Rodent Models Of Temporal Lobe Epilepsy Research Articles

Aerobic exercise alters DNA hydroxymethylation levels in an experimental rodent model of temporal lobe epilepsy

The therapeutic potential of aerobic exercise in mitigating seizures and cognitive issues in temporal lobe epilepsy (TLE) is recognized, yet the underlying mechanisms are not well understood. Using a rodent TLE model induced by Kainic acid (KA), we investigated the impact of a single bout of exercise (i.e., acute) or 4 weeks of aerobic exercise (i.e., chronic). Blood was processed for epilepsy-associated serum markers, and DNA methylation (DNAme), and hippocampal area CA3 was assessed for gene expression levels for DNAme-associated enzymes. While acute aerobic exercise did not alter serum Brain-Derived Neurotrophic Factor (BDNF) or Interleukin-6 (IL-6), chronic exercise resulted in an exercise-specific decrease in serum BDNF and an increase in serum IL-6 levels in epileptic rats. Additionally, whole blood DNAme levels, specifically 5-hydroxymethylcytosine (5-hmC), decreased in epileptic animals following chronic exercise. Hippocampal CA3 5-hmC levels and ten-eleven translocation protein (TET1) expression mirrored these changes. Furthermore, immunohistochemistry analysis revealed that most 5-hmC changes in response to chronic exercise were neuron-specific within area CA3 of the hippocampus. Together, these findings suggest that DNAme mechanisms in the rodent model of TLE are responsive to chronic aerobic exercise, with emphasis on neuronal 5-hmC DNAme in the epileptic hippocampus.

Refinement of the Barnes and Morris water maze protocols improves characterization of spatial cognitive deficits in the lithium-pilocarpine rat model of epilepsy

Temporal lobe epilepsy (TLE) often causes cognitive impairment, especially a decline in spatial memory. Reductions in spatial memory and learning are also common in rodent models of TLE. The Morris water maze and the Barnes maze are the standard methods for evaluating spatial learning and memory in rodents. However, animals with TLE may exhibit agitation, distress, and fail to follow the paradigmatic context of these tests, making the interpretation of experimental data difficult. This study optimized the procedure of the Morris water maze and the Barnes maze to evaluate spatial learning and memory in rats with the lithium-pilocarpine TLE model (LPM rats). It was demonstrated that LPM rats required a mandatory and prolonged habituation stage for both tests. Therefore, the experimental rats performed relatively well on these tests. Nevertheless, LPM rats exhibited a slower learning process compared to the control rats. LPM rats also showed a reduction in spatial memory formation. This was more pronounced in the Barnes maze. Also, LPM rats utilized a sequential strategy for searching in the Barnes maze and were incapable of developing a more efficient spatial search strategy that is common in control animals. The Barnes maze may be a better choice for assessing search strategies, learning deficits, and spatial memory in rats with TLE when choosing between the two tests. This is because of the risk of unexpected seizure occurrence during the Morris water maze tests, and the potential risks for animal welfare.

GluK2 Is a Target for Gene Therapy in Drug-Resistant Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) is characterized by recurrent seizures generated in the limbic system, particularly in the hippocampus. In TLE, recurrent mossy fiber sprouting from dentate gyrus granule cells (DGCs) crea an aberrant epileptogenic network between DGCs which operates via ectopically expressed GluK2/GluK5-containing kainate receptors (KARs). TLE patients are often resistant to anti-seizure medications and suffer significant comorbidities; hence, there is an urgent need for novel therapies. Previously, we have shown that GluK2 knockout mice are protected from seizures. This study aims at providing evidence that downregulating KARs in the hippocampus using gene therapy reduces chronic epileptic discharges in TLE. We combined molecular biology and electrophysiology in rodent models of TLE and in hippocampal slices surgically resected from patients with drug-resistant TLE. Here, we confirmed the translational potential of KAR suppression using a non-selective KAR antagonist that markedly attenuated interictal-like epileptiform discharges (IEDs) in TLE patient-derived hippocampal slices. An adeno-associated virus (AAV) serotype-9 vector expressing anti-grik2 miRNA was engineered to specifically downregulate GluK2 expression. Direct delivery of AAV9-anti grik2 miRNA into the hippocampus of TLE mice led to a marked reduction in seizure activity. Transduction of TLE patient hippocampal slices reduced levels of GluK2 protein and, most importantly, significantly reduced IEDs. Our gene silencing strategy to knock down aberrant GluK2 expression demonstrates inhibition of chronic seizure in a mouse TLE model and IEDs in cultured slices derived from TLE patients. These results provide proof-of-concept for a gene therapy approach targeting GluK2 KARs for drug-resistant TLE patients. ANN NEUROL 2023;94:745-761.

Enhanced excitability of the hippocampal CA2 region and its contribution to seizure activity in a mouse model of temporal lobe epilepsy

The hippocampal CA2 region, an area important for social memory, has been suspected to play a role in temporal lobe epilepsy (TLE) because of its resistance to degeneration observed in neighboring CA1 and CA3 regions in both humans and rodent models of TLE. However, little is known about whether alterations in CA2 properties promote seizure generation or propagation. Here, we addressed the role of CA2 using the pilocarpine-induced status epilepticus model of TLE. Exvivo electrophysiological recordings from acute hippocampal slices revealed a set of coordinated changes that enhance CA2 PC intrinsic excitability, reduce CA2 inhibitory input, and increase CA2 excitatory output to its major CA1 synaptic target. Moreover, selective chemogenetic silencing of CA2 pyramidal cells caused a significant decrease in the frequency of spontaneous seizures measured in vivo. These findings provide the first evidence that CA2 actively contributes to TLE seizure activity and may thus be a promising therapeutic target.

Bioinformatic analysis identifies key transcriptome signatures in temporal lobe epilepsy.

AimsTo identify transcriptome signatures underlying epileptogenesis in temporal lobe epilepsy (TLE).MethodsRobust rank aggregation analysis was used to integrate multiple microarrays in rodent models of TLE and determine differentially expressed genes (DEGs) in acute, latent, and chronic stages. Functional annotation and protein‐protein interaction analysis were performed to explore the potential functions of the DEGs and identify hub genes with the highest intramodular connectivity. The association between hub genes and hippocampal sclerosis/seizure frequency was analyzed using publicly available RNA‐sequencing datasets from TLE patients. We subsequently established a pilocarpine‐induced status epilepticus (SE) model in rats and validated mRNA expression of hub genes by quantitative reverse transcription PCR (qRT‐PCR).ResultsThe DEGs in the acute, latent, and chronic phases of TLE in animal models were prominently enriched in inflammatory response. Hub genes identified in the acute phase mainly participated in biological processes including inflammation, blood‐brain barrier damage, and cell adhesion. The hub genes in the latent phase were related to microglia/macrophage activation (Emr1 and Aif1) and phagocytosis (Cd68, Tyrobp, and Lyz). In the chronic phase, the hub genes were associated with activation of complements and microglia/macrophages. We further found that some hub genes identified in human TLE, such as Tlr2, Lgals3, and Stat3, were positively correlated with seizure frequency. Other hub genes, including Lgals3 and Serpine1, were associated with hippocampus sclerosis. qRT‐PCR analysis confirmed that the mRNA levels of hub genes in rat hippocampus were significantly up‐regulated after SE induction.ConclusionsOur integrated analysis identified hub genes in different stages of epilepsy. The functional annotations suggest that the activation and phagocytic activities of microglia/macrophages may play critical roles in epileptogenesis of TLE.

Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

Temporal lobe epilepsy (TLE) is a prevalent neurological disorder with many patients experiencing poor seizure control with existing anti-epileptic drugs. Thus, novel insights into the mechanisms of epileptogenesis and identification of new drug targets can be transformative. Changes in ion channel function have been shown to play a role in generating the aberrant neuronal activity observed in TLE. Previous work demonstrates that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability and are mislocalized within CA1 pyramidal cells in a rodent model of TLE. The subcellular distribution of HCN channels is regulated by an auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), and disruption of this interaction correlates with channel mislocalization. However, the molecular mechanisms responsible for HCN channel dysregulation in TLE are unclear. Here we investigated whether changes in TRIP8b phosphorylation are sufficient to alter HCN channel function. We identified a phosphorylation site at residue Ser237 of TRIP8b that enhances binding to HCN channels and influences channel gating by altering the affinity of TRIP8b for the HCN cytoplasmic domain. Using a phosphospecific antibody, we demonstrate that TRIP8b phosphorylated at Ser237 is enriched in CA1 distal dendrites and that phosphorylation is reduced in the kainic acid model of TLE. Overall, our findings indicate that the TRIP8b-HCN interaction can be modulated by changes in phosphorylation and suggest that loss of TRIP8b phosphorylation may affect HCN channel properties during epileptogenesis. These results highlight the potential of drugs targeting posttranslational modifications to restore TRIP8b phosphorylation to reduce excitability in TLE.

The validation of Short Interspersed Nuclear Elements (SINEs) as a RT-qPCR normalization strategy in a rodent model for temporal lobe epilepsy.

BackgroundIn gene expression studies via RT-qPCR many conclusions are inferred by using reference genes. However, it is generally known that also reference genes could be differentially expressed between various tissue types, experimental conditions and animal models. An increasing amount of studies have been performed to validate the stability of reference genes. In this study, two rodent-specific Short Interspersed Nuclear Elements (SINEs), which are located throughout the transcriptome, were validated and assessed against nine reference genes in a model of Temporal Lobe Epilepsy (TLE). Two different brain regions (i.e. hippocampus and cortex) and two different disease stages (i.e. acute phase and chronic phase) of the systemic kainic acid rat model for TLE were analyzed by performing expression analyses with the geNorm and NormFinder algorithms. Finally, we performed a rank aggregation analysis and validated the reference genes and the rodent-specific SINEs (i.e. B elements) individually via Gfap gene expression.ResultsGeNorm ranked Hprt1, Pgk1 and Ywhaz as the most stable genes in the acute phase, while Gusb and B2m were ranked as the most unstable, being significantly upregulated. The two B elements were ranked as most stable for both brain regions in the chronic phase by geNorm. In contrast, NormFinder ranked the B1 element only once as second best in cortical tissue for the chronic phase. Interestingly, using only one of the two algorithms would have led to skewed conclusions. Finally, the rank aggregation method indicated the use of the B1 element as the best option to normalize target genes, independent of the disease progression and brain region. This result was supported by the expression profile of Gfap.ConclusionIn this study, we demonstrate the potential of implementing SINEs -notably the B1 element- as a stable normalization factor in a rodent model of TLE, independent of brain region or disease progression.

Spike–wave discharges in adult Sprague–Dawley rats and their implications for animal models of temporal lobe epilepsy

Spike–wave discharges (SWDs) are thalamocortical oscillations that are often considered to be the EEG correlate of absence seizures. Genetic absence epilepsy rats of Strasbourg (GAERS) and Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) exhibit SWDs and are considered to be genetic animal models of absence epilepsy. However, it has been reported that other rat strains have SWDs, suggesting that SWDs may vary in their prevalence, but all rats have a predisposition for them. This is important because many of these rat strains are used to study temporal lobe epilepsy (TLE), where it is assumed that there is no seizure-like activity in controls. In the course of other studies using the Sprague–Dawley rat, a common rat strain for animal models of TLE, we found that approximately 19% of 2- to 3-month-old naive female Sprague–Dawley rats exhibited SWDs spontaneously during periods of behavioral arrest, which continued for months. Males exhibited SWDs only after 3months of age, consistent with previous reports (Buzsáki et al., 1990). Housing in atypical lighting during early life appeared to facilitate the incidence of SWDs.Spike–wave discharges were often accompanied by behaviors similar to stage 1–2 limbic seizures. Therefore, additional analyses were made to address the similarity. We observed that the frequency of SWDs was similar to that of hippocampal theta rhythm during exploration for a given animal, typically 7–8Hz. Therefore, activity in the frequency of theta rhythm that occurs during frozen behavior may not reflect seizures necessarily. Hippocampal recordings exhibited high frequency oscillations (>250Hz) during SWDs, suggesting that neuronal activity in the hippocampus occurs during SWDs, i.e., it is not a passive structure. The data also suggest that high frequency oscillations, if rhythmic, may reflect SWDs. We also confirmed that SWDs were present in a common animal model of TLE, the pilocarpine model, using female Sprague–Dawley rats. Therefore, damage and associated changes to thalamic, hippocampal, and cortical neurons do not prevent SWDs, at least in this animal model. The results suggest that it is possible that SWDs occur in rodent models of TLE and that investigators mistakenly assume that they are stage 1–2 limbic seizures. We discuss the implications of the results and ways to avoid the potential problems associated with SWDs in animal models of TLE.

Predictors of pharmacoresistant epilepsy: Pharmacoresistant rats differ from pharmacoresponsive rats in behavioral and cognitive abnormalities associated with experimentally induced epilepsy

Patients with intractable temporal lobe epilepsy (TLE) exhibit an increased risk of psychiatric comorbidity, including depression, anxiety, psychosis, and learning disorders. Furthermore, a history of psychiatric comorbidity has been suggested as a predictor of lack of response to therapy with antiepileptic drugs (AEDs) in patients with epilepsy. However, clinical studies on predictors of pharmacoresistant epilepsy are affected by several confounding variables, which may complicate conclusions. In the present study, we evaluated whether behavioral alterations in epileptic rats are different in AED nonresponders versus responders. For this purpose, we used an animal model of TLE in which AED responders and nonresponders can be selected by prolonged treatment of epileptic rats with phenobarbital (PB). Behavioral and cognitive abnormalities were compared between responders and nonresponders as well as between epileptic rats and nonepileptic controls in a battery of tests. Fifteen epileptic rats with spontaneous recurrent seizures (SRS) either responding (11 rats) or not responding (4 rats) to PB were used for this study. The nonresponders differed markedly in behavioral and cognitive abnormalities from responders and nonepileptic controls in tests of anxiety (open field, elevated-plus maze test), behavioral hyperexcitability (approach-response, touch-response, pick-up tests), and learning and memory (Morris water maze). Our hypothesis that AED-resistant rats will show more severe behavioral and cognitive changes than AED-responsive rats was confirmed by the present experiments. The data substantiate that rodent models of TLE are useful to delineate predictors of pharmacoresistant epilepsy.

Striking Differences in Individual Anticonvulsant Response to Phenobarbital in Rats with Spontaneous Seizures after Status Epilepticus

More than one third of patients with epilepsy have inadequate control of seizures with drug therapy, but mechanisms of intractability are largely unknown. Because of this large number of pharmacoresistant patients with epilepsy, the existing process of antiepileptic drug (AED) discovery and development must be reevaluated with a focus on preclinical models of therapy-resistant epilepsy syndromes such as mesial temporal lobe epilepsy (TLE). However, although various rodent models of TLE are available, the pharmacoresponsiveness of most models is not well known. In the present study, we used a post-status epilepticus model of TLE to examine whether rats with spontaneous recurrent seizures (SRSs) differ in their individual responses to phenobarbital (PB). Status epilepticus was induced in Sprague-Dawley rats by prolonged electrical stimulation of the basolateral amygdala. Once the rats had developed SRSs, seizure frequency and severity were determined by continuous EEG/video recording over a 6-week period (i.e., a predrug control period of 2 weeks, followed by PB treatment for 2 weeks, and a postdrug control period of 2 weeks). PB was administered twice daily at maximal tolerated doses. Analysis of plasma drug concentrations showed that drug concentrations within the therapeutic range (10-40 microg/ml) were maintained in all rats throughout the period of treatment. In six (55%) of 11 rats, complete control of seizures was achieved, and another rat exhibited a >90% reduction of seizure frequency. These seven rats were considered responders. The remaining four (36%) rats showed either no response at all (n=3) or only moderate reduction in seizure frequency and were therefore considered nonresponders. Plasma drug concentrations did not differ between these two groups of rats. These data demonstrate that, similar to patients with epilepsy, rats with SRSs markedly differ in their individual responses to AED treatment. Pharmacoresistant rats selected by prolonged drug treatment from groups of rats with SRSs may provide a unique model to study mechanisms of pharmacoresistance and to identify novel AEDs for treating seizures of patients currently not controlled with existing therapies.