Medicine (RMH) - Theses
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ItemGenetic and molecular determinants of acquired and genetic epilepsy( 2016)Introduction: Despite the differences in the pathophysiological mechanisms and clinical features of both genetic and acquired epilepsies, dysfunction of both voltage- and ligand-gated ion channels have been highlighted as major contributors of the epileptic phenotype. In this thesis, the molecular mechanism by which T-type Ca2+ channels, AMPAR and TARPs lead to the development of epilepsy was evaluated. In addition, whole genome sequencing was utilised to identify novel candidate genes that could play a role in the epilepsy phenotype. Results: Anti-epileptogenic effects of selective blocking of T-type calcium channels in models of acquired epilepsy. The anti-epileptogenic effects of Z944, a novel, potent and highly selective T-type Ca2+ was used in the amygdala kindling and post-status epilepticus (SE) model. In the amygdala kindling model, Z944 a novel, potent and highly selective T-type Ca2+, did not suppress seizures in fully kindled rats. However, treatment with Z944 delayed de progression of kindling. In the post-SE model, treatment with Z944 after SE massively reduced the number of spontaneous seizures in comparison to vehicle and levetiracetam treated animals. Moreover, treatment with Z944 showed a strong comorbidity modifier effect in depressive like behaviour and may improve cognition after SE. Effects of the T-type calcium channel CaV3.2 R1584P mutation on seizure susceptibility in congenic rats. The CaV3.2 R1584P mutation was not enough to cause absence seizures in a seizure resistant background in the congenic animals but may be associated with the anxiety phenotype. However, the genetic background of the NEC congenic might contain genes that may suppress the pro-epileptic effect of the R1584P mutation. Role of AMPAR and TARPs in the pathogenesis of genetic generalised epilepsy and acquired epilepsy. The results of this chapter indicate a temporal association between the increased TARPs, Stargazin, γ3, γ4, γ5 and γ8, mRNA expression and the development of absence seizures in GAERS. In the post-SE epilepticus model, there was a significant reduction in mRNA expression in the TARPs γ3 and γ8 in the hippocampus 13 weeks after SE. Whole genome sequencing the GAERS and NEC rat strains. TThrough the proposed screen construct variants were identified in high-seizing F2 rats in the following genes; Cacna1h which codes for the CaV3.2 T-type Ca2+ channel, and F1LVI7_RAT and LOC300024. Similarly, non-seizing F2 rats are heterozygous for the mutations in RGD1308133, D3ZPQ1_RAT, Mkl and BiK. In the NEC and F2 non-seizing variants found in Abat, Cyp11b3 and Cyp11b2. Moreover, the flexibility of this method means that it can be applicable with other models of genetic and acquired epilepsies. Conclusions: Here is shown that blocking T-type Ca2+ channels with Z944 has encouraging positive preclinical evidence for disease-modifying in epileptogenesis and in epilepsy behavioural comorbidities that may be possible to translate to a clinical trial. Moreover, the analysis of T-type Ca2+ channels, AMPAR, TARPs and the discovery of novel potential molecular targets using whole genome sequencing uncovers overlap among genetic and acquired epilepsy. Importantly, the findings in this thesis could lead to discoveries that expand our knowledge of epileptogenesis, opening the door for the development of novel therapies.
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ItemAntidepressant pharmacotherapy in epilepsy: the effects of chronic fluoxetine and citalopram treatments in a rat model of epileptogenesis( 2012)Introduction: In patients with epilepsy there is a high incidence of comorbid psychiatric illnesses, especially mood and anxiety disorders, which have been associated with lower quality of life, impaired function and an elevated risk of suicide. In fact, the occurrence of these illnesses in patients with epilepsy has been reported to be a stronger predictor of quality of life than epilepsy variables such as illness duration or seizure frequency. In addition, there is evidence that the depressed state itself may predispose to seizures and epilepsy. For these reasons, effective management of depressive and anxiety symptoms and syndromes in epilepsy is essential. Selective serotonin reuptake inhibitors (SSRIs) are commonly used to treat depression in epilepsy, therefore it is important to consider their impact on epilepsy. To date, many studies have suggested that SSRI are safe for use in epilepsy, but the majority of these studies administered SSRIs only acutely or for short periods, and investigated effects only on acute seizure endpoints. There is no indication of the effect that chronic SSRI treatment may have on epileptogenesis and the associated neurobiological changes that continue after seizures emerge. This thesis aimed to investigate the effects of chronic SSRI treatment in a rat model of epileptogenesis, as well as investigating common neurobiological substrates of SSRI treatment and epileptogenesis that may also influence the disorder. It was hypothesised that chronic SSRI treatment would slow the rate of kindling epileptogenesis, as well as mitigate effects on common neurobiological substrates. Methods: The amygdala kindling model was used to assess the effects of chronic SSRI treatment (with fluoxetine or citalopram) on epileptogenesis. 9-11 week old male Wistar rats were surgical implanted with a bipolar electrode into the left amygdala for electrical kindling and a subcutaneously implanted osmotic pump filled with fluoxetine (10mg/kg/day, n=19) or vehicle (50% DMSO, n=22) or citalopram (10mg/kg/day, n=26) or vehicle (50% DMSO, n=22), comprising two separate cohorts. All rats were given 30 stimulations and then kindling rate, seizure duration and seizure threshold before and after kindling were monitored. Effects on anxiety- and depressive-like behaviours were also investigated after kindling using two well-validated tests, the elevated plus maze and forced swim test respectively, as well as assessing the corticosterone response to stress and dentate gyrus neurogenesis. Results: The key finding of this study was that rats chronically treated with SSRIs, either fluoxetine or citalopram, demonstrated accelerated rates of kindling epileptogenesis, showing a more rapid progression through the different stages of kindling compared to vehicle treated rats. The increase in seizure duration was also accelerated in the early stages of kindling in both cohorts of SSRI treated rats, however seizure threshold was not significantly different between vehicle and fluoxetine or vehicle and citalopram treated rats, either before or after kindling. This indicates that while epileptogenesis itself progressed at a faster rate during chronic SSRI treatment, accelerating the increase in seizure severity and duration, the local excitability and the threshold at which a seizure occurred was not affected by SSRI treatment. In order to investigate potential mechanisms underlying this, neurobiological alterations common to epileptogenesis and SSRI treatment were also investigated. Behavioural analyses found that both fluoxetine and citalopram treatments did not affect anxiety- or depressive-like behaviours, while kindling increased anxiety-like behaviour, but only in the fluoxetine treated cohort. Dentate gyrus neurogenesis was not significantly affected by kindling or drug treatment while stress-induced corticosterone levels were significantly reduced only by fluoxetine treatment. These investigations do not suggest that these alterations are associated with accelerating kindling rate during chronic SSRI treatment, however how these are affected during or immediately after kindling was not investigated. Conclusions: Chronic treatment with fluoxetine and citalopram, at clinically relevant doses, accelerated kindling epileptogenesis in rodents. This highlights the need to investigate the effects of SSRI treatment on epileptogenesis over time in both animal models and people with epilepsy, rather than focusing solely on acute seizure time points. While the investigations in this study do not suggest that alterations in behaviour, neurogenesis or neuroendocrine responses are associated with accelerating kindling rate during chronic SSRI treatment, how these are affected during or immediately after kindling was not investigated. Therefore, future studies should further investigate the mechanisms underlying the effects of SSRIs on epileptogenesis at appropriate time points, such as during kindling epileptogenesis and also in complementary animal models of epilepsy, such as post-status epilepticus and post-traumatic models. It is essential to treat the depressive symptoms that manifest in people with epilepsy; however whether these medications affect the course of epilepsy and how they may do so should become priority areas for future research.