Inhibition of microglial activation with minocycline at the intrathecal level attenuates sympathoexcitatory and proarrhythmogenic changes in rats with chronic temporal lobe epilepsy

The incidence of sudden unexpected death in epilepsy (SUDEP) is highest in people with chronic and drug resistant epilepsy. Chronic spontaneous recurrent seizures cause cardiorespiratory autonomic dysfunctions. Pituitary adenylate cyclase-activating polypeptide (PACAP) is neuroprotective, whereas microglia produce both pro- and anti- inflammatory effects in the CNS. During acute seizures in rats, PACAP and microglia produce sympathoprotective effect at the intermediolateral cell column (IML), whereas their action on the presympathetic rostral ventrolateral medulla (RVLM) neurons mediates proarrhythmogenic changes. We evaluated the effect of PACAP and microglia at the IML on sympathetic nerve activity (SNA), cardiovascular reflex responses, and electrocardiographic changes in the post-status epilepticus (SE) model of acquired epilepsy, and control rats. Chronic spontaneous seizures in rats produced tachycardia with profound proarrhythmogenic effects (prolongation of QT interval). Antagonism of microglia, but not PACAP, significantly reduced the SNA and the corrected QT interval in post-SE rats. PACAP and microglia antagonists did not change baroreflex and peripheral or central chemoreflex responses with varied effect on somatosympathetic responses in post-SE and control rats. We did not notice changes in microglial morphology or changes in a number of M2 phenotype in epileptic nor control rats in the vicinity of RVLM neurons. Our findings establish that microglial activation, and not PACAP, at the IML accounts for higher SNA and proarrhythmogenic changes during chronic epilepsy in rats. This is the first experimental evidence to support a neurotoxic effect of microglia during chronic epilepsy, in contrast to their neuroprotective action during acute seizures

Altered cardiac structure and function is related to seizure frequency in a rat model of chronic acquired temporal lobe epilepsy

Objective: This study aimed to prospectively examine cardiac structure and function in the kainic acid-induced post-status epilepticus (post-KA SE) model of chronic acquired temporal lobe epilepsy (TLE), specifically to examine for changes between the pre-epileptic, early epileptogenesis and the chronic epilepsy stages. We also aimed to examine whether any changes related to the seizure frequency in individual animals. Methods: Four hours of SE was induced in 9 male Wistar rats at 10 weeks of age, with 8 saline treated matched control rats. Echocardiography was performed prior to the induction of SE, two- and 10-weeks post-SE. Two weeks of continuous video-EEG and simultaneous ECG recordings were acquired for two weeks from 11 weeks post-KA SE. The video-EEG recordings were analyzed blindly to quantify the number and severity of spontaneous seizures, and the ECG recordings analyzed for measures of heart rate variability (HRV). PicroSirius red histology was performed to assess cardiac fibrosis, and intracellular Ca2+ levels and cell contractility were measured by microfluorimetry. Results: All 9 post-KA SE rats were demonstrated to have spontaneous recurrent seizures on the two-week video-EEG recording acquired from 11 weeks SE (seizure frequency ranging from 0.3 to 10.6 seizures/day with the seizure durations from 11 to 62 s), and none of the 8 control rats. Left ventricular wall thickness was thinner, left ventricular internal dimension was shorter, and ejection fraction was significantly decreased in chronically epileptic rats, and was negatively correlated to seizure frequency in individual rats. Diastolic dysfunction was evident in chronically epileptic rats by a decrease in mitral valve deceleration time and an increase in E/E` ratio. Measures of HRV were reduced in the chronically epileptic rats, indicating abnormalities of cardiac autonomic function. Cardiac fibrosis was significantly increased in epileptic rats, positively correlated to seizure frequency, and negatively correlated to ejection fraction. The cardiac fibrosis was not a consequence of direct effect of KA toxicity, as it was not seen in the 6/10 rats from separate cohort that received similar doses of KA but did not go into SE. Cardiomyocyte length, width, volume, and rate of cell lengthening and shortening were significantly reduced in epileptic rats. Significance: The results from this study demonstrate that chronic epilepsy in the post-KA SE rat model of TLE is associated with a progressive deterioration in cardiac structure and function, with a restrictive cardiomyopathy associated with myocardial fibrosis. Positive correlations between seizure frequency and the severity of the cardiac changes were identified. These results provide new insights into the pathophysiology of cardiac disease in chronic epilepsy, and may have relevance for the heterogeneous mechanisms that place these people at risk of sudden unexplained death

Neurodegenerative pathways as targets for acquired epilepsy therapy development

There is a growing body of clinical and experimental evidence that neurodegenerative diseases and epileptogenesis after an acquired brain insult may share common etiological mechanisms. Acquired epilepsy commonly develops as a comorbid condition in patients with neurodegenerative diseases such as Alzheimer's disease, although it is likely much under diagnosed in practice. Progressive neurodegeneration has also been described after traumatic brain injury, stroke, and other forms of brain insults. Moreover, recent evidence has shown that acquired epilepsy is often a progressive disorder that is associated with the development of drug resistance, cognitive decline, and worsening of other neuropsychiatric comorbidities. Therefore, new pharmacological therapies that target neurobiological pathways that underpin neurodegenerative diseases have potential to have both an anti-epileptogenic and disease-modifying effect on the seizures in patients with acquired epilepsy, and also mitigate the progressive neurocognitive and neuropsychiatric comorbidities. Here, we review the neurodegenerative pathways that are plausible targets for the development of novel therapies that could prevent the development or modify the progression of acquired epilepsy, and the supporting published experimental and clinical evidence

Data sets for manuscript by Leung et al., entitled "INHERENT SUSCEPTIBILITY TO ACQUIRED EPILEPSY IN SELECTIVELY-BRED RATS INFLUENCES THE ACUTE RESPONSE TO TRAUMATIC BRAIN INJURY"

Final data sets (Excel format) of raw data used in manuscript by Leung WL et al. (2023), published in the Journal of Neurotrauma, generated by researchers at Monash University Department of Neuroscience (Central Clinical School).  </p

Effects of the T-type calcium channel CaV3.2 R1584P mutation on absence seizure susceptibility in GAERS and NEC congenic rats models

Rationale: Low-voltage-activated or T-type Ca2+ channels play a key role in the generation of seizures in absence epilepsy. We have described a homozygous, gain of function substitution mutation (R1584P) in the CaV3.2 T-type Ca2+ channel gene (Cacna1h) in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). The non-epileptic control (NEC) rats, derived from the same original Wistar strains as GAERS but selectively in-breed not to express seizures, are null for the R1584P mutation. To study the effects of this mutation in rats who otherwise have a GAERS or NEC genetic background, we bred congenic GAERS-Cacna1hNEC (GAERS null for R1584P mutation) and congenic NEC-Cacna1hGAERS (NEC homozygous for R1584P mutation) and evaluated the seizure and behavioral phenotype of these strains in comparison to the original GAERS and NEC strains. Methods: To evaluate seizure expression in the congenic strains, EEG electrodes were implanted in NEC, GAERS, GAERS-Cacna1hNEC without the R1584P mutation, and NEC-Cacna1hGAERS with the R1584P mutation rats. In the first study, continuous EEG recordings were acquired from week 4 (when seizures begin to develop in GAERS) to week 14 of age (when GAERS display hundreds of seizures per day). In the second study, the seizure and behavioral phenotype of GAERS and NEC-Cacna1hGAERS strains were evaluated during young age (6 weeks of age) and adulthood (16 weeks of age) of GAERS, NEC, GAERS-Cacna1hNEC and NEC-Cacna1hGAERS. The Open field test (OFT) and sucrose preference test (SPT) were performed to evaluate anxiety-like and depressive-like behavior, respectively. This was followed by EEG recordings at 18 weeks of age to quantify the seizures, and spike-wave discharge (SWD) cycle frequency. At the end of the study, the whole thalamus was collected for T-type calcium channel mRNA expression analysis. Results: GAERS had a significantly shorter latency to first seizures and an increased number of seizures per day compared to GAERS-Cacna1hNEC. On the other hand, the presence of the R1584P mutation in the NEC-Cacna1hGAERS was not enough to generate spontaneous seizures in their seizure-resistant background. 6 and 16-week-old GAERS and GAERS-Cacna1hNEC rats showed anxiety-like behavior in the OFT, in contrast to NEC and NEC-Cacna1hGAERS. Results from the SPT showed that the GAERS developed depressive-like in the SPT compared to GAERS-Cacna1hNEC, NEC, and NEC-Cacna1hGAERS. Analysis of the EEG at 18 weeks of age showed that the GAERS had an increased number of seizures per day, increased total seizure duration and a higher cycle frequency of SWD relative to GAERS-Cacna1hNEC. However, the average seizure duration was not significantly different between strains. Quantitative real-time PCR showed that the T-type Ca2+ channel isoform CaV3.2 channel expression was significantly increased in GAERS compared to NEC, GAERS-Cacna1hNEC and NEC-Cacna1hGAERS. The presence of the R1584P mutation increased the total ratio of CaV3.2 + 25/−25 splice variants in GAERS and NEC-Cacna1hGAERS compared to NEC and GAERS-Cacna1hNEC. Discussion: The data from this study demonstrate that the R1584P mutation in isolation on a seizure-resistant NEC genetic background was insufficient to generate absence seizures, and that a GAERS genetic background can cause seizures even without the mutation. However, the study provides evidence that the R1584P mutation acts as a modulator of seizures development and expression, and depressive-like behavior in the SPT, but not the anxiety phenotype of the GAERS model of absence epilepsy

Evaluating whole genome sequence data from the Genetic Absence Epilepsy Rat from Strasbourg and its related non-epileptic strain.

The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbreed Wistar rat strain widely used as a model of genetic generalised epilepsy with absence seizures. As in humans, the genetic architecture that results in genetic generalized epilepsy in GAERS is poorly understood. Here we present the strain-specific variants found among the epileptic GAERS and their related Non-Epileptic Control (NEC) strain. The GAERS and NEC represent a powerful opportunity to identify neurobiological factors that are associated with the genetic generalised epilepsy phenotype.We performed whole genome sequencing on adult epileptic GAERS and adult NEC rats, a strain derived from the same original Wistar colony. We also generated whole genome sequencing on four double-crossed (GAERS with NEC) F2 selected for high-seizing (n = 2) and non-seizing (n = 2) phenotypes.Specific to the GAERS genome, we identified 1.12 million single nucleotide variants, 296.5K short insertion-deletions, and 354 putative copy number variants that result in complete or partial loss/duplication of 41 genes. Of the GAERS-specific variants that met high quality criteria, 25 are annotated as stop codon gain/loss, 56 as putative essential splice sites, and 56 indels are predicted to result in a frameshift. Subsequent screening against the two F2 progeny sequenced for having the highest and two F2 progeny for having the lowest seizure burden identified only the selected Cacna1h GAERS-private protein-coding variant as exclusively co-segregating with the two high-seizing F2 rats.This study highlights an approach for using whole genome sequencing to narrow down to a manageable candidate list of genetic variants in a complex genetic epilepsy animal model, and suggests utility of this sequencing design to investigate other spontaneously occurring animal models of human disease

Evaluating whole genome sequence data from the Genetic Absence Epilepsy Rat from Strasbourg and its related non-epileptic strain

Objective The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbreed Wistar rat strain widely used as a model of genetic generalised epilepsy with absence seizures. As in humans, the genetic architecture that results in genetic generalized epilepsy in GAERS is poorly understood. Here we present the strain-specific variants found among the epileptic GAERS and their related Non-Epileptic Control (NEC) strain. The GAERS and NEC represent a powerful opportunity to identify neurobiological factors that are associated with the genetic generalised epilepsy phenotype. Methods We performed whole genome sequencing on adult epileptic GAERS and adult NEC rats, a strain derived from the same original Wistar colony. We also generated whole genome sequencing on four double-crossed (GAERS with NEC) F2 selected for high-seizing (n = 2) and non-seizing (n = 2) phenotypes. Results Specific to the GAERS genome, we identified 1.12 million single nucleotide variants, 296.5K short insertion-deletions, and 354 putative copy number variants that result in complete or partial loss/duplication of 41 genes. Of the GAERS-specific variants that met high quality criteria, 25 are annotated as stop codon gain/loss, 56 as putative essential splice sites, and 56 indels are predicted to result in a frameshift. Subsequent screening against the two F2 progeny sequenced for having the highest and two F2 progeny for having the lowest seizure burden identified only the selected Cacna1h GAERS-private protein-coding variant as exclusively co-segregating with the two high-seizing F2 rats. Significance This study highlights an approach for using whole genome sequencing to narrow down to a manageable candidate list of genetic variants in a complex genetic epilepsy animal model, and suggests utility of this sequencing design to investigate other spontaneously occurring animal models of human disease

An Integrated Multi-Omic Network Analysis Identifies Seizure-Associated Dysregulated Pathways in the GAERS Model of Absence Epilepsy

Absence epilepsy syndromes are part of the genetic generalized epilepsies, the pathogenesis of which remains poorly understood, although a polygenic architecture is presumed. Current focus on single molecule or gene identification to elucidate epileptogenic drivers is unable to fully capture the complex dysfunctional interactions occurring at a genetic/proteomic/metabolomic level. Here, we employ a multi-omic, network-based approach to characterize the molecular signature associated with absence epilepsy-like phenotype seen in a well validated rat model of genetic generalized epilepsy with absence seizures. Electroencephalographic and behavioral data was collected from Genetic Absence Epilepsy Rats from Strasbourg (GAERS, n = 6) and non-epileptic controls (NEC, n = 6), followed by proteomic and metabolomic profiling of the cortical and thalamic tissue of rats from both groups. The general framework of weighted correlation network analysis (WGCNA) was used to identify groups of highly correlated proteins and metabolites, which were then functionally annotated through joint pathway enrichment analysis. In both brain regions a large protein-metabolite module was found to be highly associated with the GAERS strain, absence seizures and associated anxiety and depressive-like phenotype. Quantitative pathway analysis indicated enrichment in oxidative pathways and a downregulation of the lysine degradation pathway in both brain regions. GSTM1 and ALDH2 were identified as central regulatory hubs of the seizure-associated module in the somatosensory cortex and thalamus, respectively. These enzymes are involved in lysine degradation and play important roles in maintaining oxidative balance. We conclude that the dysregulated pathways identified in the seizure-associated module may be involved in the aetiology and maintenance of absence seizure activity. This dysregulated activity could potentially be modulated by targeting one or both central regulatory hubs

Variant tallies compared to BN rat reference (RGSC v3.4 b4).

<p>Variant tallies compared to BN rat reference (RGSC v3.4 b4).</p

Annotations for indel variants observed in one strain and not the other, excluding indels overlapping non-reference sequence.

<p>Annotations for indel variants observed in one strain and not the other, excluding indels overlapping non-reference sequence.</p