mphil thesis tharaka dassanayake

8/9/2019 MPhil Thesis Tharaka Dassanayake

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Cognitive effects of

organophosphorus insecticide poisoning

studied using reaction time,

evoked potentials and

event-related potentials

Waidyaratne Dassanayake Mudiyanselage Tharaka Lagath

Dassanayake

Master of Philosophy June 2007


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Cognitive effects of

organophosphorus insecticide poisoning

studied using reaction time,

evoked potentials and

event-related potentials

Waidyaratne Dassanayake Mudiyanselage Tharaka Lagath

Dassanayake

MBBS (Sri Lanka)

Department of Physiology

A thesis submitted to the Faculty of Medicine in fulfilment of the

requirements for the Degree of

Master of Philosophy

of the

University of Peradeniya

Sri Lanka

June 2007


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i

Declaration

I hereby certify that the work reported in this thesis was carried out by me under the

supervision of Dr. V. S. Weerasinghe, Dr. U. Dangahadeniya and Professor Nimal

Senanayake.

It describes the results obtained from my own research work except where due reference

has been made in the text. No part of this thesis has been presented for any other degree

in this or any other University.

Date:................................... ...................................................

Dr. W. D. M. T. L. Dassanayake

Certified by:

1. Supervisor: Dr. V. S. Weerasinghe Date: ………………..

……………………………………

2. Supervisor: Dr. U. Dangahadeniya Date: ……………….

………………………………….

3. Supervisor: Prof. N. Senanayake Date: ……………….

..………………………………….


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ii

Acknowledgements

Although this thesis presents my own research work, I would not be able to complete it

without the guidance and the support of many individuals and organisations to which I

must express my sincere gratitude.

I am very much obliged to my academic supervisor and Head of the Department of

Physiology Dr. Vajira Weerasinghe, for introducing me into scientific research and the

field of neurophysiology. He always encouraged me and rendered intellectual and

technical guidance from the day of inception of the thinking behind this work. Prof.

Nimal Senanayake sharpened my thinking by critically evaluating my work and giving

invaluable advice throughout the study. I am fortunate to have an exemplary researcher

like him to supervise my research degree. As an academic supervisor, Dr. Udaya

Dangahadeniya always welcomed me and provided prompt advice and guidance when I

was in need. I am much indebted to him.

I am thankful to my teachers and senior colleagues in the Department of Physiology;

Prof. Malini Udupihille, Prof. Premalatha Balasooriya, Dr. Jayantha Rajaratne, Dr.

Shamila Rajaratne, Dr. Anula Kariyawasam, Dr. Indu Nanayakkara, Dr. Anoja

Ariyasinghe and Dr. Sudheera Kalupahana for their encouraging me and sharing my

departmental commitments during the period of this study. Dr. Indu Nanayakkara and Dr.

Sudheera Kalupahana provided valuable suggestions in writing up the thesis.


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This project would not have been possible without the financial and intellectual support

of the South Asian Clinical Toxicology Collaboration (SACTRC). A very special thank

should go to Prof. Andrew Dawson, who provided numerous opportunities for me to

present my work in the scientific community. He was a great person to work with. I am

also thankful to all the colleagues of the SACTRC research team for supporting me in

many ways.

I must also acknowledge the Staff of the Poisoning Management Unit, the Neurology

Ward and the Medical Wards, Teaching Hospital, Peradeniya who supported me in many

ways in recruiting the study participants. I am especially grateful to Dr. Keerthi Kularatne

who advised and helped me in clinical assessment of the patients.

I greatly appreciate all the patients and the volunteers who consented to participate in the

study without any hesitation.

Finally, I deeply admire my wife Dewasmika and my mother who energized my work

with their understanding, encouragement and love.


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paracetamol overdose (n=11). The second part was a prospective study where the OP

poisoned patients were followed up after six months of poisoning and the findings were

compared with their immediate post cholinergic phase measurements. The tests used to

assess visuomotor information processing were simple visual reaction time, recognition

visual reaction time, visual evoked potentials (VEP) and motor evoked potentials. The

term “cognitive processing time (CPT)” was used to denote the time taken from initial

cortical perception of a stimulus to initiation of descending motor impulses. CPT of each

type of visual reactions was calculated by subtracting the sum of the visual impulse

duration and the motor impulse duration from reaction time (CPT = Reaction time –

(P100 VEP latency + total motor conduction time)). Auditory P300 cognitive event-

related potential (ERP) was recorded, quantified and analysed to assess cognitive

processing of auditory information.

Results: Patients with OP insecticide poisoning showed significant delays in CPT of

simple visual reactions (CPTSVR ) (p=0.037), CPT of recognition visual reactions

(CPTRVR ) (p=0.024) and P300 latency (p=0.003) compared to healthy controls. The

patients also had a significant impairment in CPTSVR (p=0.017), CPTRVR (p=0.002) and

P300 latency (p=0.009) compared to the control group with paracetamol overdose.

Comparison of the initial and follow-up findings of the patients revealed that the

impairment in CPTSVR (p=0.527) and P300 latency (p=0.867) remained unchanged even

six months after poisoning. However, CPTRVR showed a significant improvement

(p=0.012). Visual and motor conduction latencies or P300 amplitude were similar

between the groups and between the two assessments of the patients with OP poisoning.


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Conclusions: OP insecticide poisoning appears to slow the cognitive processes involved

in visuomotor information processing and auditory stimulus evaluation. These effects

persist beyond the clinical recovery from the cholinergic phase, and the deficits in

auditory stimulus evaluation and cognitive processing in simple visual reactions appear to

be persistent even six months after exposure. These findings are compatible with the

cognitive deficits observed in some previous human studies. The neural substrates of the

affected cognitive processes are largely compatible with the topography of the

neuropathological lesions that have been observed in OP exposed experimental animals.


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Table of contents

Declaration ………………………………………………………………… …………... i

Acknowledgements ……………………………………………………………………... ii

Abstract …………………………………………………………………………………. iv

List of tables …………………………………………………………………………... xii

List of figures …………………………………………………………………………. xiii

List of abbreviations …………………………………………………………………... xvi

Section 1

INTRODUCTION …………………………………………………………………….. 1

1.1.

Organophosphorus insecticide poisoning ……………………………………... 1

1.1.1. Epidemiology ..………………………………………………………… 1

1.1.2. Pathophysiology ..……………………………………………………… 3

1.2. Behaviour and cognition ..………………………………………………………. 5

1.3. Cognitive processing of sensory information and integration between

cortical areas ……………………………………………………………………. 8

1.3.1.

Visual perception ..…………………………………………………… 12

1.3.2. Auditory perception ..………………………………………………… 15

1.4. Measurement of information processing in human brain ……………..………. 17

1.4.1. Reaction time ..……………………………………………………….. 17

1.4.2.

Event-related potentials (ERPs) and P300 ..………………………….. 21


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1.5. Delayed cognitive effects of acute OP poisoning: evidence from previous

studies and their limitations …………………………………………………… 25

1.6. Rationale, aims and objectives of the present study ………………………….. 29

Section 2

METHODOLOGY ………………………………………………………………….... 31

2.1. Study design………………………………………………………………….… 31

2.2. Ethical considerations …………………………………………………….…… 32

2.3. Subjects ………………………………………………………………………... 33

2.3.1. Test group ……………………………………………………………... 33

2.3.1.1.Inclusion criteria …………………………………………………… 33

2.3.1.2.Exclusion criteria …………………………………………………... 33

2.3.2. Healthy control group ………………………………………………….. 34


2.3.2.2.Exclusion criteria ………………………………………………….. 34

2.3.3. Hospitalised control group ……………………………………………... 34


2.3.3.2.Exclusion criteria …………………………………………………... 34

2.4. Background information ………………………………………………………. 35

2.4.1. Test group ……………………………………………………………... 35

2.4.2. Healthy controls ……………………………………………………...... 36

2.4.3.

Hospitalised controls ………………………………………………….... 36


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2.5. Assessment of cognitive processing …………………………………………. 40

2.5.1.

Basis of assessment paradigms ……………………………………….. 40

2.5.2. Equipment …………………………………………………………….. 42

2.5.3. Details of the techniques ……………………………………………… 44

2.5.4.

Testing protocol ………………………………………………………. 53

2.6. Data analysis …………………………………………………………………. 58

2.6.1. Calculated outcome variables …………………………………………. 58

2.6.2. Statistical analysis ……………………………………………………... 59

Section 3

RESULTS …………………………………………………………………………….. 61

3.1. Data analysis ………………………………………………………………….. 61

3.1.1. Test group …………………..…………………………………………. 61

3.1.2. Healthy control group ..………………………………………………... 61

3.1.3. Hospitalised control group …………………………………………….. 62

3.2. Episode of poisoning, management measures and complications ……………. 64

3.2.1.

Test group …………………………………………………………....... 64

3.2.2.

Hospitalised control group …………………………………………….. 67

3.3. Intergroup comparison of measures of cognitive processing, visual and

motor components ……………………………………………………………. 68

3.3.1. Visual reaction time …………………………………………………… 69

3.3.2.

Components of visual reaction time …………………………………... 72


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3.3.2.1.Visual conduction …………………………………………………. 72

3.3.2.2.Motor component ………………………………………………….. 73

3.3.2.3.CPT of visual reactions ……………………………………………. 76

3.3.2.3.1. CPT(SVR) …………………………………………………… 76

3.3.2.3.2.

CPT(RVR) …………………………………………………… 76

3.3.3. P300 ERP ……………………………………………………………… 78

3.3.4. Summary of intergroup comparison of measures of information

processing ………………………………………………………………... 81

3.3.5. Correlation between measures of cognitive processing ……………….. 84

3.4. Follow up assessment of the patients with OP insecticide poisoning ………… 86

3.4.1. Visual reaction time ……………………………………………………. 86

3.4.2. Components of visual reaction time …………………………………… 87

3.4.2.1.Visual conduction ………………………………………………….. 87

3.4.2.2.Motor component ………………………………………………….. 88

3.4.2.3.CPT of visual reactions ……………………………………………. 89

3.4.3. P300 ERP ………………………………………………………………. 90

3.4.4. Summary of the comparison of initial and follow up assessment of the

parameters of cognitive processing, afferent and efferent conduction ….... 92

Section 4

DISCUSSION ………………………………………………………………………… 94

4.1.

Effects of OP on visual reaction time …………………………………………….. 95


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4.2. Importance of analysis of individual components of visual reactions ………......... 96

4.3.

Cognitive processes and the flow of information in visuomotor reactions ….......... 97

4.4. Long-term effects of OP pesticide poisoning on visuomotor information

processing ………………………………………………………………………... 101

4.5.

Effects of OP poisoning on P300 event-related potential ………………………... 106

4.6. OP induced brain damage: corroborating evidence from human studies

and animal experiments ………………………………………………………….. 109

4.7. Limitations of the study ……………………………………………………......... 111

Section 5

CONCLUSIONS AND FUTURE DIRECTIONS ………………………………… 114

List of references ……………………………………………………………………... 116

Appendices …………………………………………………………………………… 138


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xii

List of tables

3.1. Comparison of age: Test group and healthy control group ………………………. 62

3.2.

Age distribution of organophosphorus poisoned patients, matched hospitalised

control group and healthy controls ……………………………………………… 63

3.3.

The type of organophosphorus (OP) insecticide ingested by patients …………... 65

3.4.

Numbers of subjects whose measurements were taken in tests of information

processing ……………………………………………………………………….. 69

3.5. Comparison of the measures of information processing: test group (initial

assessment) vs. healthy controls ………………………………………………… 82

3.6.

Comparison of the measures of information processing: hospitalised controls

vs. matched test group subjects and healthy controls …………………………… 83

3.7. Correlation between parameters of cognitive processing in all study participants.. 84

3.8. Comparison of parameters of information processing of the test group patients

in the initial post-cholinergic phase and the follow up ………………………….. 93

4.1. Neuropsychological findings of the epidemiological studies on chronic effects

of acute OP pesticide poisoning ………………………………………………… 103


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xiii

List of figures

1.1. Chemical structure of organophosphorus compounds ……………………………. 4

1.2. Neuropsychological classification of dimensions of behaviour and cognition …... 6

1.3. Generic scheme of information processing in sensory pathways ………………… 9

2.1.

Selection and comparison of study samples ……………………………………... 39

2.2. Calculation of cognitive processing time ………………………………………… 41

2.3. Magstim Model 200TM magnetic stimulator with circular 90mm coil …………... 43

2.4. Signal averaging machines ……………………………………………………… 43

2.5. Electrode placement in VEP recording ………………………………………….. 46

2.6. A normal VEP waveform ………………………………………………………… 47

2.7. Magnetic field and the induced current produced by the current flowing

through the coil of the magnetic stimulator ……………………………………… 49

2.8. A standard MEP waveform ……………………………………………………… 50

2.9. Electrode placement in recording P300 component of ERPs …………………… 52

2.10.

A normal P300 ERP waveform ………………………………………………… 53

2.11.

Reaction time test ………………………………………………………………. 55

2.12. Recording auditory P300 event-related potential …………………………….... 55

2.13. Recording pattern reversal visual evoked potentials ………………………….. 57

2.14. Transcranial magnetic stimulation and motor evoked potential recording …… 57


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3.1. Age distribution of the test group and healthy controls ………………………….. 62

3.2.

Number of patients that showed each cholinergic feature ………………………. 67

3.3. VRT in the test group and healthy controls …………………………………....... 71

3.4. VRT in the test subjects, matched hospitalised controls and healthy controls ….. 71

3.5.

VEPs of a patient with OP poisoning …………………………………………… 72

3.6. Distribution of P100 latency …………………………………………………….. 73

3.7. MEPs in a patient with OP poisoning …………………………………………… 74

3.8. Motor conduction times in patients and healthy controls ……………………….. 75

3.9. Motor conduction times in test subjects, matched hospitalised controls

and matched healthy controls ……………………………………………………. 75

3.10. CPT(SVR) and CPT(RVR) in the test group and healthy control group …………... 77

3.11. CPT(SVR) and CPT(RVR): test subjects, matched hospitalised controls and

healthy controls ………………………………………………………………….. 78

3.12. Grand average ERP waveforms for target tones .……………………………… 79

3.13. Distribution of P300 latencies …………………………………………………. 80

3.14. Distribution of P300 amplitudes ………………………………………………. 81

3.15. Correlation between CPT(SVR) and CPT(RVR) …………………………………... 85

3.16.

Correlation between CPT(RVR) and P300 latency ……………………………… 85

3.17. Changes in VRT from initial post-cholinergic phase assessment to the

follow up assessment ……………………………………………………………. 87

3.18. Changes in P100 VEP latency from initial post-cholinergic phase

assessment to the follow up assessment …………………………………………. 88


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3.19. Measures of motor conduction in the first assessment and the follow up in the

patients with OP poisoning ……………………………………………………… 89

3.20. CPT of visual reactions in the patients in the first assessment and

the follow up assessment ………………………………………………………... 90

3.21.

Grand average ERP waveforms of the first assessment and the follow up

assessment of the patients with OP poisoning …………………………………... 91

3.22. P300 ERP changes in patients with OP poisoning: first assessment vs.

follow up assessment ……………………………………………………………. 92


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xvi

List of abbreviations

AChE – acetylcholinesterase

ACh – acetylcholine

CI – confidence intervals

CMCT – central motor conduction time

CPT – cognitive processing time

CPTRVR – cognitive processing time of recognition visual reactions

CPTSVR – cognitive processing time of simple visual reactions

dB – decibels

EP – evoked potential

ERP – cognitive event-related potential

FDS – Flexor digitorum superficialis

Hz – Hertz

IQR – inter-quartile range

M – magnocellular

MEP – motor evoked potential

ms – milliseconds

MT – movement time

n – number of subjects

OP – organophosphorus


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P – parvocellular

PET - positron emission tomography

PMCT –peripheral motor conduction time

r – Pearson’s correlation coefficient

RAS - reticular activating system

RRT – recognition reaction time

RT – reaction time

RVRT – recognition visual reaction time

SE – standard error of the mean

SRT – simple reaction time

SVRT – simple visual reaction time

TMCT – total motor conduction time

TMS – transcranial magnetic stimulation

V1 – primary visual cortex

V2 – secondary visual cortex

VEP – visual evoked potential

WHO – World Health Organisation

µV – microvolt


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Section 1

INTRODUCTION

1.1. Organophosphorus insecticide poisoning

1.1.1. Epidemiology

Production of synthetic pesticide formulations has a history of over half-a-century.

In year 2000 the global production was 3.75 million metric tons (Tilman et al., 2001).

About one-fourth of formulated pesticides are used in the developing countries

(Jeyaratnam, 1984). Organophosphorus (OP) compounds have been the principal means

of agricultural pest control throughout the world since 1980s (Stephens, 1995).

In 1992, World Health Organization (WHO) reported that roughly three million

pesticide poisonings occur annually resulting in 220,000 deaths worldwide. Poisonings

are far more frequent in the developing world in comparison to the developed countries.

Ninety-nine per cent of more than 200,000 pesticide-related deaths occur in the

developing world (Jeyaratnam, 1990). Ingestion of pesticides, particularly OP


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compounds, is a widely prevalent method of deliberate self-harm in agricultural

communities across Asia. According to WHO reports in 2002, the estimated annual death

rate from self-harm in the Asian region is half-a-million (World Health Organisation

2002). Of these, ingestion of pesticides accounts for 300,000 deaths (Eddleston, 2000).

About two-thirds of the instances (i.e. 200,000 deaths) the causative pesticide is an OP

insecticide (Eddleston, 2000; Eddleston and Phillips, 2004).

Like in many other Asian countries, pesticide poisoning is a major clinical and

public health problem in Sri Lanka (Jeyaratnam et al., 1982; Eddleston et al., 1998; Van

der Hoek et al., 1998; Fernando & Hewagalage, 1999). According to the Annual Health

Bulletin year 2000, pesticide poisoning is the leading cause of death in many agricultural

districts (Ministry of Health, 2001). Although pesticides are widely used in agricultural

communities, most clinically documented acute poisonings in Sri Lanka are not due to

occupational overexposure, but episodes of deliberate ingestion. In a study carried out in

North-Central province, out of 526 pesticide-related poisonings 68% cases were self-

inflicted, 19% were spraying-related and 13% were accidental (Van der Hoek et al.,

1998). Young adults form the major proportion of deliberate self-poisoning victims (de

Alwis, 1988; Van der Hoek et al., 1998). Occupational exposure leading to intoxication

through inhalation and skin contact is also seen in agricultural communities. Pesticide

sprayers who do not use protective gear are particularly at risk.


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Following the common pattern of the Asian region (Eddleston, 2000; Eddleston and

Phillips, 2004), OP insecticides are responsible for the majority of pesticide poisonings in

Sri Lanka. By late 1970s and early 80s, OP compounds became the main causative agents

in pesticide poisoning associated hospital admissions in many parts of Sri Lanka

[Colombo 1976: 26 out of 46 (55%), Peradeniya 1984/85: 92 out of 117 (79%) and Jaffna

259 (89%) out of 291] (Senanayake, 1986). In the following decade the islandwide

hospital statistics showed that about 75% of poisoning related admissions and 85% of

deaths from poisoning were caused by OP insecticides (Senanayake, 1998).

1.1.2. Pathophysiology

All OP compounds share a common chemical structure (figure 1.1). The basic

components include a phosphorus atom that forms a double bond with either oxygen (P =

O) or sulphur (P = S), and three side chains. Group X differs widely in individual OP

agents and to a great extent determines the physical and chemical characteristics of the

compounds (Erdman, 2004). The R 1 and R 2 side chains are typically alkoxy groups, but

may be aliphatic or aromatic hydrocarbons. The biological action of OP depends on the

phosphorylating ability of the OP, which in turn is determined by the electrophilicity of

the phosphorus atom. Electrophilicity is mostly determined by the substituent groups in

the OP molecule. In vivo activity of the OP depends also on the lipid solubility of the OP

compound. Highly lipid soluble OP compounds can cross the biological membranes and

the blood brain barrier, to reach the neural tissues where the OP can exert its main action.


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Figure 1.1: Chemical structure of organophosphorus compounds.

OP insecticides inhibit acetylcholinesterase (AChE), the enzyme which degrades

the neurotransmitter acetylcholine (ACh) released by cholinergic nerve endings.

Resulting accumulation of ACh leads to different effects depending on the site. It causes

depolarisation block of the nicotinic cholinergic receptors at the neuromuscular junction.

Cholinergic overactivity in the autonomic nervous system principally causes

parasympathetic stimulation through muscarinic cholinergic receptors. Accumulation of

ACh also affects the cholinergic neural circuits in the central nervous system. Classically,

three main syndromes of acute OP poisoning have been identified, viz. acute cholinergic

crisis, intermediate syndrome and delayed polyneuropathy. The latter two are seen only

in a proportion of the victims who develop acute cholinergic crisis. To a large extent, the

amount and the type of the OP compound determines the occurrence of intermediate

syndrome and delayed polyneuropathy.

In addition to these manifestations, research over last few decades suggests the

possibility of delayed sequelae of OP on ‘higher functions’ of the nervous system. The


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observations have variously referred in literature as “neurobehavioural effects”,

“neuropsychological abnormalities” or “neuropsychiatric changes” (Abou-donia, 2003).

Particularly, the cognitive sequelae have drawn attention of researchers engaged in

toxicology as well as in many other fields including neurology, neuropsychology and

psychophysiology.

1.2. Behaviour and cognition

Behaviour can be conceptualised in three dimensions, viz. cognition, emotionality

and executive functions (Figure 1.2) (Lezak, 1995).

1. Cognition is defined as ‘the information handling aspect of behaviour’. In general

terms, cognition is described as knowing, perceiving or conceiving as a faculty

distinct from emotion and volition (Oxford Dictionary, 1994).

2. Emotionality concerns with feelings and motivation.

3. Executive functions deal with the ways in which the mental operations are

executed. The observable motor activity comes under this domain.

Cognitive functions may be studied under four faculties (Figure 1.2):

1. Receptive functions encompass abilities to select, acquire and integrate

information. Major part of sensory information perception falls into this category.


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2. Memory and learning refers to encoding, storage, mental representations and

retrieval of information.

3. Thinking, at least in a neuropsychological perspective, can be classified under the

dimension of cognition. It involves voluntary mental organisation and

reorganisation of information. In this view, any cognitive operation that relates

two or more bits of information explicitly (e.g. adding two numbers) or implicitly

(e.g. judging an action as ‘good’ or ‘bad’) can be regarded as a thinking process.

4. Expressive functions encompass the mental operations dealing with means

through which information is communicated or acted upon (e.g. speech, writing

and drawing, gestures and manipulating). Thus, the expressive aspect of cognition

forms the immediate mental substrate for executive functions of behaviour.

Figure 1.2: Neuropsychological classification of dimensions of behaviour and cognition.


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However, the mental tasks cannot be strictly compartmentalised in any one of these

functional subdivisions of cognition. Even in ‘simple’ tasks such as face-recognition,

many of them operate in unison. The initial cortical processing involves analysis of

perceptual properties of the visual stimulus such as lines, colour of the face and the

spatial relationship of its parts. This mainly demands receptive functional resources. At a

higher level of recognition, stored memory traces are retrieved to match the perceptual

representations of the face. These latter stages of processing attribute a semantic value for

initial visual signals (Gazzaniga et al., 1998). Therefore, visual recognition necessitates

two facets of cognition (viz. receptive functions and memory) to work in harmony.

Nevertheless this classification of behaviour and cognition permits easier practical

observation, measurement and description of normal and abnormal behaviour, and

constitutes a framework for organisation of behavioural data.

‘ Activity variables’ determine the efficiency of the above dimensions of behaviour.

Some neuropsychologists categorise consciousness and attention under activity variables

(Lezak, 1995). For instance, solving an arithmetic problem involves numerous

interactions between receptive functions, memory and thinking. When a person is fully

conscious and directs maximum attention towards the task, he can integrate the above

dimension of cognition and solve the problem more accurately and faster than when he is

less attentive. Activity variables do not have any specific behavioural outcomes or end

products of their own. Thus there are no ‘pure’ behavioural tests to measure activity

variables. Behavioural correlates of activity variables are always assessed through


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performance of a task related to other behavioural domains mentioned earlier. Thus, a

given cognitive process involves activation of one or more faculties of cognition, the rate

of the process being influenced by activity variables.

1.3. Cognitive processing of sensory information and integration between cortical

areas

Information processed during cognitive operations are either retrieved from

memory stores, or acquired from the external environment as sensory inputs. Sensory

signals generated by external stimuli are carried to brain via afferent neuronal pathways.

These signals cross several synapses and reach the cerebral cortex. Synaptic transmission

permits the afferent signals to be modified to a certain extent by pre- and/or post-synaptic

influences. Thalamus, with its specific sensory nuclei, plays a major role as a multimodal

relay station where sensory perception occurs at an elementary level. On their way to

cortical areas, sensory pathways send inputs to the reticular activating system (RAS) of

the brain stem. RAS is involved in maintaining the arousal state of the individual.

Thalamocortical fibres project to primary sensory cortical areas. Higher-order perceptual

processing begins in these modality-specific (vision, hearing, somatic sensations etc.)

primary cortical areas. An abstract schematic representation of this initial information

processing is illustrated in figure 1.3.


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Figure 1.3: Generic scheme of information processing in sensory pathways. (RAS:

reticular activating system).

Primary sensory areas are found in different parts of the cerebral cortex, depending

on the sensory modality they subserve:

• Primary visual area: striate cortex of the occipital lobe

• Primary auditory area: superior temporal gyrus

• Primary somatosensory cortex: postcentral gyrus


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Billions of neurons in primary sensory cortical areas register raw sensory

information in the form of a ‘map’. These representations per se do not have a semantic

value. Primary sensory cortical representations are transmitted to adjacent secondary

association areas which integrate these raw perceptions, increasing the complexity of

sensory information. The cells in secondary association areas are also modality specific.

Only a stimulus of the appropriate modality can fire neurons in secondary association

areas.

Tertiary association areas are considered responsible for integration of mental

representations of more than one modality (Lezak, 1995). Posterior association cortex is a

main area where this supramodal integration of perceptual functions takes place.

Therefore it is also called heteromodal or multimodal cortex. Having a parieto-temporo-

occipital distribution, it is in close proximity with somatosensory, auditory and visual

cortical regions. Inputs from many sensory organs can fire the neuronal populations of

posterior association cortex. Patients with lesions in posterior parietal cortex may develop

constructional disorders where multimodal integration of visuospatial information is

deficient (Black & Bernard, 1984). Transfer of impulses between cortical regions is not

unidirectional. Feedback loops from secondary and tertiary association areas back-

innervate more elementary sensory areas. The association areas also have numerous

connections with subcortical structures.


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As information moves from one stage to the next, the cortical representations

become perceptually elaborate and semantically sophisticated. Findings of single cell

recording experiments support this. For example, a simple flash of light can stimulate

primary visual cortical neurons. In contrast, cells of the association areas of inferior

temporal cortex do not fire on exposure to simple lines or spots of light. Rather, they

respond to complex stimuli such as an elaborate drawing of a hand (Desimone et al.,

1990). Thus, visual association areas in the inferior temporal cortex appear to play a role

is object recognition rather than simple perception of a quantum of light.

Multimodal representations synthesised in association areas are transmitted to

limbic and frontal association areas, to be activated into feelings, thoughts and actions

(Pandya & Yeterian, 1985, 1990). Prefrontal division of the frontal lobe receives

information about the external environment from postcentral structures. According to one

estimate, around 60% of these neuronal pathways come from tertiary association areas

and 25% from secondary association areas (Strub & Black, 1988). Prefrontal cortex also

receives inputs form limbic system about the internal status of the body. Convergence of

many types of information from multiple sources (memory storage, visceral arousal

centres, sensory association areas etc.) enables the prefrontal cortex to process and

integrate information at the highest order or greatest complexity. The prefrontal division

interacts with the secondary motor association area in the premotor division and

subsequently with the primary motor cortex, to bring about executive functions of

behaviour such as movements, gestures, speech etc.


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As discussed earlier, receptive functions do not operate in isolation. There are

numerous complex interactions with memory systems occupying multiple loci including

the diencephalon, hippocampus, striatum, amygdala and many parts of the neocortex.

Two-way information transfer nourishes both these faculties of cognition. Retrieval of

information from the memory stores helps higher order semantic interpretation of

perceptual cues, whilst perceptual processing enables encoding and consolidation of new

external information to strengthen memory networks. In humans, visual and auditory

perception provide large amount of external information for higher-order processing.

1.3.1. Visual perception

Visual stimuli evoke sensory neural impulses that travel through afferent visual

pathways to lateral geniculate nuclei and then to the primary visual cortex in the occipital

lobes. Cerebral neural networks process these signals further to identify their perceptual

and semantic properties. In the present study, processing of the visual representations

beyond the primary visual cortex is regarded as cognitive processing of visual

information.

Visual perception involves concurrent, parallel analysis of various attributes of the

stimulus (colour, shape, orientation, motion etc.) through different subsystems

(Gazzaniga et al., 1998). This parallel processing begins in the lateral geniculate nuclei,


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where magnocellular (M) neurons and parvocellular (P) neurons analyse different

features of the stimulus. This M-P distinction is maintained at primary (V1 or

Brodmann’s area 17) and secondary (V2 or Broadmann’s area 18) visual cortical regions

as demonstrated by histological studies (Bear et al., 1996). M pathways are sensitive to

changes in contrast and motion. As shown in cytochrome oxidase histological staining

techniques, P pathway has two subsystems viz. blobs and interblobs in V1, which

correspond to thin strips and interstrips respectively, in V2. Blobs and thin strips in P

pathway are mainly sensitive to colour. Interblobs and interstrips are mainly sensitive to

location and orientation of the stimulus. Additional evidence for parallel processing in

visual perception comes from single cell recording studies (Hubel & Wiesel, 1968;

Maunsell & Van Essen, 1983), visual search task experiments (Treisman & Gelade,

1980), and more recently, from positron emission tomography (PET) studies (Zeki,

1993).

The specialisation hypothesis of visual perception is based on the above

observations. It suggests that in a given moment, there are many maps of the same image

in many visual areas. But each map differs according to the type of information (e.g.

colour, shape, orientation etc.) they represent. Higher level visual processing engage

subcortical pathways extending form V1 to adjacent parietal and temporal cortices.

Superior longitudinal fasciculus (occipito-parietal pathway) contains axons extending to

posterior parietal cortex. Inferior longitudinal fasciculus (occipito-temporal pathway)

contains axons terminating in inferior temporal cortex.


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Mishkin and Ungerleider first proposed a functional model involving these two

pathways, to explain extraction of two fundamentally different properties of visual

information (Mishkin et al., 1982a; 1982b; 1983). According to them, the ventral,

occipito-temporal pathway is involved in recognising what we are looking at (i.e. object

recognition), and hence called “what” pathway. Dorsal, occipito-parietal tracts are

specialised in spatial perception where we identify the location of an object and the

spatial relationship of objects in the visual field. Therefore the original researchers called

it “where” pathway. This “what – where” dichotomy of visual perception is supported by

electrophysiological evidence (Robinson et al.,1978; Desimone, 1991; Ito et al. 1995),

behavioural experiments on animals (Pohl, 1973) and PET studies of normal human brain

(Haxby et al., 1994).

The purpose of the analysis of individual visual features is to recognise the objects

in the visual field. According to the Warrington’s two-stage model, analysis of visual

features helps categorisation of the object we see according to its physical properties (i.e.

colour, shape etc.), and it is called perceptual categorisation. Patients who are unable to

perform this perceptual categorisation are incapable of recognising an object as the same

one when viewed under different lighting conditions or from different viewpoints

(Gazzaniga et al., 1998). This condition is called appreciative agnosia. At a higher level

of processing, the perceptual representation of the object undergoes semantic

categorisation, where the perceptual representation is given a meaningful functional

value. Those who are unable to perform this semantic categorisation suffer from


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associative agnosia. When they are shown a set of objects and asked to choose two

objects which are similar in terms of their function they tend to select two which are

similar in physical appearance (Warrington & Taylor, 1978).

At these higher levels of visual information processing where the demands are to

recognise the object, perceptual representations interact with long-term memory traces of

the known objects. In other words, multiple dimensions of cognition (figure 1.2) viz.

receptive functions, memory and learning and even thinking, begin to interact strongly

with one another.

1.3.2. Auditory perception

Auditory afferents enter the brainstem via cochlear nerves. Forming synapses in

cochlear nuclei, superior olivary nucleus, inferior colliculus and medial geniculate

nucleus, the neuronal pathways reach the primary auditory cortex (Brodmann’s area 41)

in the superior temporal gyrus. Secondary auditory cortex occupies the Brodmann’s areas

42 and 43. Neurons in the auditory areas are arranged to form a tonotopic map. For

instance, cells in a given area of the primary auditory cortex are maximally sensitive to a

particular frequency. The frequency of maximal sensitivity changes little by little from

one cell to the adjacent ones, so that one can map the auditory area into a frequency

gradient. Therefore the ‘receptive field’ of a neuron involved in auditory processing is


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expressed as a frequency range (in contrast to a visual cortical cell, of which the receptive

field encompasses a specific area of the visual field, which can be expressed in spatial

dimensions).

Basilar membrane near the base of the cochlea is maximally sensitive to high

frequency sounds, whilst low frequency sounds mainly activate the apex of the cochlea.

The tuning specificity of auditory receptive fields becomes more refined as the stimulus

moves through the system. For instance, a cell in cochlea that is maximally sensitive to

5000Hz stimulus, responds to stimuli ranging from 2000Hz to 10000Hz. A cell in

auditory cortex that responds maximally to a stimulus of 5000Hz may respond to stimuli

of 4000Hz to 6000Hz, which is a much narrower range.

The computational goals of audition are similar to those of vision. The two goals

are to recognize the nature of the sound (“what?”) and the location of the sound source

(“where?”). Scientific evidence on mechanisms of auditory information processing is

much scarce in comparison to those of visual information processing. Auditory cortex is

involved in determining the nature of the auditory stimulus (“what is the stimulus?”) as

well as direction from which sound emanates. However evidence based on the

experimental animal model barn owl, suggests that to a certain extent, the question

“where is the stimulus?” is solved at the level of brain stem and the auditory cortex

(Konishi, 1993). Medial superior olivary nucleus plays a major role in detecting the time

lag between acoustic signals entering the two ears.


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1.4. Measurement of information processing in human brain

The rapidity of human information processing and scarcity of knowledge on the

neural correlates of higher-order information processing makes studying the neural

processes of cognition difficult. However, the discipline of mental chronometry, which

has origins that date back to more than a century, seeks to measure the time-course of

mental operations in the human brain. Mental chronometric tasks have been used

extensively in cognitive neuroscience to elucidate mechanisms underlying cognitive

processing. From late 1800s to 1950s, assessment of temporal nature of information

processing was built almost entirely around a single behavioural method: measuring and

comparing reaction times during simple cognitive tasks. Later, with the development of

electrophysiological techniques, evoked potentials (EPs) and cognitive event-related

potentials (ERPs) started to contribute to elucidation of the time-course of information

processing in the nervous system. Present study uses visual reaction time (VRT) tests, EP

and ERP techniques to quantitatively assess the cognitive processing of visual and

auditory information processing in victims of OP poisoning.

1.4.1. Reaction time

Reaction time (RT) is defined as the time that passes from arousal of a sensory

organ to a motor reaction. Depending on task requirements and complexity, RT tests are

widely used to evaluate the time-course of various cognitive processes (Gazzaniga et al.,


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1998). All types of reaction tasks demand analysis of certain perceptual and/or semantic

properties of a presented stimulus (e.g. colour, shape, position in space, tone, meaning of

a word etc.), and execution of an appropriate motor response. The analysis of perceptual

and semantic properties of the stimulus involves receptive functions, memory updating

and thinking, whereas selection and execution of motor response involve expressive

functions of cognition.

In simple reaction time (SRT) experiments, there is only one stimulus (e.g. white

flash) and one response (e.g. pressing a button). Therefore the responder does not have to

concentrate on specific characteristics of the stimulus (e.g. whether it is a white flash or a

red flash) and just need to respond to the stimulus as quickly as possible. If the stimulus

is of visual modality the test is a simple visual reaction time (SVRT) test.

Neuropsychologists use SRT as a measure of attention (Lezak, 1995).

In recognition reaction time (RRT) experiments, there are some stimuli that should

be responded to, and others that should get no response. For example, in a recognition

visual reaction time (RVRT) test, either a white flash or a red flash may appear

randomly, and the requirement is to respond only to white flashes as soon as possible.

Thus there is still only one correct response. Before responding the individual has to

detect the stimulus and also discriminate the target stimulus (i.e. white flash) from the

distracting stimulus (i.e. red flash).


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Pioneer RT studies were those of F.C. Donders in 1868. He showed that SRT is

shorter than RRT. Laming (1968) in his studies, determined that SRT averaged 220ms

and RRT averaged 384ms. According to Bellis (1933), pressing a key in response to a

light (i.e. SVRT) is around 220ms in males, and about 260ms in females (Kosinski,

2000). This is in line with many studies that concluded that complex stimulation

paradigms, as in RRT experiments, elicit slower RTs (Brebner, 1980, Teichner et al.,

1974). Over the last century, RT has been used to assess psychomotor speed in variety of

neurological and psychiatric illnesses (e.g. mental retardation, dementias, depression,

schizophrenia, drug dependence) and in performance oriented groups (e.g. sportsmen).

Being a behavioural test, RT includes not only cognitive processing of information

but also sensory and motor components of the task. Welford (1968) defined three

components of a psychomotor reaction: ‘first, the time taken by the stimulus to activate

the sense organ and for impulses to travel from it to the brain; second, the central

processes concerned with identifying the signal and initiating a response to it; and third,

the time required to energize the muscles and to produce an overt recorded response’

(Danev et al., 1971). With invent of electrophysiological techniques, there are direct

ways to assess the sensory and motor components.

1. Sensory Component:

This can be visual, auditory etc., depending on the modality of the stimulus. The

duration of this component is the time period between stimulation of the sensory


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receptors and arrival of the afferent neuronal impulse to the primary sensory (visual,

auditory or somatosensory) cortex. If the stimulus is visual, the light strikes the retina,

stimulates photoreceptors and initiates the afferent nerve impulses that reach the primary

visual cortex. Transmission of impulses along the visual pathways can be studied using

visual evoked potential (VEP) techniques. In VEP recordings P100 component is a

waveform that peaks around a latency of 100ms. Research evidence suggests that it is

generated by the neuronal activity of the striate cortex when a visual impulse reaches the

occipital cortex (Seki et al. 1996). Thus, P100 latency can substitute the duration of

afferent component of a visual reaction.

2. Cognitive Processing:

This comprises the processes that occur in between stimulation of the

corresponding sensory (visual/auditory) cortex and initiation of the motor signal in the

area of the primary motor cortex that control the responding part of the body (e.g. hand,

finger etc.). In this process, more meaningful mental representations of the stimulus are

linked with neural generators of the appropriate motor response. Thus perception of the

sensory stimulus comes under receptive functions of cognition whereas selection of the

appropriate response can be categorised under thinking and expressive functions of

cognition. In comparison to simple reactions, cognitive processing takes a longer time in

recognition reactions because the brain needs to discriminate between two or more types

of stimuli and decide on the appropriate motor response.


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3. Motor component:

Once the desired motor response (e.g. flexion of the index finger to press a response

button) is planned, corresponding area of the primary motor cortex is stimulated. The

efferent signals travel along the corticospinal tracts and stimulate the corresponding

anterior horn cells and then transmit along the peripheral nerve to generate the endplate

potentials at the fibres of the target muscles. The time taken for this motor conduction

can be measured with motor evoked potential (MEP) studies, where the primary motor

cortex is stimulated and the MEPs are recorded at the skeletal muscles. The duration

between motor cortical stimulation and the onset of MEP denotes the duration of the

motor component of the psychomotor reaction. Motor cortical stimulation can be

performed by transcranial magnetic stimulation (TMS) which is a safe and non-invasive

procedure (Barker et al., 1985).

The sensory and motor components involve the same neuronal pathways and their

durations are expected to be the same in all types of RT tests. This implies that the

differences in RTs are due to differences in cognitive component (Miller, 2001).

1.4.2. Event-related potentials (ERPs) and P300

Recording the average event-related electrical potentials from scalp electrodes

became a research tool in the 1960s with the advent of analogue and then digital


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computers to accomplish the recording and averaging. The principal use of ERPs is to

determine the time course of cognitive processes in the human brain (Bressler, 2002).

The physiological basis of the cortical ERPs lies in fields of potential generated by

interacting neurons. Activity of the neural circuits of the brain generates electrical fields

which can be recorded over the scalp as potential differences on a time scale. The

potential at a given site over the scalp changes every millisecond, reflecting the ever-

changing activity of underlying neural circuits. This electrical activity is contributed to by

numerous mental processes which cannot be identified in isolation. Thus, in order to

observe the neural activity related to a particular cognitive process two main

requirements have to be fulfilled.

First, the electrical potentials related to the cognitive process of interest, should be

captured during the short time duration it occurs. In ERP tests this is done by applying an

external event, the properties of which are analysed by the brain. For example, if the tone

of a presented auditory stimulus needs to be analysed, this stimulus evaluation process

generates electrical activity in cerebral neural circuits within the next fraction of a

second. Thus if acquisition of the electrical activity begins at the onset of the stimulus

and continue for next second or so, the electrical potentials related to the mental process

of stimulus evaluation can be captured. In other words, in order to capture a particular

brain process, the recording epoch has to be time-locked with the stimulus that evokes the

cognitive process. However, there are so many unrelated neural activities going on even


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during this small time span, and this ‘noise’ (as labelled in electrophysiology), may mask

the captured mental process of interest.

Thus, secondly, the captured mental process needs to be highlighted or isolated .

This highlighting is done through averaging many time-locked epochs. Unrelated

electrical activity, having no fixed temporal relationship with the stimulus and the

recorded time epoch, cancels out with repeated averaging, retaining the potentials of the

mental process of interest in the recording.

ERP components could be systematically related to sensory and motor stages of

information processing. Present study focuses on the auditory ERP waveform P300,

which is generated in analysing auditory stimuli. P300 was first demonstrated by Sutton

and colleagues in 1965 (Sutton, et al., 1965). It is a positive ERP deflection which peaks

around a latency of 300 - 600ms, when a person is exposed to randomly timed two

different stimuli of the same modality, and required to respond to the infrequent (rare)

stimulus (also called the target stimulus). Thus the paradigm that is used to elicit P300 is

called the ‘oddball paradigm’. The response to the target stimulus need not necessarily be

a motor response. Even mentally counting the target stimuli evoke P300.

Behavioural correlates of P300 indicate that it reflects a consequence of orienting

and attending to task-relevant events. Therefore P300 latency is considered a measure of


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‘stimulus evaluation time’, that is independent of selection of the response (Kutas et al.,

1977; McCarthy and Donchin, 1981).

P300 latency was found to have good test-retest reliability in normal individuals, so

that it has the stability required for clinical and research applications (Polich, 1986;

Sclare et al., 1984). However, P300 latency is affected by aging. Older subjects were

found to have longer P300 latencies than young (Marsh & Thompson, 1972; Pfefferbaum

et al., 1979, 1980a, 1980b; Ford et al., 1979, 1982a, 1982b; Beck et al., 1980).

Assessment of P300 latencies over a continuous age range have demonstrated that this

increase in latency begins at puberty and extends into the eighth decade (Brown et al.,

1983).

ERP amplitude variation is considered to reflect the variation in intensity of

activation of neural structures in the brain by the task variables (Kok, 1990). In

experimental settings that apply the simple ‘oddball’ paradigm, P300 amplitude also

depends on the probability of the target stimulus; rarer the target, greater the amplitude of

the P300 it elicits (Coles, 1995).


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1.5. Delayed cognitive effects of acute OP poisoning: evidence from previous

studies and their limitations

The first reports of chronic neurobehavioural manifestations of OP poisoning date

back to 1960s. They mainly reported symptoms such as poor memory, confusion,

anxiety, drowsiness, fatigue, depression and irritability in patients heavily exposed to OP

compounds (Dille & Smith, 1964; Gerson & Shaw, 1961; Metcalf & Homes, 1969;

Durham et al., 1965). Although these studies were of little help to reach conclusions, they

have, for the first time, highlighted the possibility of development of ‘‘chronic OP-

induced neuropsychiatric disorders’’ (Jamal, 1997). After these early studies, research on

long-term neurobehavioural effects of OP compounds pursued along two lines:

1. The occurrence of chronic neurobehavioural impairment as a consequence of an

acute OP poisoning (Savage et al., 1988; Rosenstock et al., 1991; Steenland et al.,

1994; Wesseling, et al., 2002, Stallones & Beseler, 2002)

2. The occurrence of neurobehavioural changes as a consequence of prolonged

exposure without preceding episodes of acute poisoning. Changes with chronic

low-level exposure were studied in many occupational groups including farmers

(Fiedler et al., 1997; Stallones & Beseler, 2002), farm-workers (Daniell et al.,

1992; London et al., 1997), sheep-dippers (Stephens et al., 1995) and termiticide

applicators (Steenland et al., 2000).


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Present study focuses on delayed cognitive effects of acute OP poisoning. Other

than initial case studies, only few large scale analytical epidemiological studies have

assessed the chronic neurological sequelae of acute OP poisoning (Savage et al., 1988;

Rosenstock et al., 1991; Steenland et al., 1994, Wesseling et al., 2002).

However, there are several methodological limitations and inconsistencies of the

findings in the early case studies (Gerson & Shaw, 1961; Dille & Smith, 1964; Durham et

al., 1965; Metcalf & Homes, 1969) as well as recent case-control studies (Savage et al.,

1988; Rosenstock et al., 1991; Steenland et al., 1994, Wesseling et al., 2002):

1. All these were cross-sectional studies where patients were assessed once, usually

months to years after poisoning. The large scale epidemiological studies on

delayed effects of acute poisoning (Savage et al., 1988; Rosenstock et al., 1991;

Steenland et al., 1994, Wesseling et al., 2002) present little information about the

acute clinical picture: the type and amount of OP, cholinergic manifestations and

complications.

2. All the investigators have used symptom inquiry and/or neuropsychological

testing tools to assess cognitive functions. However, all neuropsychological tests

require the subjects to perform a particular motor task. Thus the behavioural test

performance depends largely on cognitive functions as well as executive

functions. In other words, standard neurobehavioural tests (e.g. RT) cannot assess

the cognitive component in isolation, as the neurobehavioural task duration

includes the sensory and motor conduction. When these behavioural experiments


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are used to deduce conclusions on cognitive functions, it is assumed that patient’s

sensory and motor conduction are normal. However normal variations and

pathological deviations in sensory or motor conduction can affect the behavioural

test outcome, particularly in those where the speed of the performance is tested

(e.g. RT tests). In this respect, OP induced delayed neuropathy is a major

confounding factor. Delayed polyneuropathy manifests after 1-4 weeks of acute

OP poisoning, causing slowed sensory and motor conduction (Lotti & Moretto,

2005; Glynn, 2006).

3. Techniques such as electrophysiology and functional neuroimaging, which assess

brain function at a more fundamental level, were hardly ever used in the previous

studies to elicit chronic cognitive effects of acute OP pesticide poisoning except

in few, where EEG recordings were analyzed (Metcalf & Holmes, 1969; Savage

et al., 1988). So far, OP related ERP changes have been reported only in three

studies: two of them evaluated ERP changes in chronic subclinical exposure in

pesticide applicators (Teo et al., 1987; Misra et al., 1994) and the other one

assessed ERP in victims of OP warfare agent sarin (Murata et al., 1997). There

are no studies that evaluated the delayed effects of acute OP insecticide poisoning

on ERP.

4. Cognitive processing test performance depends on many long-term/stable (e.g.

age, sex, alcohol abuse etc.) confounding factors, and short-term modifying

factors (level of arousal, recent alcohol/tobacco intake, sleep deprivation etc.)

(International Programme on Chemical Safety, 2001). In the previous research


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reports, authors have described the long-term confounding factors. However,

information is insufficient to ascertain whether the investigators have taken steps

to minimise the influence of short-term modifying factors before testing the

subjects.

5. Patients have complained more neuropsychiatric symptoms than controls in all

four epidemiological analytical studies (Savage et al., 1988; Rosenstock et al.,

1991; Steenland et al., 1994, Wesseling et al., 2002). However neurological

examination did not reveal any abnormalities in poisoned subjects. Reviewers

have questioned the validity of the positive symptomatology, suggesting that it

may be due to recall bias: patients who were exposed to pesticides are more

motivated to recall or report symptoms (Kamel & Hoppin, 2004).

6. Findings of neuropsychological tests are also inconsistent among different

studies. On one hand, the ambiguity of the results of these studies may be

attributed to variations in sampling criteria and methodological differences from

one study to another. On the other hand, some subsequent authors have

questioned the objectivity of the testing methods and the sensitivity of the existing

objective testing tools in identifying any subtle impairment in higher brain

functions (Steenland et al., 2000; Kamel & Hoppin, 2004). However, one

common feature is that all studies have revealed abnormalities in at least one

aspect of visual information processing.


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1.6. Rationale, aims and objectives of the present study

Present study was designed hypothesising that acute OP insecticide poisoning

impairs cognitive processing of visual and auditory stimuli, even after recovery form the

cholinergic phase of poisoning. In contrast to cross-sectional studies conducted by

previous researchers on patients several months after exposure, we enrolled the patients

during the acute stage of poisoning. Initial assessment was done in the immediate post-

cholinergic phase and a follow up assessment was done six moths after poisoning.

Furthermore, selection criteria and study protocols were designed to minimize the effects

of long-term confounding factors and short-term modifying factors that affect cognitive

performance. The effect of age and sex on the measures of cognitive processing was

particularly considered and hence the patients were individually matched with the control

groups for age and sex.

Particular attention was given to the psychophysiological indicators of cognitive

processing of visual and auditory information. We aimed at isolating the cognitive

component of visual reactions, which is not possible with standard RT measurements.

The term ‘Cognitive Processing Time (CPT)’ is used in this study to specify the duration

of the cognitive component of a visual reaction. This component of psychomotor function

has not been studied to date. As the sensory and the motor components are outside the

cognitive domain and vary even among healthy individuals, CPT is a more refined and

accurate measure (in comparison to RT itself) of cognitive processing of information. We


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used ERPs to assess cognitive processing of auditory information. With more refined and

quantitative tests which closely correlate with actual neuronal processes (viz. cognitive

processing time in RT tasks, EPs and ERPs), main concerns were to maximize the

objectivity of the findings and to identify the exact mental processes affected.

Objectives:

Specific objectives of this study were to compare the psychophysiological measures

of cognitive processing of visual information (viz. cognitive processing time of simple

visual reactions and cognitive processing time of recognition visual reactions) and

cognitive processing of auditory information (viz. P300 latency and amplitude),

1. between patients with OP insecticide poisoning (on recovery from the cholinergic

phase) and matched controls

2. between immediate post-cholinergic phase and six months after poisoning in the

patients.


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Section 2

METHODOLOGY

2.1. Study design

This study consisted of two parts.

First part was a case-control study where parameters of cognitive processing were

compared in patients with OP insecticide poisoning and matched control groups. This

phase included three groups: the test group and two control groups.

1. Test group: These were the patients with acute OP poisoning. They were tested twice:

initially in the immediate post-cholinergic stage of poisoning, and then six months after

poisoning.

2. Healthy controls: Healthy individuals from the community, who did not have any

exposure to OP compounds.

3. Hospitalised controls: This control group consisted of patients hospitalised with

paracetamol overdose. This second group was added to match the general wellbeing and

the psychological status of the test and the control groups at the time of

neurophysiological assessment. The drug paracetamol was chosen as it has no known

direct action on cognitive functions.


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The second part was a follow up study where the patients with OP poisoning were

tested six months after poisoning. The follow up results of the patients were compared

with their initial values.

The study was carried out in medical wards, poisoning management unit and the

clinical neurophysiology laboratory, Teaching Hospital, Peradeniya. Data were collected

over a period of 31 months, from July 2004 to February 2007. Patients admitted to the

medical wards and the poisoning management unit during this period were assessed for

eligibility for the study.

2.2. Ethical considerations

The study design and protocols complied with the World Medical Association

Declaration of Helsinki. Informed written consent was obtained from all participants (see

appendix 1 for patient information sheet and the consent form). Ethical clearance was

granted by Research and Ethical Review Subcommittee, Faculty of Medicine, University

of Peradeniya, Sri Lanka, on 18/06/2004.


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2.3. Subjects

2.3.1. Test group

All patients admitted to the hospital with suspected OP poisoning were assessed for

eligibility.

2.3.1.1. Inclusion criteria:

1) Reliable history of OP exposure

2) Any of the clinical features of cholinergic over-activity: fasciculation, miosis,

bradycardia, excessive sweating / salivation, dyspnoea/lung signs, impaired

consciousness

2.3.1.2. Exclusion criteria:

1) Chronic exposure to OP compounds (e.g. occupational exposure in farmers, pesticide

applicators etc.)

2) Other pre-existing (diagnosed) neurological illnesses, which can affect cognitive or

motor functions

3) Gross visual or hearing impairment where the patient is unable to identify visual and

auditory stimuli

4) Long-term intake of medications which have the potential to alter cognitive functions

5) Consumption of alcohol: more than the upper limit of the safe range (men >

21units/wk, women > 14units/wk. One unit = equivalent of 8 grams of alcohol)


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2.3.2. Healthy control group


1) Age- and sex-matched individuals with similar social background: For the social and

educational factors to be comparable, patients were instructed to bring a relative or a

friend of the same age and sex, on attending cognitive function tests. In the instances they

were unable to do that, appropriate volunteers were tested. The age of the matched

control was within 3 years of the age of the test group subject.


1) Past history of OP poisoning

2) All the exclusion criteria applied for the test group (criteria 1 to 5 under 2.3.1.2.)

2.3.3. Hospitalised controls


1) Age- and sex-matched individuals hospitalised with paracetamol overdose. The age of

the matched control was within 3 years of the age of the test group subject.


1) Clinical features of hepatic encephalopathy

2) Past history of OP poisoning

3) All the exclusion criteria applied for the patient group (criteria 1 to 5 under 2.3.1.2.)


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2.4. Background information

Before selecting the participants for cognitive tests, background information were

collected using structured data sheets (see appendix 2). This aimed at assessing the

eligibility (criteria under 3.3 above) for the study, finding out the details of poisoning,

and identifying any confounding factors.

2.4.1. Test group

Clinical interview, examination and inward assessment targeted the information

related to following areas (see appendix 2.1):

1. Personal details: Name, sex, age, postal address, occupation, educational status and

computer familiarity

2. Details of the episode acute poisoning: type of OP, amount, route of exposure, mode

(intentional/accidental)

3. Signs characteristic of OP poisoning: on admission and during hospital-stay

4. Management measures: gastric lavage, atropine, pralidoxime, intensive care and other

supportive treatment

5. Any major complications: e.g. respiratory/cardiac arrest, seizures, shock etc.

6. Probable confounding factors and other performance modifying factors:

• prediagnosed visual/neurological/cognitive impairment

• pre-diagnosed psychiatric illnesses


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• alcohol, tobacco and other addictive drug use: duration, regularity, amount per

day/week

2.4.2. Healthy controls

Background information included personal details and probable confounding

factors and other performance modifying factors stated under the information of the test

group (areas 1 and 6 above) ( Appendix 2.2).

2.4.3. Hospitalised controls

Background information collected was similar to those of the test group:

1. Personal details: Name, sex, age, postal address, occupation, educational status and

computer familiarity.

2. Details of paracetamol overdose: amount, mode (intentional/accidental) if intentional,

the reason

3. Clinical features: on admission and during hospital-stay

4. Management measures: antidotes (N-acetylcysteine), supportive treatment, intensive

care

5. Any major complications: e.g. liver failure, hepatic encephaolpathy

6. Probable confounding factors and other performance modifying factors stated above

under information of the test group (see appendix 2.3).


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All the patients with intentional poisoning were referred to the hospital psychiatrist.

The psychiatric assessment and the diagnosis were also included in the record.

After assessment of background information, the eligible subjects were selected to

perform tests of cognitive functions. Numbers of recruited subjects and the design of

inter-group comparison are illustrated in figure 2.1. From July 2004 to September 2006,

67 patients admitted to Teaching Hospital, Peradeniya with a history of OP poisoning

were assessed for eligibility for the study. Of these, nine did not meet the inclusion

criteria either because the evidence of OP exposure was unreliable or because they did

not show clinical features of intoxication. Of the 58 who fulfilled the inclusion criteria,

two were excluded due to chronic occupational exposure to OP and seven were excluded

due to excessive intake of alcohol. These nine patients were males. Of the remaining 49,

five could not be tested as they left the hospital prematurely. Accordingly, 44 patients (28

males and 16 females) were assessed after recovery form cholinergic phase (i.e. 1st visit),

usually on the day of discharge from the hospital. Recovery from acute poisoning was

considered complete when the patients were free of all signs and symptoms of the acute

cholinergic syndrome, and free of medication such as atropine. There was at least 24-

hour gap between the last dose of atropine/benzodiazepines and cognitive function tests.

The median duration between exposure and cognitive testing was of 10.5 days (Range: 3

to 47 days).


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The healthy control group that comprised 43 individuals matched for age and sex

with the patients on one-to-one basis (healthy control number-64 was matched with the

patient number-18 and number-26). Healthy controls were tested on an appointment

basis.

Recruitment of the hospitalised control group (i.e. patients with paracetamol

poisoning) was started in April 2006. Patients admitted to the hospital with paracetamol

poisoning were enrolled in the hospitalised control group only if the patient could be

matched for sex and age (to nearest 3 years) with a member of the test group.

Accordingly, the hospitalised control group comprised 11 subjects (three males and nine

females) who could be matched individually for age and sex with 11 patients in the test

group. The hospitalised control group was also compared with 11 healthy individuals

matched with the 11 OP poisoned patients drawn from the test group. This helped to

determine whether psychological conditions associated with self harm and hospitalisation

(which was present in patients with paracetamol overdose, but absent in healthy

individuals) affects the test performance. Hospitalised control group was tested after

recovery from the acute stage, on the day of discharge from the ward. The median

duration between ingestion and testing was 5 days (range: 3 to 6 days).


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67 patients with suspected OP

poisoning assessed for eligibility

58 fulfilled

inclusion criteria

9 excluded:

2: chronic occupational exposure

7: excessive alcohol intake

49 eligible patients

5 did not participate

44 assessed for

cognitive functions

on recovery from

cholinergic phase

43 healthy

controls

11 hospitalisedcontrols

30 assessed for

cognitive fuctions

after 6 months

14 loss of follow up

Age-and-sex-

matched

11 patients drawnfrom test group

Age-and-sex-

matched

Figure 2.1: Selection and comparison of study samples.


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The subjects were instructed and made to stick to the following criteria before

attending the cognitive function tests, so that it was possible to minimize the short-term

factors modifying the cognitive processing speed (International Programme on Chemical

Safety, 2001):

1. Abstain from alcohol on the day of testing and the day before

2. Abstain from smoking and drinking coffee on the day of the test

3. Having a sleep of at least 6 hours in the night before the test

Subjects were inquired for contraindications for magnetic stimulation (intracranial

metal implants, cardiac pacemakers) but none had any contraindication. A neurological

examination and a visual acuity test were performed on the day of cognitive assessment.

2.5. Assessment of cognitive processing

After neurological examination, subjects participated in the test battery devised to

assess cognitive processing of visual information and auditory information.

2.5.1.

Basis of the assessment paradigms

Visual information processing: Studying the cognitive processing of visual information

was based on measurement of the cognitive component of visual reactions. We denoted

the cognitive component of visual reactions as ‘cognitive processing time’ (CPT), and


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quantified the CPT using combined VRT test and EP studies. The collective duration of

the sensory component, cognitive component and the motor component of the visual

reaction was measured using VRT tests. VEPs were used to measure the afferent

conduction time of the visual stimulus, and MEPs to measure the motor component of the

reaction time. P100 VEP latency was considered the sensory component. We stimulated

motor cortical neurons using transcranial magnetic stimulation (TMS) and recorded the

MEPs of the muscle that responds in the visual reaction. Then we measured the duration

between cortical stimulation and the onset of MEPs, which is the total motor conduction

time (TMCT). This was considered the length of the motor component of the visual

reaction. Then CPT of the visual reaction was calculated by subtracting the sum of the

sensory component and the motor component from the VRT (figure 2.2).

Figure 2.2: Calculation of cognitive processing time (CPT). VRT: visual reaction time.

TMCT: total motor conduction time.


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Auditory information processing: Assessment of cognitive processing of auditory

information was based on findings of ERPs which are electrophysiological correlates of

cognition. Applying the auditory oddball paradigm, we recorded ERPs that reflect the

auditory stimulus evaluation process of the neural circuits.

2.5.2. Equipment

1. A computer-based test (SermionTM) was used to measure SVRT and RVRT. Visual

stimuli were displayed on a 14-inch colour monitor.

2. A Magstim Model 200TM magnetic stimulator with a circular 90mm coil (Type 9784)

was used for TMS and spinal stimulation (figure 2.3).

3. Medtronic Keypoint® signal averaging machines were used to record and average EPs

and ERPs (figures 2.4A & 2.4B). MEPs were recorded with pre-gelled surface

electrodes. 10mm gold-plated cup electrodes were used for VEP and ERP recording.


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Figure 2.3: Magstim Model 200TM magnetic stimulator with circular 90mm coil.

Figure 2.4: Signal averaging machines: (A) Medtronic KeypointTM (used to record visual

evoked potentials and motor evoked potentials). (B) Medtronic Keypoint PortableTM

(used to record ERPs).

A B

8/9/2019 MPhil Thesis Tharaka Dassan

mphil thesis tharaka dassanayake

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