Unilateral Intrahippocampal Injection Research Articles

Granule Cell Layer Morphology and Wnt Signaling in Temporal Lobe Epilepsy

Background and Hypothesis: Temporal lobe epilepsy (TLE) is the most common human seizure disorder and can develop after neurologic insults such as trauma or infection. No treatment exists to prevent the development of epilepsy during this critical period. Epileptogenesis is characterized by pathological neuronal network remodeling in the hippocampal dentate gyrus (DG). Previously, we found that Wnt pathway signaling is dysregulated in the kainate (KA) mouse model of TLE, such that Wnt antagonism exacerbated epileptogenic DG remodeling. We hypothesize that Wnt agonism will mitigate pathological DG remodeling of the granule cell layer (GCL) during epileptogenesis.Project Methods: TLE was induced by unilateral intrahippocampal KA injection in POMC-eGFP transgenic mice, while controls received saline. Mice received injections of vehicle or Wnt agonist Chir99021 daily. eGFP+ immature dentate granule cells were characterized by confocal microscopy. GCL width and immature dentate granule neuronal migration in the ictal/ipsilateral and peri-ictal/contralateral DG were quantified. Quantitative analyses were performed to compare means of the 4 groups.Results: We found that GCL width significantly increased in the ictal zone 2 weeks after seizure induction in both KA groups and was not mitigated by Chir treatment. Immature dentate granule cell migration also increased in the ictal zone in the KA groups and was not altered by Chir treatment. In the peri-ictal zone, GCL width and cell migration were unchanged across KA and saline control mice.Conclusion and Potential Impact: The Wnt agonist Chir99201 did not appear to alter GCL morphology in control or KA mice. It is likely that Wnt signaling may impact neuronal functioning rather than morphology in DG remodeling, and this will be explored through future electrophysiological studies. The Wnt pathway remains a potential therapeutic target in the prevention of the development of epilepsy.

Pronounced antiseizure activity of the subtype-selective GABAA positive allosteric modulator darigabat in a mouse model of drug-resistant focal epilepsy.

AimDarigabat is an α2/3/5 subunit‐selective positive allosteric modulator of GABAA receptors that has demonstrated broad‐spectrum activity in several preclinical models of epilepsy as well as in a clinical photoepilepsy trial. The objective here was to assess the acute antiseizure effect of darigabat in the mesial temporal lobe epilepsy (MTLE) mouse model of drug‐resistant focal seizures.MethodsThe MTLE model is generated by single unilateral intrahippocampal injection of low dose (1 nmole) kainic acid in adult mice, and subsequent epileptiform activity is recorded following implantation of a bipolar electrode under general anesthesia. After a period of epileptogenesis (~4 weeks), spontaneous and recurrent hippocampal paroxysmal discharges (HPD; focal seizures) are recorded using intracerebral electroencephalography. The number and cumulated duration of HPDs were recorded following administration of vehicle (PO), darigabat (0.3–10 mg kg−1, PO), and positive control diazepam (2 mg kg−1, IP).RESULTSDarigabat dose‐dependently reduced the expression of HPDs, demonstrating comparable efficacy profile to diazepam at doses of 3 and 10 mg kg−1.CONCLUSIONSDarigabat exhibited a robust efficacy profile in the MTLE model, a preclinical model of drug‐resistant focal epilepsy. A Phase II proof‐of‐concept placebo‐controlled, adjunctive‐therapy trial (NCT04244175) is ongoing to evaluate efficacy and safety of darigabat in patients with drug‐resistant focal seizures.

Granule cell dispersion in two mouse models of temporal lobe epilepsy and reeler mice is associated with changes in dendritic orientation and spine distribution.

Temporal lobe epilepsy is characterized by hippocampal neuronal death in CA1 and hilus. Dentate gyrus granule cells survive but show dispersion of the compact granule cell layer. This is associated with decrease of the glycoprotein Reelin, which regulates neuron migration and dendrite outgrow. Reelin-deficient (reeler) mice show no layering, their granule cells are dispersed throughout the dentate gyrus. We studied granule cell dendritic orientation and distribution of postsynaptic spines in reeler mice and two mouse models of temporal lobe epilepsy, namely the p35 knockout mice, which show Reelin-independent neuronal migration defects, and mice with unilateral intrahippocampal kainate injection. Granule cells were Golgi-stained and analyzed, using a computerized camera lucida system. Granule cells in naive controls exhibited a vertically oriented dendritic arbor with a small bifurcation angle if positioned proximal to the hilus and a wider dendritic bifurcation angle, if positioned distally. P35 knockout- and kainate-injected mice showed a dispersed granule cell layer, granule cells showed basal dendrites with wider bifurcation angles, which lost position-specific differences. Reeler mice lacked dendritic orientation. P35 knockout- and kainate-injected mice showed increased dendritic spine density in the granule cell layer. Molecular layer dendrites showed a reduced spine density in kainate-injected mice only, whereas in p35 knockouts no reduced spine density was seen. Reeler mice showed a homogenous high spine density. We hypothesize that granule cells migrate in temporal lobe epilepsy, develop new dendrites which show a spread of the dendritic tree, create new spines in areas proximal to mossy fiber sprouting, which is present in p35 knockout- and kainate-injected mice and loose spines on distal dendrites if mossy cell death is present, as it was in kainate-injected mice only. These results are in accordance with findings in epilepsy patients.

Seizure activity triggers tau hyperphosphorylation and amyloidogenic pathways.

Although epilepsies and neurodegenerative disorders show pathophysiological similarities, their direct functional associations are unclear. Here, we tested the hypothesis that experimental seizures can induce tau hyperphosphorylation and amyloidogenic modifications over time, with intersections with neuroinflammation. We used a model of mesial temporal lobe epilepsy (MTLE) where unilateral intrahippocampal injection of kainic acid (KA) in C57BL/6mice elicits epileptogenesis and spontaneous focal seizures. We used a model of generalized status epilepticus (SE) obtained by intraperitoneal KA injection in C57BL/6mice. We performed analyses and cross-comparisons according to a schedule of 72 h, 1 week, and 8 weeks after KA injection. In experimental MTLE, we show AT100, PHF1, and CP13 tau hyperphosphorylation during epileptogenesis (72h-1week) and long-term (8weeks) during spontaneous seizures in the ipsilateral hippocampi, the epileptogenic zone. These pathological modifications extended to the contralateral hippocampus, a seizure propagating zone with no histological lesion or sclerosis. Two kinases, Cdk5 and GSK3β, implicated in the pathological phosphorylation of tau, were activated. In this MTLE model, the induction of the amyloidogenic pathway (APP, C99, BACE1) was prominent and long-lasting in the epileptogenic zone. These Alzheimer's disease (AD)-relevant markers, established during seizure progression and recurrence, reciprocated an enduring glial (GFAP, Iba1) inflammation and the inadequate activation of the endogenous, anti-inflammatory, glucocorticoid receptor system. By contrast, a generalized SE episode provoked a predominantly transient induction of tau hyperphosphorylation and amyloidogenic markers in the hippocampus, along with resolving inflammation. Finally, we identified overlapping profiles of long-term hippocampal tau hyperphosphorylation by comparing MTLE to J20mice, the latter a model relevant to AD. MTLE and a generalized SE prompt persistent and varying tau hyperphosphorylation or amyloidogenic modifications in the hippocampus. In MTLE, an AD-relevant molecular trajectory intertwines with neuroinflammation, spatiotemporally involving epileptogenic and nonlesional seizure propagating zones.

Loss of efferent projections of the hippocampal formation in the mouse intrahippocampal kainic acid model

Unilateral intrahippocampal injection of kainic acid is used as a model of medial temporal lobe epilepsy and provides a platform to study the mechanisms of epilepsy. Here, we used an AAV-9 EYFP-tagged viral vector as an anterograde tracer, injected into the dorsal and ventral hippocampus after kainic acid injection, to map out the efferent hippocampal projections after the development of spontaneous seizures in this model. The purpose of the study was to identify the extent of changes in hippocampal efferent system in several brain regions that receive significant inputs from the hippocampus. Loss of efferent hippocampal fibers was greatest in the retrosplenial cortex where neuronal loss was also observed. Loss of fibers was also observed in the fornix without any specific effect in the lateral mammillary nuclei. Although expected, these observations provide further evidence of the broader network effects as a result of hippocampal cell loss.

Neurovascular multiparametric MRI defines epileptogenic and seizure propagation regions in experimental mesiotemporal lobe epilepsy.

Improving the identification of the epileptogenic zone and associated seizure-spreading regions represents a significant challenge. Innovative brain-imaging modalities tracking neurovascular dynamics during seizures may provide new disease biomarkers. With use of a multi-parametric magnetic resonance imaging (MRI) analysis at 9.4 Tesla, we examined, elaborated, and combined multiple cellular and cerebrovascular MRI read-outs as imaging biomarkers of the epileptogenic and seizure-propagating regions. Analyses were performed in an experimental model of mesial temporal lobe epilepsy (MTLE) generated by unilateral intra-hippocampal injection of kainic acid (KA). In the ipsilateral epileptogenic hippocampi, tissue T1 and blood-brain barrier (BBB) permeability to gadolinium were increased 48-72hours post-KA, as compared to sham and contralateral hippocampi. BBB permeability endured during spontaneous focal seizures (4-6weeks), along with a significant increase of apparent diffusion coefficient (ADC) and blood volume fraction (BVf). Simultaneously, ADC and BVf were augmented in the contralateral hippocampus, a region characterized by electroencephalographic seizure spreading, discrete histological neurovascular cell modifications, and no tissue sclerosis. We next asked whether combining all the acquired MRI parameters could deliver criteria to classify the epileptogenic from the seizure-spreading and sham hippocampi in these experimental conditions and over time. To differentiate sham from epileptogenic areas, the automatic multi-parametric classification provided a maximum accuracy of 97.5% (32 regions) 48-72hours post-KA and of 100% (60 regions) at spontaneous seizures stage. To differentiate sham, epileptogenic, and seizure-spreading areas, the accuracies of the automatic classification were 93.1% (42 regions) 48-72hours post-KA and 95% (80 regions) at spontaneous seizure stage. Combining multi-parametric MRI acquisition and machine-learning analyses delivers specific imaging identifiers to segregate the epileptogenic from the contralateral seizure-spreading hippocampi in experimental MTLE. The potential clinical value of our findings is critically discussed.

Seizure activity and brain damage in a model of focal non-convulsive status epilepticus.

Focal non-convulsive status epilepticus (FncSE) is a common emergency condition that may present as the first epileptic manifestation. In recent years, it has become increasingly clear that de novo FncSE should be promptly treated to improve post-status outcome. Whether seizure activity occurring during the course of the FncSE contributes to ensuing brain damage has not been demonstrated unequivocally and is here addressed. We used continuous video-EEG monitoring to characterise an acute experimental FncSE model induced by unilateral intrahippocampal injection of kainic acid (KA) in guinea pigs. Immunohistochemistry and mRNA expression analysis were utilised to detect and quantify brain injury, 3-days and 1-month after FncSE. Seizure activity occurring during the course of FncSE involved both hippocampi equally. Neuronal loss, blood-brain barrier permeability changes, gliosis and up-regulation of inflammation, activity-induced and astrocyte-specific genes were observed in the KA-injected hippocampus. Diazepam treatment reduced FncSE duration and KA-induced neuropathological damage. In the contralateral hippocampus, transient and possibly reversible gliosis with increase of aquaporin-4 and Kir4.1 genes were observed 3days post-KA. No tissue injury and gene expression changes were found 1-month after FncSE. In our model, focal seizures occurring during FncSE worsen ipsilateral KA-induced tissue damage. FncSE only transiently activated glia in regions remote from KA-injection, suggesting that seizure activity during FncSE without local pathogenic co-factors does not promote long-lasting detrimental changes in the brain. These findings demonstrate that in our experimental model, brain damage remains circumscribed to the area where the primary cause (KA) of the FncSE acts. Our study emphasises the need to use antiepileptic drugs to contain local damage induced by focal seizures that occur during FncSE.

Role of pERK1/2-NFκB signaling in the neuroprotective effect of thalidomide against cerebral ischemia reperfusion injury in rats

In the present investigation, we tested the hypothesis that suppression of the phospho-extracellular signal regulated kinase (pERK1/2)-nuclear factor kappa (NFκ)-B signaling, subsequent to tumor necrosis factor-α (TNF-α) inhibition, underlies thalidomide (TLM) mediated neuroprotection. Male Wistar rats (250–280 g) were divided into five groups: (1) sham; (2) negative control receiving TLM (5μg/1μl/site) and 3 groups of ischemia-reperfusion (IR) injury rats pretreated with: (3) vehicle (DMSO 100%); (4) TLM (5μg/1μl/site) or (5) PD98059 (0.16μg/1μl/site). IR rats were subjected to occlusion of both common carotid arteries for 45 min followed by reperfusion for 24 h. Drugs and/or vehicles were administered by unilateral intrahippocampal injection after removal of the carotid occlusion and at the beginning of the reperfusion period. IR rats exhibited significant infarct size, histopathological damage, memory impairment, motor incoordination and hyperactivity. Unilateral intra-hippocampal TLM ameliorated these behavioral deficits along with the following ex vivo hippocampal effects: (i) abrogation of the IR-evoked elevations in hippocampal TNF-α, pERK1/2, NFκB, BDNF, iNOS contents and (ii) partial restoration of the reduced anti-inflammatory cytokine IL-10 and p-nNOS S852. These neurochemical effects, which were replicated by the pERK1/2 inhibitor PD98059, likely underlie the reductions in c-Fos and caspase-3 levels as well as the anti-apoptotic effect of TLM in the IR model. These results suggest a crucial anti-inflammatory role for pERK1/2 inhibition in the salutary neuronal and behavioral effects of TLM in a model of brain IR injury.

Wogonin preventive impact on hippocampal neurodegeneration, inflammation and cognitive defects in temporal lobe epilepsy.

Previous studies demonstrated that the pathophysiological changes after temporal lobe epilepsy (TLE) such as oxidative stress, inflammatory reaction contribute to cognitive defect and neuronal damage. The present study was conducted to evaluate the anticonvulsant effect of wogonin ameliorates kainate-induced TLE, and to investigate the mechanism underlying these effects. Rats were divided into control, wogonin, kainate, and wogonin-pretreated kainate groups. The rat model of TLE was induced by unilateral intrahippocampal injection of 0.4 ug/ul of kainate. The results showed that the cognitive function in TLE rats was significantly impaired, and wogonin treatment improved cognitive function in the Morris water maze (MWM). H & E staining and TUNEL staining showed obvious damage in the hippocampus of TLE rats, and wogonin alleviated the damage. To evaluate the oxidative stress, the expression of MDA and GSH in plasma were detected. Nrf-2 and HO-1 mRNA expression in the hippocampus were detected. The levels of MDA in plasma increased in TLE rats, and the levels of GSH in plasma and Nrf-2, HO-1 in the brain decreased. Treatment with wogonin alleviated these changes. We also detected the mRNA expression of inflammatory mediators like IL-1β, TNF-α, and NF kB in the brain. The inflammatory reaction was significantly activated in the brain of TLE rats, and wogonin alleviated neuroinflammation. We detected the mRNA expression of Bcl-2, Bax, caspase-3, in the hippocampus. The levels of Bcl-2 decreased in TLE rats, Bax and caspase-3 increased, while wogonin alleviated these changes. The present study indicated that wogonin exerted a noticeable neuroprotective effect in kainate-induced TLE rats.

A positive allosteric modulator of mGluR5 promotes neuroprotective effects in mouse models of Alzheimer's disease

Alzheimer's Disease (AD) is the most prevalent neurodegenerative disorder. Despite advances in the understanding of its pathophysiology, none of the available therapies prevents disease progression. Excess glutamate plays an important role in excitotoxicity by activating ionotropic receptors. However, the mechanisms modulating neuronal cell survival/death via metabotropic glutamate receptors (mGluRs) are not completely understood. Recent data indicates that CDPPB, a positive allosteric modulator of mGluR5, has neuroprotective effects. Thus, this work aimed to investigate CDPPB treatment effects on amyloid-β (Aβ) induced pathological alterations in vitro and in vivo and in a transgenic mouse model of AD (T41 mice). Aβ induced cell death in primary cultures of hippocampal neurons, which was prevented by CDPPB. Male C57BL/6 mice underwent stereotaxic surgery for unilateral intra-hippocampal Aβ injection, which induced memory deficits, neurodegeneration, neuronal viability reduction and decrease of doublecortin-positive cells, a marker of immature neurons and neuronal proliferation. Treatment with CDPPB for 8 days reversed neurodegeneration and doublecortin-positive cells loss and recovered memory function. Fourteen months old T41 mice presented cognitive deficits, neuronal viability reduction, gliosis and Aβ accumulation. Treatment with CDPPB for 28 days increased neuronal viability (32.2% increase in NeuN+ cells) and reduced gliosis in CA1 region (Iba-1+ area by 31.3% and GFAP+ area by 37.5%) in transgenic animals, without inducing hepatotoxicity. However, it did not reverse cognitive deficit. Despite a four-week treatment did not prevent memory loss in aged transgenic mice, CDPPB is protective against Aβ stimulus. Therefore, this drug represents a potential candidate for further investigations as AD treatment.

A pericyte-glia scarring develops at the leaky capillaries in the hippocampus during seizure activity.

Inflammatory cerebrovascular damage occurs in epilepsy. Here, we tested the hypothesis that a pericyte-glia scar forms around the outer wall of hippocampal capillaries in a model of temporal lobe epilepsy associated with hippocampal sclerosis. We studied the participation of stromal cells expressing platelet-derived growth factor receptor beta (PDGFRβ) and extracellular matrixmodifications tothe perivascularscar during epileptogenesis. We used NG2DsRed/C57BL6 mice and induced status epilepticus (SE) followed by epileptogenesis and spontaneous recurrent seizures (SRS) by means of unilateral intrahippocampal injection of kainic acid (KA). For pharmacological assessment, we used organotypic hippocampal cultures (OHCs) where ictal electrographic activity was elicited by KA or bicuculline. NG2DsRed pericytes, GFAP astroglia, and IBA1 microglia are reactive and converge to form a pericapillary multicellular scar in the CA hippocampal regions during epileptogenesis and at SRS. The capillaries are leaky as indicated by fluorescein entering the parenchyma from the peripheral blood. Concomitantly, PDGFRβ transcript and protein levels were significantly increased. Within the regional scar, a fibrotic-like PDGFRβ mesh developed around the capillaries, peaking at 1week post-SE and regressing, but not resolving, at SRS. Abnormal distribution or accumulation of extracellular matrix collagens III/IV occurred in the CA regions during seizure progression. PDGFRβ/DAPI cells were in direct contact with or adjacent to the damaged NG2DsRed pericytes at the capillaryinterface, consistent with the notion of stromal cell reactivity or fibroblast formation. Inducing electrographic activity in OHCs was sufficient to augment PDGFRβ reactivity around the capillaries. The latter effect was pharmacologically mimicked by treating OHCs with the PDGFRβ agonist PDGF-BB and it was diminished by the PDGFRβ inhibitor imatinib. The reported multicellular activation and scar are traits of perivascularinflammation and hippocampal sclerosis in experimental epilepsy, with an implication for neurovascular dysfunction. Modulation of PDGFRβ could be exploited to target inflammation in thischronic disease setting.

Expression of brain‐derived neurotrophic factor and structural plasticity in the dentate gyrus and CA2 region correlate with epileptiform activity

Hippocampal sclerosis is a hallmark of mesial temporal lobe epilepsy (MTLE), comprising gliosis and neuronal loss in the hippocampus. However, dentate granule cells and CA2 pyramidal cells (PCs) survive, as they share physiological characteristics that may render them less sensitive to hyperexcitation in MTLE. Here, we asked whether both engage similar molecular plasticity mechanisms to support their resilience in MTLE. We chose brain-derived neurotrophic factor (BDNF), correlated the expression with activity, and used neuropeptide Y (NPY) and principal cell dispersion as plasticity readout. Adult male mice received a unilateral intrahippocampal kainate injection to induce status epilepticus (SE) and bilateral electrodes into the dentate gyrus and CA2 for in vivo recordings and quantification of epileptiform activity. To assess the time course of Bdnf mRNA expression in these regions, we performed fluorescence in situ hybridization, complemented by immunohistochemistry for NPY and quantification of principal cell dispersion. We show that Bdnf expression was transiently up-regulated during SE in the granule cell layer (GCL) and CA2 and, after a slight reduction at 2 days, increased persistently in both regions ipsilaterally. Intrahippocampal recordings revealed a threshold for the duration of SE to induce these changes. Recurrent epileptiform activity developed in the ipsilateral dentate gyrus and CA2 over time and was correlated with Bdnf mRNA levels, although more pronounced in the dentate gyrus. The dispersion of the GCL and CA2 correlated with Bdnf mRNA expression. NPY protein expression was only increased in granule cells and mossy fibers, remaining unchanged in CA2. Our study reveals differential molecular plasticity changes in granule cells and CA2 PCs despite many similarities (epileptiform activity, somatic mossy fiber input, dispersion). These findings contribute to the understanding of common as well as individual characteristics of the cell populations underlying the epileptic hippocampal network.

Contribution of early Alzheimer's disease-related pathophysiology to the development of acquired epilepsy.

Aberrant epileptic activity is detectable at early disease stages in Alzheimer's disease (AD) patients and in AD mouse models. Here, we investigated in young ArcticAβ mice whether AD-like pathology renders neuronal networks more susceptible to the development of acquired epilepsy induced by unilateral intrahippocampal injection of kainic acid (IHK). In this temporal lobe epilepsy model, IHK induces a status epilepticus followed after two weeks by spontaneous recurrent seizures (SRS). ArcticAβ mice exhibited more severe status epilepticus and early onset of SRS. This hyperexcitable phenotype was characterized in CA1 neurons by decreased synaptic strength, increased kainic acid-induced LTP and reduced frequency of spontaneous inhibitory currents. However, no difference in neurodegeneration, neuroinflammation, axonal reorganization or adult neurogenesis was observed in ArcticAβ mice compared to wild-type littermates following IHK-induced epileptogenesis. Neuropeptide Y (NPY) expression was reduced at baseline and its IHK-induced elevation in mossy fibres and granule cells was attenuated. However, although this alteration might underlie premature seizure onset, neutralization of soluble Aβ species by intracerebroventricular Aβ-specific antibody application mitigated the hyperexcitable phenotype of ArcticAβ mice and prevented early SRS onset. Therefore, the development of seizures at early stages of AD is mediated primarily by Aβ species causing widespread changes in synaptic function.

Longitudinal imaging of Microglia-Astrocyte activation in mouse mesial temporal lobe epilepsy with TSPO PET to identify the best therapeutic time windows

Objectives Mesiotemporal lobe epilepsy (MTLE) is the most common type of drug-resistant partial epilepsy with a specific history which begins by a status epilepticus due to a neurological insult followed by a silent period before the recurrent seizures begin. Once established, these seizures can become resistant to medications. Along this period a specific lesion is often found, described as a unilateral hippocampal sclerosis (HS). Currently it is still challenging to determine when and which drugs can be most effective along the constitution of this epileptic process. Neuroinflammation has been reported to play an important role in pathophysiological mechanisms in epilepsy, and therefore brain inflammation probes can be potent epilepsy biomarkers. The Translocator Protein 18 kDa (TSPO) expression is associated with neuroinflammatory processes involving reactive astrocytes and microglia. A unilateral intrahippocampal kainate injection mouse model can reproduce the defining features of human temporal lobe epilepsy with unilateral hippocampal sclerosis and the pattern of chronic pharmacoresistant temporal seizures. We hypothesized that TSPO PET imaging using 18F-DPA-714 TSPO-targeted PET could be a highly potent longitudinal biomarker of epilepsy and could be of interest to determine the therapeutic windows in epilepsy and follow response to treatment in the mouse model of MTLE. Methods The model was induced by unilateral injection of kainic acid into the right dorsal hippocampus of male C57/Bl6 mice. μPET/CT scanning was performed before model induction and along the development of the HS at 7 days, 14 days, 1 month and 6 months. In vitro autoradiography and immunohistofluorescence were performed on additional mice at each time point. Results TSPO PET uptake reached the peak at 7 days and mostly correlated with microglial activation whereas after 14 days, reactive astrocytes were shown to be the main cells expressing TSPO which would be related to the increased PET uptake. Our studies suggest that mTOR signal pathways play an important role in the modulation of astrocyte activities. Our hypothesis is that the neurotoxicity during ictogenesis (aggravation phase) could be due to persistent mTOR reactive signaling in astrocytes. Conclusion : on this knowledge of TSPO expression over time in the MTLE model, TSPO PET imaging could be a valuable tool to evaluate efficacy of mTOR treatments.

Longitudinal positron emission tomography imaging of glial cell activation in a mouse model of mesial temporal lobe epilepsy: Toward identification of optimal treatment windows.

Mesiotemporal lobe epilepsy is the most common type of drug-resistant partial epilepsy, with a specific history that often begins with status epilepticus due to various neurological insults followed by a silent period. During this period, before the first seizure occurs, a specific lesion develops, described as unilateral hippocampal sclerosis (HS). It is still challenging to determine which drugs, administered at which time point, will be most effective during the formation of this epileptic process. Neuroinflammation plays an important role in pathophysiological mechanisms in epilepsy, and therefore brain inflammation biomarkers such as translocator protein 18 kDa (TSPO) can be potent epilepsy biomarkers. TSPO is associated with reactive astrocytes and microglia. A unilateral intrahippocampal kainate injection mouse model can reproduce the defining features of human temporal lobe epilepsy with unilateral HS and the pattern of chronic pharmacoresistant temporal seizures. We hypothesized that longitudinal imaging using TSPO positron emission tomography (PET) with 18 F-DPA-714 could identify optimal treatment windows in a mouse model during the formation of HS. The model was induced into the right dorsal hippocampus of male C57/Bl6 mice. Micro-PET/computed tomographic scanning was performed before model induction and along the development of the HS at 7 days, 14 days, 1 month, and 6 months. In vitro autoradiography and immunohistofluorescence were performed on additional mice at each time point. TSPO PET uptake reached peak at 7 days and mostly related to microglial activation, whereas after 14 days, reactive astrocytes were shown to be the main cells expressing TSPO, reflected by a continuing increased PET uptake. TSPO-targeted PET is a highly potent longitudinal biomarker of epilepsy and could be of interest to determine the therapeutic windows in epilepsy and to monitor response to treatment.

Faster flux of neurotransmitter glutamate during seizure - Evidence from 13C-enrichment of extracellular glutamate in kainate rat model.

The objective is to examine how the flux of neurotransmitter glutamate from neurons to the extracellular fluid, as measured by the rate of 13C enrichment of extracellular glutamate (GLUECF), changes in response to seizures in the kainate-induced rat model of temporal-lobe epilepsy. Following unilateral intrahippocampal injection of kainate, GLUECF was collected by microdialysis from the CA1/CA3 region of awake rats, in combination with EEG recording of chronic-phase recurrent seizures and intravenous infusion of [2,5-13C]glucose. The 13C enrichment of GLUECF C5 at ~ 10 picomol level was measured by gas-chromatography mass-spectrometry. The rate of 13C enrichment, expressed as the increase of the fractional enrichment/min, was 0.0029 ± 0.0001/min in frequently seizing rats (n = 4); this was significantly higher (p < 0.01) than in the control (0.00167 ± 0.0001/min; n = 6) or in rats with infrequent seizures (0.00172 ± 0.0001/min; n = 6). This result strongly suggests that the flux of the excitatory neurotransmitter from neurons to the extracellular fluid is significantly increased by frequent seizures. The extracellular [12C + 13C]glutamate concentration increased progressively in frequently seizing rats. Taken together, these results strongly suggest that the observed seizure-induced high flux of glutamate overstimulated glutamate receptors, which triggered a chain reaction of excitation in the CA3 recurrent glutamatergic networks. The rate of 13C enrichment of extracellular glutamine (GLNECF) at C5 was 0.00299 ± 0.00027/min in frequently seizing rats, which was higher (p < 0.05) than in controls (0.00227 ± 0.00008/min). For the first time in vivo, this study examined the effects of epileptic seizures on fluxes of the neurotransmitter glutamate and its precursor glutamine in the extracellular fluid of the hippocampus. The advantages, limitations and the potential for improvement of this approach for pre-clinical and clinical studies of temporal-lobe epilepsy are discussed.

The Ying and Yang of Adenosine A1 and A2A Receptors on ERK1/2 Activation in a Rat Model of Global Cerebral Ischemia Reperfusion Injury.

Adenosine impacts cerebral ischemia reperfusion (IR) through the inhibitory A1 and the excitatory A2 receptors. The present study aimed at investigating the contrasting role of pERK1/2 inmediating adenosine A1R (protective) versus A2AR (deleterious)effects in IR. Male Wistar rats subjected to bilateral carotid occlusion (45min) followed by reperfusion (24h) exhibited increased pERK1/2 activity, downstream from DAG pathway, along with increases in hippocampal glutamate, c-Fos, NF-κB, TNF-α, iNOS, TBARS, cytochrome c, caspase-3, BDNF, Nrf2, and IL-10 contents. Further, hippocampal microglial reactivity, glial TNF-α, and BDNF expression were observed. Although unilateral intrahippocampal injection of either the A1R agonist CHA or the A2AR agonist CGS21680 increased pERK1/2, only CHA mitigated histopathological and behavioral deficits along with reducing glutamate, microglial activation, c-Fos, TNF-α, iNOS, TBARS, cytochrome c and caspase-3 and elevating Nrf2 and IL-10 levels in IR rats. These results yield insight into the double-faceted nature of pERK1/2 in mediating protective and deleterious effects of A1R and A2AR signaling, respectively, against IR injury.

Differential Effects of Antiepileptic Drugs on Focal Seizures in the Intrahippocampal Kainate Mouse Model of Mesial Temporal Lobe Epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common form of drug-refractory epilepsy. Most of the morphological and electrophysiological features of human MTLE can be reproduced in a mouse by a unilateral intrahippocampal injection of kainate (MTLE mouse model). The effects of antiepileptic drugs (AEDs) on the occurrence of recurrent focal hippocampal seizures in this model remain to be specified. Here, we addressed the pharmacological reactivity of this model to the most commonly used AEDs. Using depth electroencephalographical (EEG) recordings, we tested the dose-response effects of acute injection of nine AEDs on the occurrence of hippocampal paroxysmal discharges (HPDs) as well as on ictal and interictal power spectra in the MTLE mouse model. Valproate, carbamazepine, and lamotrigine dose dependently suppressed HPDs and modified the general behavior and/or EEG activity. Levetiracetam and pregabalin suppressed HPDs at high doses but without any behavioral nor interictal EEG changes. Finally, phenobarbital, tiagabine, vigabatrin, and diazepam suppressed HPDs in a dose-dependent manner at doses devoid of obvious behavioral effects. The MTLE mouse model displays a differential sensitivity to AEDs with a greater efficacy of drug that facilitates GABAergic transmission. This model provides an efficient tool to identify new treatment for drug-resistant forms of focal epilepsies.

Rapid changes in expression of class I and IV histone deacetylases during epileptogenesis in mouse models of temporal lobe epilepsy

A prominent role of epigenetic mechanisms in manifestation of epilepsy has been proposed. Thus altered histone H3 and H4 acetylation has been demonstrated in experimental models of temporal lobe epilepsy (TLE). We now investigated changes in the expression of the class I and class IV histone deacetylases (HDAC) in two complementary mouse TLE models. Unilateral intrahippocampal injection of kainic acid (KA) induced a status epilepticus lasting 6 to 24h, development of spontaneous limbic seizures (2 to 3days after KA injection) and chronic epilepsy, as revealed by telemetric recordings of the EEGs. Mice were killed at different intervals after KA injection and expression of HDAC mRNAs was investigated by in situ hybridization. We observed marked decreases in the expression of HDACs 1, 2 and 11 (by up to 75%) in the granule cell and pyramidal cell layers of the hippocampus during the acute status epilepticus (2 to 6h after KA injection). This was followed by increased expression of all class I HDAC mRNAs in all principal cell layers of the hippocampus after 12 to 48h. In the chronic phase, 14 and 28days after KA, only modest increases in the expression of HDAC1 mRNA were observed in granule and pyramidal cells. Immunohistochemistry using an antibody detecting HDAC2 revealed results consistent with the mRNA data and indicates also expression in glial cells on the injection side. Similar changes as seen in the KA model were observed after a pilocarpine-induced status epilepticus except that decreases in HDACs 2, 3 and 8 were also seen at the chronic 28day interval.The prominent decreases in HDAC expression during status epilepticus are consistent with the previously demonstrated increased expression of numerous proteins and with the augmented acetylation of histone H4. It is suggested that respective putative gene products could facilitate proconvulsive as well as anticonvulsive mechanisms. The increased expression of all class I HDACs during the “silent phase”, on the other hand, may be related to decreased histone acetylation, which could cause a decrease in expression of certain proteins, a mechanism that could also promote epileptogenesis. Thus, addressing HDAC expression may have a therapeutic potential in interfering with a status epilepticus and with the manifestation of TLE.

The Yin and Yan of Adenosine A1 and A2A Receptors in Global Cerebral Ischemia Reperfusion Injury

Global cerebral ischemia reperfusion (IR) following cardiac arrest, shock and asphyxia can affect wide brain areas where new therapies are imperative. Moreover, many intracellular pathways mediating tissue response to IR are still elusive. Adenosine is a neuromodulator that greatly impacts IR through the inhibitory A1 and excitatory A2 receptors. Studies indicate that both adenosine A1 agonists and A2A antagonists are neuroprotective during ischemic insults. However, many of the signaling pathways affecting such responses are largely unknown. In the current study, bilateral carotid occlusion for 45 min. followed by 24h reperfusion period resulted in histopathological damage as well as memory impairment, motor incoordination and increased locomotor activity. Such events were attenuated by unilateral intra‐hippocampal injection of both the selective A1 agonist N6‐cyclohexyl adenosine (CHA) and the A2A antagonist 7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐triazolo[1,5‐c]pyrimidine (SCH58261). On the cellular level, IR increased glutamate, pERK1/2, c‐Fos, NF‐kB, TNF‐α, cytochrome c, caspase‐3, BDNF, Nrf2 and IL‐10 contents. Both CHA and SCH58261signifiantly reduced glutamate, c‐Fos, TNF‐α, cytochrome c, caspase‐3 and increased Nrf‐2 and IL‐10 contents. While only SCH58261 reduced pERK1/2, NF‐kB and BDNF contents. By the same token, iNOS and TBARS levels were increased following IR and attenuated by both CHA and SCH58261 treatments. Accordingly, these results lend insight to the neuroprotective effects of A1 and A2A agonists and antagonists respectively where pERK1/2 and NF‐kB pathways might be involved in adenosine mediated attenuation of inflammation, apoptosis and oxidative stress.