The Development of Neurodegeneration and Behavioural Alterations following Lithium/Pilocarpine ...

349 Pages·2011·6.88 MB·English

THE DEVELOPMENT OF NEURODEGENERATION AND
BEHAVIOURAL ALTERATIONS FOLLOWING
LITHIUM/PILOCARPINE-INDUCED STATUS
EPILEPTICUS IN RATS
by
Crystal Maureen Dykstra
A thesis submitted in conformity with the requirements
for the degree of doctor of philosophy
Institute of Medical Science
University of Toronto
© Copyright by Crystal Maureen Dykstra (2011)
THE DEVELOPMENT OF NEURODEGENERATION AND
BEHAVIOURAL ALTERATIONS AFTER
LITHIUM/PILOCARPINE-INDUCED STATUS EPILEPTICUS IN
RATS
Crystal Maureen Dykstra
Doctor of Philosophy
Institute of Medical Science
University of Toronto
2011
The lithium/pilocarpine model of epilepsy mimics mesial temporal lobe epilepsy with
hippocampal sclerosis (MTLE-HS) in humans. Systemic injection of pilocarpine in lithium
chloride (LiCL) pretreated adult rats results in an acute episode of severe continuous seizure
activity (status epilepticus, SE). SE causes a latent period, whereby the animal appears
neurologically normal, with subsequent development of spontaneous recurrent seizures (SRSs).
Neuropathological changes that occur during the latent period are believed to contribute to the
epileptic condition. The present thesis characterized the development of neuronal death and
behavioural alterations in rats after SE induced by the repeated low-dose pilocarpine procedure
(RLDP), and investigated the causal relationship between these two processes.
Our data demonstrated that the RLDP procedure for the induction of SE results in widespread
neurodegeneration and behavioural alterations comparable to the pilocarpine and low-dose
pilocarpine (LDP) procedures. However, the advantage to using this protocol was strain-
dependent as it reduced mortality in Wistar, but not in Long Evans Hooded (LEH), rats.
Stereological analysis of neurons (stained for the neuronal specific marker [NeuN]) at various
times (1 hr to 3 months) following SE showed that different brain regions within the
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hippocampus, amygdala, thalamus and piriform cortex exhibited differential rates of neuronal
loss, with the majority of SE-induced neuronal death present by 24 hours. SE resulted in
decreased exploratory behavior as assessed in the open field test, increased aggression to
handling, increased hyperreactivity as assessed in the touch-response test, and anxiolytic effects
as measured in the elevated-plus maze. Furthermore, deficits in search strategies used, as well as
impaired spatial learning and memory, contributed to poor Morris water maze (MWM)
performance. Partial neuroprotection within the hippocampus (by tat-NR2B9c) had no effect on
the number of rats developing SRSs or on behavioural alterations; this argues against a causal
relationship between neurodegeneration within this region, genesis of SRSs, and behavioural
morbidity.

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Acknowledgments
This thesis would not have been possible without the support of many people. First and
foremost, I offer my sincerest gratitude to Dr. James Gurd, whose encouragement, guidance and
support from the initial to the final level enabled me to complete this thesis. I greatly appreciate
his patience and knowledge whilst allowing me the room to work in my own way. I would like
to extend my gratitude to Dr. Bill Milgram, who was abundantly helpful and offered invaluable
assistance, support and guidance. I would also like to thank my program advisory committee
members, Drs. Mac Burnham and Mike Tymianski, for their time, guidance and constructive
criticism.
There are many individuals who have offered their time and expertise in training me in specific
laboratory techniques. I would like to thank Dr. Gwen Ivy for teaching me histological
techniques that were invaluable for this work and for sharing her lab space. I would like to
express my gratitude to Raymond Or for training on the microscopes, and to Candace Ikeda-
Douglas for introducing me to the lithium/pilocarpine seizure model. I am grateful to Dr.
Stephen Reid for sharing his time and expertise in animal surgeries. I am extremely grateful to
Nankie Bissoon for not only sharing her vast knowledge of experimental techniques, but also for
her guidance and friendship in the lab. I would like to extend my gratitude to Dr. Janelle
Leboutillier for her knowledge, guidance and support.
Finally, I owe my deepest gratitude to my parents for their love and support and to the rest of my
family for their encouragement.

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List of Publications
Some of the material presented in this thesis has been published. This is to certify that I, Crystal
Dykstra, carried out the research documented in the following publications:

Dykstra CM, Ratnam M, Gurd JW (2009) Neuroprotection after status epilepticus by targeting
protein interactions with postsynaptic density protein 95. J Neuropathol Exp Neurol 68:823-831.

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Table of Contents
Acknowledgments ........................................................................................................................ iv
List of Publications ....................................................................................................................... v
Table of Contents ......................................................................................................................... vi
List of Tables ............................................................................................................................... xv
List of Figures ............................................................................................................................. xvi
List of Appendices ...................................................................................................................... xix
List of Abbreviations .................................................................................................................. xx
Chapter 1 General Introduction .................................................................................................. 1
1.1Main features of mesial temporal lobe epilepsy with hippocampal sclerosis ..................... 1
1.2 Animal models of seizure development and epilepsy ........................................................... 6
1.2.1 Kindling............................................................................................................................. 6
1.2.2 Post-status epilepticus models ........................................................................................... 7
1.3 Background information on the pilocarpine and lithium/pilocarpine models .................. 8
1.3.1 The pilocarpine model ....................................................................................................... 8
1.3.2 Convulsive effects of pilocarpine are mediated by activation of M1 receptors ................ 9
1.3.3 The cholinergic system is involved in the initiation but not the maintenance of SE ...... 10
1.3.4 The lithium/pilocarpine model ........................................................................................ 10
1.3.5 The proconvulsive mechanisms of lithium ..................................................................... 11
1.3.6 Use of diazepam to control SE duration and reduce mortality ....................................... 12
1.4 Behavioural and clinical features of seizure development in the lithium/pilocarpine
model ....................................................................................................................................... 12
1.4.1 The acute phase ............................................................................................................... 13
1.4.1.1 Behavioural seizures during the acute phase ........................................................ 13
1.4.1.2 Electroencephalographic patterns during motor limbic seizures and SE .............. 14
1.4.1.3 Scoring of pilocarpine-induced seizures ............................................................... 14
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1.4.1.4 Effect of SE duration on mortality and neuropathology ....................................... 16
1.4.2 Epileptogenesis ............................................................................................................... 17
1.4.2.1 Duration of the latent phase .................................................................................. 17
1.4.3 The chronic phase ........................................................................................................... 19
1.4.3.1 Behaviour during the chronic phase ..................................................................... 19
1.4.3.2 Electroencephalographic patterns observed during SRSs ..................................... 19
1.5 Neuropathology ..................................................................................................................... 20
1.5.1 Neurodegeneration .......................................................................................................... 20
1.5.1.1 SE-induced neurodegeneration ............................................................................. 20
1.5.1.2 Progression and severity of neuronal loss following SE ...................................... 21
1.5.1.3 Mechanisms underlying SE-induced neuronal death ............................................ 21
1.5.1.4 Types of cell death mechanisms initiated by SE .................................................. 22
1.5.1.5 Factors determining the extent and phenotype of SE-induced neuronal death ..... 24
1.5.2 Synaptic reorganization ................................................................................................... 27
1.5.3 Reactive gliosis ............................................................................................................... 28
1.5.4 Neurogenesis ................................................................................................................... 29
1.6 Co-morbid interictal disorders in mesial temporal lobe epilepsy .................................... 30
1.6.1 The relationship between epilepsy and cognitive and interictal behavioural
alterations .......................................................................................................................... 30
1.6.2 Shared neurodevelopmental, genetic or environmental causes predispose subjects to
develop both epilepsy and co-morbid behavioural and cognitive disturbances ............... 31
1.6.3 Neuropathological changes underlying the genesis of interictal behavioural
disturbances are closely related to those mediating epileptogenesis itself ....................... 32
1.6.4 Spontaneous recurrent seizures contribute to interictal behavioural and cognitive
impairment in post-SE models .......................................................................................... 33
1.6.5 Other factors affecting severity of interictal behavioural and cognitive impairment ..... 33
1.6.6 Interictal behavioural disturbances following SE ........................................................... 34
1.6.6.1 Anxiety .................................................................................................................. 34
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1.6.6.2 Exploration ............................................................................................................ 36
1.6.6.3 Aggression ............................................................................................................ 38
1.6.7 The effect of SE on spatial learning and memory ........................................................... 38
1.6.7.1 The Morris water maze as a test of visual-spatial learning and memory .............. 39
1.6.7.2 The effect of SE on performance in the MWM task ............................................. 40
1.7 The goals of the thesis ........................................................................................................... 42
Chapter 2 Hypotheses and Specific Objectives ........................................................................ 43
2.1 Comparison of procedures for the induction of SE ............................................................ 44
2.2 The effect of recovery time on SE-induced neurodegeneration ......................................... 44
2.3 The effect of tat-NR2B9c on SE-induced neuropathology and cognitive impairment ...... 45
2.4 The effect of SE on behavioural performance in tasks assessing anxiety, exploration
and aggression ................................................................................................................... 45
2.5 The effect of SE on search strategy use in the Morris water maze .................................... 46
Chapter 3 ..................................................................................................................................... 47
A comparison between Long-Evans hooded and Wistar rats related to the induction
and severity of status epilepticus in the low-dose and repeated low-dose
lithium/pilocarpine procedures ............................................................................................. 47
3.1 Introduction ........................................................................................................................... 47
3.2 Methods .................................................................................................................................. 48
3.2.1 Animals ........................................................................................................................... 48
3.2.2 Induction of status epilepticus ......................................................................................... 48
3.2.3 Monitoring of seizure activity ......................................................................................... 49
3.2.4 Post-seizure animal care: ................................................................................................. 49
3.2.5 Histology and Stereological analysis: ............................................................................. 50
3.2.6 Drugs: .............................................................................................................................. 54
3.2.7 Statistical Analysis: ......................................................................................................... 54
3.3 Results .................................................................................................................................... 54
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3.3.1 Induction of SE in Wistars and Long Evans Hooded rats ............................................... 54
3.3.2 The effect of SE on mortality .......................................................................................... 54
3.3.3 Severity of seizures in LEH and Wistar rats ................................................................... 55
3.3.4 SE-induced neuropathology in LEH and Wistar rats following SE induced with the
RLDP procedure ............................................................................................................... 59
3.3.5 Comparison of SE-induced neuropathology resulting from the LDP and RLDP
protocols in Wistar rats ..................................................................................................... 59
3.4 Discussion ............................................................................................................................... 64
3.4.1 Differential effects of induction procedure in LEH and Wistar rats ............................... 64
3.4.2 Comparison of SE-induced neuropathology in LEH and Wistar rats following SE
induction with the RLDP procedure ................................................................................. 65
3.4.3 Comparison of the effect of the LDP and RLDP protocols on SE-induced
neurodegeneration in Wistar rats ...................................................................................... 66
3.4.4 Conclusion....................................................................................................................... 67
Chapter 4 ..................................................................................................................................... 68
Temporal profile of neuronal death following lithium/pilocarpine-induced status
epilepticus................................................................................................................................ 68
4.1 Introduction ........................................................................................................................... 68
4.2 Methods .................................................................................................................................. 70
4.2.1 Animals ........................................................................................................................... 70
4.2.2 Induction of status epilepticus ......................................................................................... 70
4.2.3 Post-seizure care .............................................................................................................. 70
4.2.4 Detection of SRSs ........................................................................................................... 70
4.2.5 Histology and Stereological analysis .............................................................................. 71
4.2.6 Fluoro-Jade B staining .................................................................................................... 74
4.2.7 Statistical Analysis: ......................................................................................................... 75
4.3 Results .................................................................................................................................... 80
4.3.1 SE induction and survival rates: ...................................................................................... 80
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4.3.2 Spontaneous seizures after lithium/pilocarpine induced SE ........................................... 80
4.3.2 Neuropathology following status epilepticus: Overview ................................................ 80
4.3.3 SE-induced neurodegeneration in the hippocampus: ...................................................... 82
4.3.5 SE-induced neurodegeneration in amygdaloid nuclei: ................................................... 98
4.3.6 SE-induced neurodegeneration in the piriform cortex: ................................................... 98
4.3.7 Detection of Fluoro-jade B stained neurons .................................................................... 98
4.4 Discussion ............................................................................................................................. 107
4.4.1 The neuropathological effect of increasing survival time following status epilepticus 107
4.4.2 The relationship between SE, SRSs and delayed neuronal death ................................. 110
4.4.3 Differences in the severity and spatial pattern of neuronal death following SE ........... 111
4.4.4 The type of cell death produced by SE ......................................................................... 112
4.4.5 Conclusion..................................................................................................................... 113
Chapter 5 ................................................................................................................................... 118
Neuroprotection following status epilepticus by targeting protein interactions with
PSD-95 ................................................................................................................................... 118
5.1 Introduction .................................................................................................................... 118
5.2 Methods ........................................................................................................................... 119
5.2.1 Induction of status epilepticus ............................................................................... 119
5.2.2 Administration of peptides ..................................................................................... 120
5.2.3 NeuN Immunohistochemistry ................................................................................ 121
5.2.4 Statistical Analysis ................................................................................................. 122
5.3 Results ............................................................................................................................. 122
5.3.1 Induction of status epilepticus ............................................................................... 122
5.3.2 SE induced by repeated low doses of pilocarpine results in neurodegeneration in
the hippocampus and piriform cortex ................................................................. 125
5.3.3 Tat-NR2B9c reduces SE-induced neurodegeneration in the hippocampus ........... 129
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