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Although granule cell dispersion (GCD) in the hippocampus is known to be an important feature associated with epileptic seizures in temporal lobe epilepsy (TLE), the endogenous molecules that regulate GCD are largely unknown. In the present study, we have examined whether there is any change in AEG-1 expression in the hippocampus of a kainic acid (KA)-induced mouse model of TLE. In addition, we have investigated whether the modulation of astrocyte elevated gene-1 (AEG-1) expression in the dentate gyrus (DG) by intracranial injection of adeno-associated virus 1 (AAV1) influences pathological phenotypes such as GCD formation and seizure susceptibility in a KA-treated mouse. We have identified that the protein expression of AEG-1 is upregulated in the DG of a KA-induced mouse model of TLE. We further demonstrated that AEG-1 upregulation by AAV1 delivery in the DG-induced anticonvulsant activities such as the delay of seizure onset and inhibition of spontaneous recurrent seizures (SRS) through GCD suppression in the mouse model of TLE, while the inhibition of AEG-1 expression increased susceptibility to seizures. The present observations suggest that AEG-1 is a potent regulator of GCD formation and seizure development associated with TLE, and the significant induction of AEG-1 in the DG may have therapeutic potential against epilepsy. [ABSTRACT FROM AUTHOR]

Activity induced transcription factor ΔFosB plays a key role in different CNS disorders including epilepsy, Alzheimer's disease, and addiction. Recent findings suggest that ΔFosB drives cognitive deficits in epilepsy and together with the emergence of small molecule inhibitors of ΔFosB activity makes it an interesting therapeutic target. However, whether ΔFosB contributes to pathophysiology or provides protection in drug-resistant epilepsy is still unclear. In this study, ΔFosB was specifically downregulated by delivering AAV-shRNA into the hippocampus of chronically epileptic mice using the drug-resistant pilocarpine model of mesial temporal epilepsy (mTLE). Immunohistochemistry analyses showed that prolonged downregulation of ΔFosB led to exacerbation of neuroinflammatory markers of astrogliosis and microgliosis, loss of mossy fibers, and hippocampal granule cell dispersion. Furthermore, prolonged inhibition of ΔFosB using a 1JunD construct to block ΔFosB signaling in a mouse model of Alzheimer's disease, that exhibits spontaneous recurrent seizures, led to similar findings, with increased neuroinflammation and decreased NPY expression in mossy fibers. Together, these data suggest that seizure-induced ΔFosB, regardless of seizureetiology, is part of a homeostatic mechanism that protects the epileptic brain from further deterioration. [ABSTRACT FROM AUTHOR]

Activity induced transcription factor ΔFosB plays a key role in different CNS disorders including epilepsy, Alzheimer’s disease, and addiction. Recent findings suggest that ΔFosB drives cognitive deficits in epilepsy and together with the emergence of small molecule inhibitors of ΔFosB activity makes it an interesting therapeutic target. However, whether ΔFosB contributes to pathophysiology or provides protection in drug-resistant epilepsy is still unclear. In this study, ΔFosB was specifically downregulated by delivering AAV-shRNA into the hippocampus of chronically epileptic mice using the drug-resistant pilocarpine model of mesial temporal epilepsy (mTLE). Immunohistochemistry analyses showed that prolonged downregulation of ΔFosB led to exacerbation of neuroinflammatory markers of astrogliosis and microgliosis, loss of mossy fibers, and hippocampal granule cell dispersion. Furthermore, prolonged inhibition of ΔFosB using a ΔJunD construct to block ΔFosB signaling in a mouse model of Alzheimer’s disease, that exhibits spontaneous recurrent seizures, led to similar findings, with increased neuroinflammation and decreased NPY expression in mossy fibers. Together, these data suggest that seizure-induced ΔFosB, regardless of seizure-etiology, is part of a homeostatic mechanism that protects the epileptic brain from further deterioration.

Although granule cell dispersion (GCD) in the hippocampus is known to be an important feature associated with epileptic seizures in temporal lobe epilepsy (TLE), the endogenous molecules that regulate GCD are largely unknown. In the present study, we have examined whether there is any change in AEG-1 expression in the hippocampus of a kainic acid (KA)-induced mouse model of TLE. In addition, we have investigated whether the modulation of astrocyte elevated gene-1 (AEG-1) expression in the dentate gyrus (DG) by intracranial injection of adeno-associated virus 1 (AAV1) influences pathological phenotypes such as GCD formation and seizure susceptibility in a KA-treated mouse. We have identified that the protein expression of AEG-1 is upregulated in the DG of a KA-induced mouse model of TLE. We further demonstrated that AEG-1 upregulation by AAV1 delivery in the DG-induced anticonvulsant activities such as the delay of seizure onset and inhibition of spontaneous recurrent seizures (SRS) through GCD suppression in the mouse model of TLE, while the inhibition of AEG-1 expression increased susceptibility to seizures. The present observations suggest that AEG-1 is a potent regulator of GCD formation and seizure development associated with TLE, and the significant induction of AEG-1 in the DG may have therapeutic potential against epilepsy.

This study evaluated the expression of reelin, an extracellular protein involved in lamination and migration of neurons, in the hippocampus of young piglets, and quantified to examine the following: (i) baseline levels within layers of the hippocampus and dentate gyrus (DG); (ii) differences between ventral and dorsal hippocampi; and (iii) changes attributable to postnatal exposure to continuous nicotine for 12 days, or intermittent hypercapnic hypoxia (IHH), with further analysis according to duration of IHH (1 vs 4 days). Additionally, we analysed whether any exposure altered DG morphology and whether it is related to altered reelin expression. Reelin was visualised via immunohistochemistry, and the number of positive reelin cells/mm2 was measured in the CA4/Hilus, layers of the DG, and the CA1. The dorsal DG had significantly more reelin within the subgranular zone compared to the ventral DG (p < 0.01). There was no difference in reelin between nicotine (n = 5) and controls (n = 5). IHH exposed piglets (n = 10) had significantly lowered reelin in the CA1 (p = 0.05), specifically the stratum pyramidale (p = 0.04) and the hippocampal fissure (p = 0.02), compared to their controls (n = 7); the duration of IHH had no effect. No exposure was associated with an alteration in DG morphology. This study shows that postnatal IHH exposure decreased reelin expression in the developing piglet hippocampal CA1, suggesting that IHH may result in altered neuronal migration. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) of the nervous system can be considered as a dynamically adaptable compartment between neuronal cells, in particular neurons and glial cells, that participates in physiological functions of the nervous system. It is mainly composed of carbohydrates and proteins that are secreted by the different kinds of cell types found in the nervous system, in particular neurons and glial cells, but also other cell types, such as pericytes of capillaries, ependymocytes and meningeal cells. ECM molecules participate in developmental processes, synaptic plasticity, neurodegeneration and regenerative processes. As an example, the ECM of the hippocampal formation is involved in degenerative and adaptive processes related to epilepsy. The role of various components of the ECM has been explored extensively. In particular, the ECM protein reelin, well known for orchestrating the formation of neuronal layer formation in the cerebral cortex, is also considered as a player involved in the occurrence of postnatal granule cell dispersion (GCD), a morphologically peculiar feature frequently observed in hippocampal tissue from epileptic patients. Possible causes and consequences of GCD have been studied in various in vivo and in vitro models. The present review discusses different interpretations of GCD and different views on the role of ECM protein reelin in the formation of this morphological peculiarity.

Abstract Chronic epilepsy has been associated with hippocampal abnormalities like neuronal loss, gliosis and granule cell dispersion. The granule cell layer of a normal human hippocampal dentate gyrus is traditionally regarded as a compact neuron-dense layer. Histopathological studies of surgically resected or autopsied hippocampal samples primarily from temporal lobe epilepsy patients, as well as animal models of epilepsy, describe variable patterns of granule cell dispersion including focal cell clusters, broader thick segments, and bilamination or “tram-tracking”. Although most studies have implicated granule cell dispersion as a specific feature of chronic epilepsy, very few “non-seizure” controls were included in these published investigations. Our retrospective survey of 147 cadaveric pediatric human hippocampi identified identical morphological spectra of granule cell dispersion in both normal and seizure-affected brains. Moreover, sections across the entire antero-posterior axis of a control cadaveric hippocampus revealed repetitive occurrence of different morphologies of the granule cell layer – compact, focally disaggregated and bilaminar. The results indicate that granule cell dispersion is within the spectrum of normal variation and not unique to patients with epilepsy. We speculate that sampling bias has been responsible for an erroneous dogma, which we hope to rectify with this investigation.

One characteristic feature of mesial temporal lobe epilepsy is granule cell dispersion (GCD), a pathological widening of the granule cell layer in the dentate gyrus. The loss of the extracellular matrix protein Reelin, an important positional cue for neurons, correlates with GCD formation in MTLE patients and in rodent epilepsy models. Here, we used organotypic hippocampal slice cultures (OHSC) from transgenic mice expressing enhanced green fluorescent protein (eGFP) in differentiated granule cells (GCs) to monitor GCD formation dynamically by live cell video microscopy and to investigate the role of Reelin in this process. We present evidence that following treatment with the glutamate receptor agonist kainate (KA), eGFP-positive GCs migrated mainly toward the hilar region. In the hilus, Reelin-producing neurons were rapidly lost following KA treatment as shown in a detailed time series. Addition of recombinant Reelin fragments to the medium effectively prevented the KA-triggered movement of eGFP-positive GCs. Placement of Reelin-coated beads into the hilus of KA-treated cultures stopped the migration of GCs in a distance-dependent manner. In addition, quantitative Western blot analysis revealed that KA treatment affects the Reelin signal transduction pathway by increasing intracellular adaptor protein Disabled-1 synthesis and reducing the phosphorylation of cofilin, a downstream target of the Reelin pathway. Both events were normalized by addition of recombinant Reelin fragments. Finally, following neutralization of Reelin in healthy OHSC by incubation with the function-blocking CR-50 Reelin antibody, GCs started to migrate without any direction preference. Together, our findings demonstrate that normotopic position of Reelin is essential for the maintenance of GC lamination in the dentate gyrus and that GCD is the result of a local Reelin deficiency.

One characteristic feature of mesial temporal lobe epilepsy is granule cell dispersion (GCD), a pathological widening of the granule cell layer in the dentate gyrus. The loss of the extracellular matrix protein Reelin, an important positional cue for neurons, correlates with GCD formation in MTLE patients and in rodent epilepsy models. Here, we used organotypic hippocampal slice cultures (OHSC) from transgenic mice expressing enhanced green fluorescent protein (eGFP) in differentiated granule cells (GCs) to monitor GCD formation dynamically by live cell video microscopy and to investigate the role of Reelin in this process. We present evidence that following treatment with the glutamate receptor agonist kainate (KA), eGFP-positive GCs migrated mainly toward the hilar region. In the hilus, Reelin-producing neurons were rapidly lost following KA treatment as shown in a detailed time series. Addition of recombinant Reelin fragments to the medium effectively prevented the KA-triggered movement of eGFP-positive GCs. Placement of Reelin-coated beads into the hilus of KA-treated cultures stopped the migration of GCs in a distance-dependent manner. In addition, quantitative Western blot analysis revealed that KA treatment affects the Reelin signal transduction pathway by increasing intracellular adaptor protein Disabled-1 synthesis and reducing the phosphorylation of cofilin, a downstream target of the Reelin pathway. Both events were normalized by addition of recombinant Reelin fragments. Finally, following neutralization of Reelin in healthy OHSC by incubation with the function-blocking CR-50 Reelin antibody, GCs started to migrate without any direction preference. Together, our findings demonstrate that normotopic position of Reelin is essential for the maintenance of GC lamination in the dentate gyrus and that GCD is the result of a local Reelin deficiency. [ABSTRACT FROM AUTHOR]

Purpose: To characterize a 10-year series of patients with mesial temporal lobe epilepsy (MTLE) and unilateral hippocampal sclerosis (HS) and determine the histopathological characteristic of the association between granule cell dispersion (GCD) and hippocampal neuronal loss.Methods: The study included 108 MTLE/HS patients. Histopathological analyses were performed in NeuN-stained hippocampal sections for HS pattern, neuronal density, dentate gyrus (DG) pathology, and granule cell layer width. Statistical tests investigated the association between DG pathologies and HS patterns, as well as the correlation of DG width with total hippocampal and subfield-specific neuronal densities.Results: Fifty-six patients (51.9%) presented right HS. All the four ILAE HS patterns were represented (90 Type 1, 11 Type 2, 2 Type 3, and 5 no-HS). Sixty-seven patients (62.0%) presented GCD, 39 (36.1%) normal DG, and 2 (1.9%) narrow DG. GCD was associated with initial precipitating injury, higher numbers of monthly focal seizures and lifetime bilateral tonic-clonic seizures, longer epilepsy duration, and older age at surgery. GCD was prevalent in all HS patterns, except for Type 2 (81.8% normal versus 18.2% GCD, p = 0.005). GCD was associated with total hippocampal and subfield-specific neuronal loss, except for CA1. DG width correlated with total hippocampal (r = -0.201, p = 0.037) and CA4 neuronal densities (r = -0.299, p = 0.002). Patients with HS Type 1 had better surgical outcomes, with 51 (61.4%) seizure-free in the first year post-surgery.Conclusions: This study confirmed that seizure control in MTLE/HS patients submitted to surgical treatment is comparable worldwide. Moreover, histopathological analyses showed an association between GCD and hippocampal neuronal loss, especially in the CA4 subfield. [ABSTRACT FROM AUTHOR]

Hippocampal sclerosis (HS) in Temporal Lobe Epilepsy (TLE) shows neuronal death in cornu ammonis (CA)1, CA3, and CA4. It is known that granule cells and CA2 neurons survive and their axons, the mossy fibers (MF), lose their target cells in CA3 and CA4 and sprout to the granule cell layer and molecular layer. We examined in TLE patients and in a mouse epilepsy model, whether MF sprouting is directed to the dentate gyrus or extends to distant CA regions and whether sprouting is associated with death of target neurons in CA3 and CA4. In 319 TLE patients, HS was evaluated by Wyler grade and International League against Epilepsy (ILAE) types using immunohistochemistry against neuronal nuclei (NeuN). Synaptoporin was used to colocalize MF. In addition, transgenic Thy1‐eGFP mice were intrahippocampally injected with kainate and sprouting of eGFP‐positive MFs was analyzed together with immunocytochemistry for regulator of G‐protein signaling 14 (RGS14). In human HS Wyler III and IV as well as in ILAE 1, 2, and 3 specimens, we found synaptoporin‐positive axon terminals in CA2 and even in CA1, associated with the extent of granule cell dispersion. Sprouting was seen in cases with cell death of target neurons in CA3 and CA4 (classical severe HS ILAE type 1) but also without this cell death (atypical HS ILAE type 2). Similarly, in epileptic mice eGFP‐positive MFs sprouted to CA2 and beyond. The presence of MF terminals in the CA2 pyramidal cell layer and in CA1 was also correlated with the extent of granule cell dispersion. The similarity of our findings in human specimens and in the mouse model highlights the importance and opens up new chances of using translational approaches to determine mechanisms underlying TLE. [ABSTRACT FROM AUTHOR]

Chronic epilepsy has been associated with hippocampal abnormalities like neuronal loss, gliosis and granule cell dispersion. The granule cell layer of a normal human hippocampal dentate gyrus is traditionally regarded as a compact neuron-dense layer. Histopathological studies of surgically resected or autopsied hippocampal samples primarily from temporal lobe epilepsy patients, as well as animal models of epilepsy, describe variable patterns of granule cell dispersion including focal cell clusters, broader thick segments, and bilamination or "tram-tracking". Although most studies have implicated granule cell dispersion as a specific feature of chronic epilepsy, very few "non-seizure" controls were included in these published investigations. Our retrospective survey of 147 cadaveric pediatric human hippocampi identified identical morphological spectra of granule cell dispersion in both normal and seizure-affected brains. Moreover, sections across the entire antero-posterior axis of a control cadaveric hippocampus revealed repetitive occurrence of different morphologies of the granule cell layer – compact, focally disaggregated and bilaminar. The results indicate that granule cell dispersion is within the spectrum of normal variation and not unique to patients with epilepsy. We speculate that sampling bias has been responsible for an erroneous dogma, which we hope to rectify with this investigation. [ABSTRACT FROM AUTHOR]

Temporal lobe epilepsy (TLE) is the most common form of localization-related epilepsy, with the highest prevalence rate in adulthood. Recently, we reported the beneficial effects of the individual treatment with flavonoids such as silibinin and morin in kainic acid (KA)-treated mouse model for TLE. In this study, we investigated whether there is a synergistic effect of co-treatment with silibinin and morin on the susceptibility to seizure, the frequency of spontaneous recurrent seizures (SRSs), and granule cell dispersion in the dentate gyrus, which could be partially controlled by treatment with each flavonoid in the animal model for TLE. Unfortunately, we did not observe any synergistic effect against the susceptibility of seizure and SRS induced by KA treatment. However, the combination of these flavonoids showed similar antiepileptic effects compared with treatment with each one individually. Therefore, although silibinin and morin are not suitable for combination therapy, our results still suggest that these flavonoids can be used as potent therapeutic compounds for preventing epileptic seizures. [ABSTRACT FROM AUTHOR]

Abnormal reorganization of the dentate gyrus and neuroinflammation in the hippocampus represent characteristic phenotypes of patients suffering from temporal lobe epilepsy. Hesperetin, a flavanone abundant in citrus fruit, is known to have protective effects by preventing inflammation and oxidative stress in neuronal cultures and in the adult murine brain. However, the protective effects of hesperetin against epileptic seizures in vivo remain unclear, despite one study reporting anticonvulsant effects in vitro. In this study, we report that oral administration of hesperetin not only delays the onset of seizures triggered by kainic acid (KA) but also contributes to the attenuation of granule cell dispersion in the KA-treated hippocampus. Moreover, we observed that hesperetin administration inhibited the expression of pro-inflammatory molecules produced by activated microglia in the hippocampus. Thus, administration of hesperetin might be beneficial for preventing epileptic seizures. [ABSTRACT FROM AUTHOR]

Epilepsies are a group of common neurological diseases exerting a strong burden on patients and society, often lacking clear etiology and effective therapeutical strategies. Early intervention during the development of epilepsy (epileptogenesis) is of great medical interest, though hampered by poorly characterized epileptogenetic processes. Using the intrahippocampal kainic acid mouse model of temporal lobe epilepsy, we investigated the functional role of the endogenous opioid enkephalin during epileptogenesis. We addressed three sequential questions: (1) How does enkephalin affect seizure threshold and how is it regulated during epileptogenesis? (2) Does enkephalin influence detrimental effects during epileptogenesis? (3) How is enkephalin linked to mitochondrial function during epileptogenesis?. In contrast to other neuropeptides, the expression of enkephalin is not regulated in a seizure dependent manner. The pattern of regulation, and enkephalin’s proconvulsive effects suggested it as a potential driving force in epileptogenesis. Surprisingly, enkephalin deficiency aggravated progressive granule cell dispersion in kainic acid induced epileptogenesis. Based on reported beneficial effects of enkephalin on mitochondrial function in hypoxic/ischemic states, we hypothesized that enkephalin may be involved in the adaptation of mitochondrial respiration during epileptogenesis. Using high-resolution respirometry, we observed dynamic improvement of hippocampal mitochondrial respiration after kainic acid-injections in wild-type, but not in enkephalin-deficient mice. Thus, wild-type mice displayed higher efficiency in the use of mitochondrial capacity as compared to enkephalin-deficient mice. Our data demonstrate a Janus-headed role of enkephalin in epileptogenesis. In naive mice, enkephalin facilitates seizures, but in subsequent stages it contributes to neuronal survival through improved mitochondrial respiration.

The most common form of epilepsy among adults is mesial temporal lobe epilepsy (mTLE), with seizures often originating in the hippocampus due to abnormal electrical activity. The gold standard for the histopathological analysis of mTLE is histology, which is a two-dimensional technique. To fill this gap, we propose complementary three-dimensional (3D) X-ray histology. Herein, we used synchrotron radiation-based phase-contrast microtomography with 1.6 μm-wide voxels for the post mortem visualization of tissue microstructure in an intrahippocampal-kainate mouse model for mTLE. We demonstrated that the 3D X-ray histology of unstained, unsectioned, paraffin-embedded brain hemispheres can identify hippocampal sclerosis through the loss of pyramidal neurons in the first and third regions of the Cornu ammonis as well as granule cell dispersion within the dentate gyrus. Morphology and density changes during epileptogenesis were quantified by segmentations from a deep convolutional neural network. Compared to control mice, the total dentate gyrus volume doubled and the granular layer volume quadrupled 21 days after injecting kainate. Subsequent sectioning of the same mouse brains allowed for benchmarking 3D X-ray histology against well-established histochemical and immunofluorescence stainings. Thus, 3D X-ray histology is a complementary neuroimaging tool to unlock the third dimension for the cellular-resolution histopathological analysis of mTLE., Competing Interests: Conflict of Interest The authors declare that they have no competing interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Microscopy Society of America.)

Highlights • We examined the expressional change of WNK3 on GCD of TLE patients with HS. • WNK3 expression significantly enhanced in dispersed dentate granule neurons. • The control of WNK3 may be a novel therapeutic target against epileptic processes. Abstract Granule cell dispersion (GCD) is a common neuropathological feature of hippocampal sclerosis (HS) in patients with temporal lobe epilepsy (TLE). However, the underlying molecular mechanism of GCD formation remains unclear. The present study aimed to investigate the expressional changes of With No Lysine protein kinase subtype 3 (WNK3), a molecule upstream of cation-chloride cotransporters with reciprocal expression in sclerosed hippocampus of TLE patients. Using immunofluorescence staining, we analyzed WNK3 immunoreactivity in hippocampal specimens from histologically normal controls and TLE patients with HS. Our results showed that WNK3 expression was significantly increased in dispersed granule neurons in hippocampal tissues from patients with TLE compared with histologically normal hippocampus. These findings demonstrate a potential association between an increased expression of WNK3 and GCD formation during the chronic phase of epilepsy. Controlling WNK3 expression may thus be a novel therapeutic target in epileptogenesis. [ABSTRACT FROM AUTHOR]

Epilepsies are a group of common neurological diseases exerting a strong burden on patients and society, often lacking clear etiology and effective therapeutical strategies. Early intervention during the development of epilepsy (epileptogenesis) is of great medical interest, though hampered by poorly characterized epileptogenetic processes. Using the intrahippocampal kainic acid mouse model of temporal lobe epilepsy, we investigated the functional role of the endogenous opioid enkephalin during epileptogenesis. We addressed three sequential questions: (1) How does enkephalin affect seizure threshold and how is it regulated during epileptogenesis? (2) Does enkephalin influence detrimental effects during epileptogenesis? (3) How is enkephalin linked to mitochondrial function during epileptogenesis?. In contrast to other neuropeptides, the expression of enkephalin is not regulated in a seizure dependent manner. The pattern of regulation, and enkephalin's proconvulsive effects suggested it as a potential driving force in epileptogenesis. Surprisingly, enkephalin deficiency aggravated progressive granule cell dispersion in kainic acid induced epileptogenesis. Based on reported beneficial effects of enkephalin on mitochondrial function in hypoxic/ischemic states, we hypothesized that enkephalin may be involved in the adaptation of mitochondrial respiration during epileptogenesis. Using high-resolution respirometry, we observed dynamic improvement of hippocampal mitochondrial respiration after kainic acid-injections in wild-type, but not in enkephalin-deficient mice. Thus, wild-type mice displayed higher efficiency in the use of mitochondrial capacity as compared to enkephalin-deficient mice. Our data demonstrate a Janus-headed role of enkephalin in epileptogenesis. In naive mice, enkephalin facilitates seizures, but in subsequent stages it contributes to neuronal survival through improved mitochondrial respiration. [ABSTRACT FROM AUTHOR]

Mesial temporal lobe epilepsy is characterized by focal, recurrent spontaneous seizures, sclerosis and granule cell dispersion (GCD) in the hippocampal formation. Changes in theta rhythm properties have been correlated with the severity of hippocampal restructuring and were suggested as a cause of memory deficits accompanying epilepsy. For severe sclerosis, it has even been questioned whether theta band oscillations persist. We asked how theta oscillations change with graded restructuring along the longitudinal hippocampal axis and whether these changes correlate with the overall severity of temporal lobe epilepsy. We recorded local field potentials in the medial entorhinal cortex and along the septo-temporal axis of the dentate gyrus at sites with different degrees of GCD in freely behaving epileptic mice. Theta frequency was decreased at all recording positions throughout the dentate gyrus and in the medial entorhinal cortex, irrespective of the extent of GCD or the rate of severe epileptic events. The frequency reduction by up to 1.7 Hz, corresponding to 1/3 octaves within the theta range, was present during rest, exploration and running. Despite the frequency reduction, theta oscillations remained coherent across the hippocampal formation and were modulated by running speed as in controls. The reduction in theta frequency thus is likely not a consequence of the local restructuring but rather a global phenomenon affecting the hippocampal formation as a whole. [ABSTRACT FROM AUTHOR]

Mesial temporal lobe epilepsy (mTLE) is the most common focal epilepsy in adults and is often refractory to medication. So far, resection of the epileptogenic focus represents the only curative therapy. It is unknown whether pathological processes preceding epilepsy onset are indicators of later disease severity. Using longitudinal multi-modal MRI, we monitored hippocampal injury and tissue reorganization during epileptogenesis in a mouse mTLE model. The prognostic value of MRI biomarkers was assessed by retrospective correlations with pathological hallmarks Here, we show for the first time that the extent of early hippocampal neurodegeneration and progressive microstructural changes in the dentate gyrus translate to the severity of hippocampal sclerosis and seizure burden in chronic epilepsy. Moreover, we demonstrate that structural MRI biomarkers reflect the extent of sclerosis in human hippocampi. Our findings may allow an early prognosis of disease severity in mTLE before its first clinical manifestations, thus expanding the therapeutic window.

Objective Hippocampal sclerosis (HS) is the most prevalent pathology in temporal lobe epilepsy (TLE) characterized by segmental neuronal cell loss in the cornu ammonis (CA) 1–4. In addition, migration of granule cells and reorganization of their axons is observed, known as granule cell dispersion (GCD) and mossy fiber sprouting (MFS). The loss of mossy fibers` (MF) target cells in CA4 and CA3 was considered to be causative for MFS. The ILAE HS (International League Against Epilepsy) classification identifies three subtypes with different cell loss patterns in CA1-4. We studied the relation of ILAE HS subtypes to GCD and MFS to corroborate clinico-pathological subgroups in a large retrospective single-center series. Material and methods Hippocampal specimen of 319 patients were screened, 214 could be used for analysis. Immunohistochemical stainings for semi-quantitative analysis of neuronal cell loss (NeuN) and MFS (synaptoporin) were performed. Presurgical data were available from patient files and seizure outcome was classified according to Engel score after surgery. Results In 39 patients (18%) no neuronal cell loss (ILAE no-HS), no GCD and no MFS was observed. In 154 patients (72%) severe neuronal cell loss was seen in CA1, CA4 and CA3 (ILAE HS 1, typical HS); in addition extensive GCD and MFS was observed. In 17 patients (8%) cell loss was seen predominantly in CA1 (ILAE HS 2); despite different cell loss pattern these hippocampi also showed GCD and MFS. In 4 patients (2%) cell loss was predominately detected in CA3 and CA4 (ILAE HS type 3), consecutively GCD and MFS were observed. Longer epilepsy duration and younger age at surgery was more often associated with ILAE HS 2 and febrile convulsions were completely absent in ILAE no-HS. Yet, seizure onset, age at initial precipitating injury and postsurgical seizure outcome did not show any significant association with ILAE HS subtypes. Conclusion GCD and MFS might develop independently from the neuronal cell loss of MF target cells. [ABSTRACT FROM AUTHOR]

Objective The most frequent finding in temporal lobe epilepsy is hippocampal sclerosis, characterized by selective cell loss of hippocampal subregions CA1 and CA4 as well as mossy fiber sprouting (MFS) towards the supragranular region and granule cell dispersion. Although selective cell loss is well described, its impact on mossy fiber sprouting and granule cell dispersion remains unclear. Materials and methods In a single center series, we examined 319 human hippocampal specimens, collected in a 15-years period. Hippocampal specimens were stained for neuronal loss, granule cell dispersion (Wyler scale I–IV, Neu-N, HE) and mossy fiber sprouting (synaptoporin-immunohistochemistry). For seizure outcome Engel score I–IV was applied. Results In Wyler I and II specimens, mossy fibers were found along their natural projection exclusively in CA4 and CA3. In Wyler III and IV, sprouting of mossy fibers into the molecular layer and a decrease of mossy fibers in CA4 and CA3 was detected. Mean granule cell dispersion was extended from 121 μm to 185 μm and correlated with Wyler III-IV as well as mossy fiber sprouting into the molecular layer. Wyler grade, mossy fiber sprouting and granule cell dispersion correlated with longer epilepsy duration, late surgery and higher preoperative seizure frequency. Parameters analyzed above did not correlate with postoperative seizure outcome. Discussion Mossy fiber sprouting might be a compensatory phenomenon of cell death of the target neurons in CA4 and CA3 in Wyler III–IV. Axonal reorganization of granule cells is accompanied by their migration and is correlated with the severity of cell loss and epilepsy duration. [ABSTRACT FROM AUTHOR]

Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer (GCL) in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSC) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate towards the hilar region where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3-6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons.

Mesio-temporal lobe epilepsy (MTLE), the most common drug-resistant epilepsy syndrome, is characterized by the recurrence of spontaneous focal seizures after a latent period that follows, in most patients, an initial insult during early childhood. Many of the mechanisms that have been associated with the pathophysiology of MTLE are known to be regulated by brain-derived neurotrophic factor (BDNF) in the healthy brain and an excess of this neurotrophin could therefore play a critical role in MTLE development. However, such a function remains controversial as other studies revealed that BDNF could, on the contrary, exert protective effects regarding epilepsy development. In the present study, we further addressed the role of increased BDNF/TrkB signaling on the progressive development of hippocampal seizures in the mouse model of MTLE obtained by intrahippocampal injection of kainate. We show that hippocampal seizures progressively developed in the injected hippocampus during the first two weeks following kainate treatment, within the same time-frame as a long-lasting and significant increase of BDNF expression in dentate granule cells. To determine whether such a BDNF increase could influence hippocampal epileptogenesis via its TrkB receptors, we examined the consequences of (i) increased or (ii) decreased TrkB signaling on epileptogenesis, in transgenic mice overexpressing the (i) TrkB full-length or (ii) truncated TrkB-T1 receptors of BDNF. Epileptogenesis was significantly facilitated in mice with increased TrkB signaling but delayed in mutants with reduced TrkB signaling. In contrast, TrkB signaling did not influence granule cell dispersion, an important feature of this mouse model which is also observed in most MTLE patients. These results suggest that an increase in TrkB signaling, mediated by a long-lasting BDNF overexpression in the hippocampus, promotes epileptogenesis in MTLE.

Expression of urokinase-type plasminogen activator (uPA) is increased after brain injury, suggesting that, like in cancer tissue, uPA plays roles in brain remodeling. Here we injured brain with intrahippocampal kainic acid (KA) injection in adult Wt and uPA−/− mice. At 20 days post-injury, uPA−/− mice had more severe loss of contralateral pyramidal (p

The hippocampus is an important brain area where abnormal morphological characteristics are often observed in patients with temporal lobe epilepsy (TLE), typically showing the loss of the principal neurons in the CA1 and CA3 areas of the hippocampus. TLE is frequently associated with widening of the granule cell layer of the dentate gyrus (DG), termed granule cell dispersion (GCD), in the hippocampus, suggesting that the control of GCD with protection of hippocampal neurons may be useful for preventing and inhibiting epileptic seizures. We previously reported that eugenol (EUG), which is an essential component of medicinal herbs and has anticonvulsant activity, is beneficial for treating epilepsy through its ability to inhibit GCD via suppression of mammalian target of rapamycin complex 1 (mTORC1) activation in the hippocampal DG in a kainic acid (KA)-treated mouse model of epilepsy in vivo. In addition, we reported that naringin, a bioflavonoid in citrus fruits, could exert beneficial effects, such as antiautophagic stress and antineuroinflammation, in the KA mouse model of epilepsy, even though it was unclear whether naringin might also attenuate the seizure-induced morphological changes of GCD in the DG. Similar to the effects of EUG, we recently observed that naringin treatment significantly reduced KA-induced GCD and mTORC1 activation, which are both involved in epileptic seizures, in the hippocampus of mouse brain. Therefore, these observations suggest that the utilization of natural materials, which have beneficial properties such as inhibition of GCD formation and protection of hippocampal neurons, may be useful in developing a novel therapeutic agent against TLE. [ABSTRACT FROM AUTHOR]

Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSCs) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus, as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate toward the hilar region, where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3-6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together, these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons. [ABSTRACT FROM AUTHOR]

Morphological abnormalities of the dentate gyrus (DG) are an important phenotype in the hippocampus of patients with temporal lobe epilepsy. We recently reported that naringin, a bioflavonoid in grapefruit and citrus fruits, exerts beneficial effects in the kainic acid (KA) mouse model of epilepsy. We found that naringin treatment reduced seizure activities and decreased autophagic stress and neuroinflammation in the hippocampus following in vivo lesion with KA. However, it remains unclear whether naringin may also attenuate seizure-induced morphological changes in the DG, collectively known as granule cell dispersion (GCD). To clarify whether naringin treatment reduces GCD, we evaluated the effects of intraperitoneal injection of naringin on GCD and activation of mammalian target of rapamycin complex 1 (mTORC1), an important regulator of GCD, following intrahippocampal injection of KA. Our results showed that naringin treatment significantly reduced KA-induced GCD and mTORC1 activation, which was confirmed by assessing the phosphorylated form of the mTORC1 substrate, 4E-BP1, in the hippocampus. These results suggest that naringin treatment may help prevent epilepsy-induced hippocampal injury by inhibiting mTORC1 activation and thereby reducing GCD in the hippocampus in vivo . [ABSTRACT FROM AUTHOR]

ABSTRACT Sprouty (Spry) proteins play a key role as negative feedback inhibitors of the Ras/Raf/MAPK/ERK pathway downstream of various receptor tyrosine kinases. Among the four Sprouty isoforms, Spry2 and Spry4 are expressed in the hippocampus. In this study, possible effects of Spry2 and Spry4 hypomorphism on neurodegeneration and seizure thresholds in a mouse model of epileptogenesis was analyzed. The Spry2/4 hypomorphs exhibited stronger ERK activation which was limited to the CA3 pyramidal cell layer and to the hilar region. The seizure threshold of Spry2/4+/− mice was significantly reduced at naive state but no difference to wildtype mice was observed 1 month following KA treatment. Histomorphological analysis revealed that dentate granule cell dispersion (GCD) was diminished in Spry2/4+/− mice in the subchronic phase after KA injection. Neuronal degeneration was reduced in CA1 and CA3 principal neuron layers as well as in scattered neurons of the contralateral CA1 and hilar regions. Moreover, Spry2/4 reduction resulted in enhanced survival of somatostatin and neuropeptide Y expressing interneurons. GFAP staining intensity and number of reactive astrocytes markedly increased in lesioned areas of Spry2/4+/− mice as compared with wildtype mice. Taken together, although the seizure threshold is reduced in naive Spry2/4+/− mice, neurodegeneration and GCD is mitigated following KA induced hippocampal lesions, identifying Spry proteins as possible pharmacological targets in brain injuries resulting in neurodegeneration. The present data are consistent with the established functions of the ERK pathway in astrocyte proliferation as well as protection from neuronal cell death and suggest a novel role of Spry proteins in the migration of differentiated neurons. © 2015 The Authors Hippocampus Published by Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms like altered histone acetylation may have a crucial role in epileptogenesis. In two mouse models of temporal lobe epilepsy, we investigated changes in the expression of class II histone deacetylases ( HDAC), a group of signal transducers that shuttle between nucleus and cytoplasm. Intrahippocampal injection of kainic acid ( KA) induced a status epilepticus, development of spontaneous seizures (after 3 days), and finally chronic epilepsy and granule cell dispersion. Expression of class II HDAC m RNAs was investigated at different time intervals after KA injection in the granule cell layers and in sectors CA1 and CA3 contralateral to the site of KA injection lacking neurodegeneration. Increased expression of HDAC5 and 9 m RNAs coincided with pronounced granule cell dispersion in the KA-injected hippocampus at late intervals (14-28 days after KA) and equally affected both HDAC9 splice variants. In contrast, in the pilocarpine model (showing no granule cell dispersion), we observed decreases in the expression of HDAC5 and 9 at the same time intervals. Beyond this, striking similarities between both temporal lobe epilepsy models such as fast decreases in HDAC7 and 10 m RNAs during the acute status epilepticus were observed, notably also in the contralateral hippocampus not affected by neurodegeneration. The particular patterns of HDAC m RNA expression suggest a role in epileptogenesis and granule cell dispersion. Reduced expression of HDACs may result in increased expression of pro- and anticonvulsive proteins. On the other hand, export of HDACs from the nucleus into the cytoplasm could allow for deacetylation of cytoplasmatic proteins involved in axonal and dendritic remodeling, like granule cell dispersion. [ABSTRACT FROM AUTHOR]

Granule cell dispersion (GCD), a structural abnormality, is characteristic of temporal lobe epilepsy (TLE). Eugenol (EUG) is an essential component of medicinal herbs and is suggested to exert anticonvulsant activity. However, it is unclear whether EUG ameliorates the abnormal morphological changes in granule cells induced by epileptic insults. In the present study, we examined whether intraperitoneal injection of EUG attenuated increased seizure activity and GCD following intrahippocampal injection of kainic acid (KA). Our results showed that EUG significantly increased the seizure threshold, resulting in delayed seizure onset, and reduced GCD in KA-induced epilepsy. Moreover, EUG treatment significantly attenuated KA-induced activation of mammalian target of rapamycin complex 1 (mTORC1), which is involved in GCD development, in the dentate gyrus (DG). These results suggest that EUG may have beneficial effects in the treatment of epilepsy through its ability to inhibit GCD via suppression of KA-induced mTORC1 activation in the hippocampal DG in vivo . [ABSTRACT FROM AUTHOR]

Recent studies in animal models have suggested that the mammalian target of rapamycin ( mTOR) signaling pathway is involved in several features of mesio-temporal lobe epilepsy ( MTLE), and that its inhibition could have therapeutic interests. However, it remains controversial whether mTOR activation is the cause or the consequence of MTLE. We previously showed in a mouse model of MTLE associated with hippocampal sclerosis that increased neuronal excitability and brain-derived neurotrophic factor ( BDNF) overexpression contribute to the development of morphological features of this form of epilepsy. Here, we addressed whether mTOR activation promotes MTLE epileptogenesis via increasing neuronal excitability and/or BDNF expression or rather mediates neuroplasticity associated with hippocampal sclerosis. In mice injected intrahippocampally with kainate (1 nmol), we showed a biphasic increase of phospho-S6 (p-S6) ribosomal protein expression, the downstream product of the mTOR signaling pathway, in the dispersed granule cell layer ( GCL) of the dentate gyrus with a second phase lasting up to 6 months. Chronic treatment with rapamycin suppressed p-S6 expression, granule cell dispersion and mossy fiber sprouting, but did not reduce cell loss, BDNF overexpression, glutamic acid decarboxylase ( GAD)67 expression or the development of hippocampal paroxysmal discharges. Neuronal inhibition by midazolam (2 × 10 mg/kg, i.p.) abolished the increased expression of p-S6 in the dispersed GCL. Our data suggest that activation of the mTOR signaling pathway results from the increased neuronal excitation that develops in the GCL and may contribute to MTLE morphological changes. However, these data do not support the role of this pathway in the development of MTLE or its inhibition as a therapy for this form of epilepsy. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) of the nervous system can be considered as a dynamically adaptable compartment between neuronal cells, in particular neurons and glial cells, that participates in physiological functions of the nervous system. It is mainly composed of carbohydrates and proteins that are secreted by the different kinds of cell types found in the nervous system, in particular neurons and glial cells, but also other cell types, such as pericytes of capillaries, ependymocytes and meningeal cells. ECM molecules participate in developmental processes, synaptic plasticity, neurodegeneration and regenerative processes. As an example, the ECM of the hippocampal formation is involved in degenerative and adaptive processes related to epilepsy. The role of various components of the ECM has been explored extensively. In particular, the ECM protein reelin, well known for orchestrating the formation of neuronal layer formation in the cerebral cortex, is also considered as a player involved in the occurrence of postnatal granule cell dispersion (GCD), a morphologically peculiar feature frequently observed in hippocampal tissue from epileptic patients. Possible causes and consequences of GCD have been studied in various in vivo and in vitro models. The present review discusses different interpretations of GCD and different views on the role of ECM protein reelin in the formation of this morphological peculiarity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Leifeld, Förster, Reiss and Hamad.)

Abstract: Purpose: We analyzed the association of granule cell dispersion (GCD) with memory performance, clinical data and surgical outcome in a series of patients with mesial temporal lobe epilepsy (MTLE) and mesial temporal sclerosis (MTS). Method: Hippocampal specimens from 54 patients with MTLE (27 patients with right MTLE and 27 with left MTLE) and unilateral MTS, who were separated into GCD and no-GCD groups and thirteen controls were studied. Quantitative neuropathological evaluation was performed using hippocampal sections stained with NeuN. Patients’ neuropsychological measures, clinical data, type of MTS and surgical outcome were reviewed. Results: GCD occurred in 28 (51.9%) patients. No correlation between GCD and MTS pattern, clinical data or surgical outcome was found. The presence of GCD was correlated with worse visuospatial memory performance in right MTLE, but not with memory performance in left MTLE. Conclusion: GCD may be related to memory impairment in right MTLE-MTS patients. However, the role of GCD in memory function is not precisely defined. [Copyright &y& Elsevier]

Abstract: Girdin is an Akt substrate and actin-binding protein. Mice with germ-line deletions of Girdin (a non-conditional knockout, (ncKO)) exhibit complete postnatal lethality accompanied by growth retardation and neuronal cell migration defects, which results in hypoplasia of the olfactory bulb and granule cell dispersion in the dentate gyrus. However, the physiological and molecular abnormalities in Girdin ncKO mice are not fully understood. In this study, we first defined the distribution of Girdin in neonates (P1) and adults (6months or older) using β-galactosidase activity in tissues from ncKO mice. The results indicate that Girdin is expressed throughout the nervous system (brain, spinal cord, enteric and autonomic nervous systems). In addition, β-galactosidase activity was detected in non-neural tissues, particularly in tissues with high tensile force, such as tendons, heart valves, and skeletal muscle. In order to identify the cellular population where the Girdin ncKO phenotype originates, newly generated Girdin flox mice were crossed with nestin promoter-driven Cre transgenic mice to obtain Girdin conditional knockout (cKO) mice. The phenotype of Girdin cKO mice was almost identical to ncKO mice, including postnatal lethality, growth retardation and decreased neuronal migration. Our findings indicate that loss of Girdin in the nestin cell lineage underlies the phenotype of Girdin ncKO mice. [Copyright &y& Elsevier]

Abstract: Activated astrocytes display a broad spectrum of properties, ranging from neuroprotection to active contribution to demise of neural tissue. To investigate if activation of astrocytes by a single, defined stimulus enhances neuroprotective properties, we tested whether injection of ciliary neurotrophic factor (CNTF) can ameliorate epilepsy-related brain damage. Intrahippocampal CNTF injection in mice induced a rapid (within 2days) and persistent (3weeks) activation of astrocytes reflected by strong upregulation of glial fibrillary acidic protein (GFAP) mRNA synthesis and GFAP immunoreactivity. Moreover, CNTF signaling via phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) was specifically activated in GFAP-positive astrocytes. CNTF-mediated activation of astrocytes 2days prior to an epileptogenic intrahippocampal injection of kainate (KA) resulted in strongly reduced cell death in the hilus and CA3 region of the hippocampus, revealed by Fluoro-Jade B staining. Granule cell dispersion, the pathological widening of the granule cell layer, was also significantly reduced 16days after KA injection. Importantly, intrahippocampal in vivo recordings 3weeks after KA injection showed that the occurrence of high frequency oscillations (fast ripples, FR), a surrogate marker for epileptic activity, was significantly reduced in CNTF+KA-injected mice as compared to KA-injected animals. However, when CNTF was applied in the chronic epileptic phase at 3weeks after KA injection, no reduction of FR activity was observed. In summary, our results indicate that the activation of astrocytes prior to an excitotoxic injury effectively reduces neuronal damage and the severity of epileptiform activity, whereas activation in the chronic phase is no longer protective. [Copyright &y& Elsevier]

Cortical dysplasia of various types, reflecting abnormalities of brain development, have been closely associated with epileptic activities. Yet, there remains considerable discussion about if/how these structural lesions give rise to seizure phenomenology. Animal models have been used to investigate the cause-effect relationships between aberrant cortical structure and epilepsy. In this article, we discuss three such models: (1) the Eker rat model of tuberous sclerosis, in which a gene mutation gives rise to cortical disorganization and cytologically abnormal cellular elements; (2) the p35 knockout mouse, in which the genetic dysfunction gives rise to compromised cortical organization and lamination, but in which the cellular elements appear normal; and (3) the methylazoxymethanol-exposed rat, in which time-specific chemical DNA disruption leads to abnormal patterns of cell formation and migration, resulting in heterotopic neuronal clusters. Integrating data from studies of these animal models with related clinical observations, we propose that the neuropathologic features of these cortical dysplastic lesions are insufficient to determine the seizure-initiating process. Rather, it is their interaction with a more subtly disrupted cortical 'surround' that constitutes the circuitry underlying epileptiform activities as well as seizure propensity and ictogenesis. [ABSTRACT FROM AUTHOR]