. Artificial noise was added to the images and reduced the SNRs to 40 ± 1 and 20 ± 1. For the phantom with a T1 value of 1500 ms and a SNR of 20 the CV increased (0.10, 0.15, 0.27 and 0.30) with shorter TRs (8000, 6000, 4000 and 3000 ms, respectively). This pattern is observed over the whole range of phantoms, where the CV is low with longer TRs (6000 and 8000 ms) compared to the shorter TRs (3000 and 4000 ms). The difference in the CV in the phantom with a T1 of 1500 ms between a TR of 6000 ms and 8000 ms was 33 % for a SNR of 20. In order to decrease the scan time a TR of 6000 ms was chosen for the sequence design.

25


A

1000 1500 2000 25000.0

0.1

0.2

0.3

0.4

0.5

SNR = 78

SNR = 40

SNR = 20

Mean T1

CV

(SD

/mea

n T

1)

B

1000 1500 2000 25000.0

0.1

0.2

0.3

0.4

0.5

SNR = 105

SNR = 40

SNR = 20

Mean T1

CV (S

D/m

ean

T1)

C

1000 1500 2000 25000.0

0.1

0.2

0.3

0.4

0.5

SNR = 114

SNR = 40

SNR = 20

Mean T1

CV

(SD

/mea

n T1

)

D

1000 1500 2000 25000.0

0.1

0.2

0.3

0.4

0.5

SNR = 116

SNR = 40

SNR = 20

Mean T1

CV (S

D/m

ean

T1)

Figure 11. The CV over mean T1 for T1 measurements on gel phantoms with different TRs of 3000, 4000, 6000 and 8000 ms from graph A to D, respectively. Circular ROIs were placed on the 8 vials as in Figure 8. The original and reduced SNRs to a ratio of 40 ± 1 and 20 ± 1 for respectively study are presented. The CV is low with longer TRs (6000 and 8000 ms) compared to the shorter TRs (3000 and 4000 ms).

4.1.4. TI evaluation In Figure 12, eight diagrams with 8-3 TI values (A-D) for the T1 assessment are shown. The CV is plotted versus mean T1 for the original and reduced SNRs. A constant TR of 6000 ms was used. For the phantom with a T1 value of 1500 ms the CV was between 0.12 - 0.15 (0.15, 0.13, 0.12, 0.14) for the T1 assessment with a SNR of 20 and TI values of 8, 7, 6 and 5, respectively. With a reduced amount of TI values, 4 and 3, the CV increased to 0.36 and 475. As a result, less than five TI points results in a large error for the T1 estimation where the CV curves increases.

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A

1000 1500 2000 25000.0

0.1

0.2

0.3

0.4

0.5

SNR = 114

SNR = 40

SNR = 20

Mean T1

CV (S

D/m

ean

T1)

B

1000 1500 2000 25000.0

0.1

0.2

0.3

0.4

0.5

SNR = 114

SNR = 40

SNR = 20

Mean T1

CV (S

D/m

ean

T1)

C

1000 1500 2000 25000.0

0.1

0.2

0.3

0.4

0.5

SNR = 114

SNR = 40

SNR = 20

Mean T1

CV (S

D/m

ean

T1)

D

1000 1500 2000 25000.0

0.1

0.2

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0.5

SNR = 114

SNR = 40

SNR = 20

Mean T1CV

(SD

/mea

n T1

)

E

1000 1500 2000 25000.0

0.1

0.2

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SNR = 114

SNR = 40

SNR = 20

Mean T1

CV

(SD

/mea

n T

1)

F

1000 1500 2000 25000

200

400

600

800

1000

SNR = 114

SNR = 40

SNR = 20

Mean T1

CV

(SD

/mea

n T

1)

Figure 12. The CV over mean T1 assessments on gel phantoms for different TI values of 8, 7, 6, 5, 4 and 3, starting with 8 TI points (A) down to 3 points (F) is shown. Circular ROIs were placed in the 8 vials (Figure 8). Noise was added to the images and decreased the original SNR to ratios of 40 ± 1 and 20 ± 1 for all experiments. Less than five TI points results in a large error for the T1 estimation where the CV curves increases (E, F). Note the large y-axis scale at the last diagram (F) with 3 TIs.

4.2. In vivo studies 4.2.1. T2 measurements

Images on eight TEs between 3.52 ms and 28.2 ms were acquired. The average T2 in rat lung was estimated to 9 ± 2 ms (22 %). Based on an assumption that keeping the SNR over 4, the sixth echo is still tolerable, thus an ETL of 6 was chosen.

4.2.2. T1 measurements

The measured mean T1 times were 1592 ± 308 ms (19%) and 1622 ± 294 ms (18%) for the right and left lung, respectively, without triggering. The results are calculated from a set of four rats and are presented in Table 4.

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Table 4.

T1 relaxation times [ms] in rodent lung with an ETL of 4

without triggering, during breathing of room air. The individual ROIs are averaged T1s for pixels within the ROI.

Subject Right lung Left lung

Average T1 in air ± SD Average T1 in air ± SD

1 1617 ± 331 1811 ± 394

2 1495 ± 337 1412 ± 182

3 1730 ± 362 1715 ± 408

4 1472 ± 200 1550 ± 193

Mean 1592 ± 308 (19 %) 1622 ± 294 (18 %)

4.2.3. T1 measurements with cardiac triggering

The mean T1 relaxation times in lung parenchyma were 1682 ± 203 ms (12 %) and 1769 ± 188 ms (11 %) for right and left lung with a new set of four rats. The individual results are presented in Table 5. Table 5.

T1 relaxation times [ms] in rodent lung with an ETL of 6 with cardiac triggering, during breathing of room air. The individual ROIs are averaged T1s for pixels within the ROI.

Subject Right lung Left lung

Average T1 in air ± SD Average T1 in air ± SD

1 1644 ± 248 1671 ± 161

2 1645 ± 159 1832 ± 209

3 1595 ± 126 1576 ± 183

4 1843 ± 278 1995 ± 200

Mean 1682 ± 203 (12 %) 1769 ± 188 (11 %)

4.2.4. T1 measurements with combined cardiac and respiratory triggering

Artifacts were still observed in the images in spite of the combined cardiac and respiratory triggering. Since the artifact was not related to cardiac and respiratory motion it was not possible to evaluate if the combined triggering technique improved the accuracy and precision of the T1 measurements.

4.2.5. Oxygen-enhanced T1 measurements No consistent oxygen induced changes were observable (see section 4.2.4.).

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5. Discussion 5.1. Polarity restoration

The developed polarity restoration technique with the IDL software worked fine. A comparison with the Solver tool was done to ensure the T1 estimation of the IDL software. The results on eight vials with different T1 times correlated very well (r = 1, p < 0.0001) which supports the T1 calculation in IDL. Moreover, sometimes the Solver has difficulties to find the appropriate start values in order to calculate a minimum. The program may need manual adjustment of the start values to get the solver going in right direction. It was necessary to make three different calculations to acquire a good accuracy and precision of T1, especially for noisy images. For images with low SNRs, the minimum value of the magnitude curve is not always the accurate point to switch the polarity. When the polarisation of a point is incorrect, the IR curve as well as the estimated T1 value will be inaccurate. By calculating three IR curves one is not restricted to a single answer. The sum of squared errors from the three plots was calculated and the curve with smallest error was selected. In that way the precision of T1 was optimized for images with different SNRs. An alternative method to the polarity restoration is to use the relative phases of the magnitude images to reduce the potential systematic errors due to noise. The sign of the transverse magnetization is in that way determined and signed magnitude images are created. This method was tested but excluded due to complications and shortage of time.

5.2. Scan parameters The SNR in vivo in lung is low and the primary aim with the parameter optimization was to increase the SNR with appropriate choices of slice thickness, NEX, bandwidth and pixel size. However, the scan parameters ought to work practically for the oxygen-induced acquisitions where two scans on the same animal are performed. Both air and oxygen measurements have to be completed during the examination to achieve oxygen-induced changes. The scan time is consequently a finite as it is not permitted anesthetizing the animals for an extended time. Thus, the SNR should be kept high and the acquisition time short. A relative thick slice thickness of 5 mm was chosen to increase the SNR, as the SNR is directly proportional to the slice thickness. A NEX of 2 was reasonable, since the SNR increases with the squared root of the NEX and motion artifacts are reduced for NEX >1. For NEX higher than 2, the scan time would be longer and impractical for the oxygen-enhanced measurements. A short TE is required for MRI lung measurements since the T2 in lung is very short, in this study a T2 of 9 ± 2 ms was measured. Preferably, the T2 decay should not influence the T1 assessments. To achieve a short TE, the bandwidth must be increased, in this case to 200 kHz. Unfortunately a higher bandwidth reduces the SNR, but this sacrifice had to be done to attain a short TE of 3.52 ms. The SNR increases with large voxels, as the SNR is proportional to the voxel size. Nevertheless, to manage separating objects in in vivo images the pixel size has to be less than the size of the examined object. When ROIs were placed in the in vivo images, contributions from large vessels were avoided. The diameter of a vessel in rat lung was measured to 1-3

29


mm2. With the selected pixel sizes of 0.36 mm/pixel, 0.25 mm/pixel for the frequency and phase resolutions, it was possible to separate the parenchyma from the vessels. A scan time of 8.5 minutes per TI value and six acquisitions resulted in a total scan time of about one hour. For oxygen-induced measurements this time would be doubled since one air and one oxygen measurement is required, resulting in a total acquisition of at least two hours.

5.3. Phantom studies 5.3.1. SNR reduction

The use of reducing the SNR by adding artificial noise to the gel phantom images was essential to achieve relevant information. It would not be possible studying the original phantom data with a SNR around 100 to prepare the parameters for the sequence used in vivo, since no significant conclusions can be drawn from the images.

5.3.2. TR evaluation A constant TR of 6000 ms was chosen since it was the most suitable parameter regarding the scan time and CV (Figure 11). The longest TR, 8000 ms, had a 33 % lower CV than a TR of 6000 ms for the T1 assessment. However a TR of 8000 ms would also increase the scan time with 33 % in comparison with a TR of 6000 ms. For a TR of 4000 ms the CV would increase with 80 % in contrast to 6000 ms and was therefore excluded. The acquisition time should be kept low for the oxygen-induced measurements in vivo with a good accuracy and precision of T1; therefore the TR of 6000 ms was selected. For each TR the range of TI values were changed. This could possibly affect the result of the TR evaluation since additional TI points were used for the longer TRs (Table 2). It would be interesting to examine the TR evaluation with a constant range of TIs.

5.3.3. TI evaluation Reducing the number of TI values from 8 to 5, resulted in no large change of the CV (0.12 – 0.15) (Figure 12). The CV increased with more than a 100 % when 4 TI values were used for a SNR of 20. Therefore, 5-6 TI values should be enough to sample for the T1 calculation regarding the scan time and accuracy and precision of T1. One interesting issue is the influence of the position of TI values chosen for the T1 calculation. In this study the starting points of 8 TI values were selected by intuition. However, the choices of TI values may be critical for the T1 precision. Which are the “perfect” points to sample for the inversion curve? The more points the better, but if you are restricted to only six points where should they be positioned at the curve? In the beginning of the IR curve, around the x-axis, or in the last part where the highest signal is achieved? A speculation is that the noise contribution is the same over the whole span of inversion times and the points should be chosen equidistantly over the IR curve. If the T1 is previously known, the “perfect” points can be calculated with the T1 equation [1-2exp(-TI/T1)], where perfect pulses are used. By starting with two endpoints, say 100 and 5000 ms for a constant TR of 6000 ms the rest of the TIs can be calculated. By knowing the signals for each TI, one can go backwards and calculate the TI for each signal. The equidistantly TIs in signal would

30


in that case be 100, 438, 868, 1458, 2401 and 5000 ms. Future studies will be done to see if the choice of TIs will affect the T1 assessment.

5.4. In vivo studies 5.4.1. T2 measurements

The sequence to measure T2 was not optimal, since the RARE sequence could only have eight echoes. However, the aim was not to accurately measure T2, but only to get an approximation of how long ETL that could be used. Because of the outflowing blood from the slice, the T2 most likely was underestimated, meaning that T2 should be longer in reality which resulted in a safe decision of the ETL.

5.4.2. T1 measurements with cardiac triggering For the T1 measurements, use of cardiac triggering was necessary to increase the accuracy and precision of T1. The precision improved with up to 8 % in lung parenchyma. The subject number four was an outsider and differed from the other three animals with a longer T1 due to imperfect trigger signal. But it is not totally clear why it differed and thus, will be further investigated.

5.4.1. Oxygen-enhanced T1 measurements with combined triggering A detailed examination was performed to characterize the artifact (ghosting). The artifact was only observed in certain acquisitions and it was not stable by time. We attribute the ghosting effect to shortcomings of the hardware. No conclusions can therefore be drawn about the oxygen-enhanced measurements since the magnitude of the artifact contribution was too large to detect any oxygen-induced changes. Further investigations will be performed.

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Acknowledgements I would like to express my gratitude to my supervisor Lars E. Olsson for giving me the great opportunity to work with this project, and all the excellent help and support. I would also like to thank Frank Risse for the guidance through the programming work and the valuable discussions improving my thesis, Jelena Pesic for the assistance and handling with the animals in the lab and Sven Månsson for the support with the MR sequence.

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