Extracellular matrix protein SC1/hevin in the hippocampus following pilocarpine-induced status epilepticus

Pilocarpine-induced status epilepticus (SE) mimics many features of temporal lobe epilepsy and is a useful model to study neural changes that result from prolonged seizure activity. In this study, distribution of the anti-adhesive extracellular matrix protein SC1 was examined in the rat hippocampus following SE. Western blotting showed decreased levels of SC1 protein in the week following SE. Immunohistochemistry demonstrated that the decrease in overall SC1 protein levels was reflected by a reduction of SC1 signal in granule cells of the dentate gyrus. Interestingly, levels of SC1 protein in neurons of the seizure-resistant CA2 sector of the hippocampus did not change throughout the seizure time course. However, at 1 day post-SE, a subset of neurons of the hippocampal CA1, CA3, and hilar regions, which are noted for extensive neuronal degeneration after SE, exhibited a transient increase in SC1 signal. Neurons exhibiting enhanced SC1 signal were not detected at 7 days post-SE. The cellular stress response was also examined. A prominent induction of heat-shock protein (Hsp70) and Hsp27 was detected following SE, while levels of constitutively expressed Hsp40, Hsp90, Hsp110, and Hsc70 showed little change at the time points examined. The subset of neurons that demonstrated a transient increase in SC1 colocalized with the cellular stress marker Hsp70, the degeneration marker Fluoro-Jade B, and the neuron activity marker activity-regulated cytoskeleton-associated protein (Arc). Taken together, these findings suggest that SC1 may be a component of the 'matrix response' involved in remodeling events associated with neuronal degeneration following neural injury.     

Exposure to Mozart music reduces cognitive impairment in pilocarpine-induced status epilepticus rats

Patients with temporal lobe epilepsy (TLE) often display cognitive deficits. However, current epilepsy therapeutic interventions mainly aim at how to reduce the frequency and degree of epileptic seizures. Recovery of cognitive impairment is not attended enough, resulting in the lack of effective approaches in this respect. In the pilocarpine-induced temporal lobe epilepsy rat model, memory impairment has been classically reported. Here we evaluated spatial cognition changes at different epileptogenesis stages in rats of this model and explored the effects of long-term Mozart music exposure on the recovery of cognitive ability. Our results showed that pilocarpine rats suffered persisting cognitive impairment during epileptogenesis. Interestingly, we found that Mozart music exposure can significantly enhance cognitive ability in epileptic rats, and music intervention may be more effective for improving cognitive function during the early stages after Status epilepticus. These findings strongly suggest that Mozart music may help to promote the recovery of cognitive damage due to seizure activities, which provides a novel intervention strategy to diminish cognitive deficits in TLE patients.     

Alterations in hyperpolarization-activated cyclic nucleotidegated cation channel (HCN) expression in the hippocampus following pilocarpine-induced status epilepticus

To understand the effects of HCN as potential mediators in the pathogenesis of epilepsy that evoke long-term impaired excitability; the present study was designed to elucidate whether the alterations of HCN expression induced by status epilepticus (SE) is responsible for epileptogenesis. Although HCN1 immunoreactivity was observed in the hippocampus, its immunoreactivities were enhanced at 12 hrs following SE. Although, HCN1 immunoreactivities were reduced in all the hippocampi at 2 weeks, a re-increase in the expression at 2-3 months following SE was observed. In contrast to HCN1, HCN 4 expressions were un-changed, although HCN2 immunoreactive neurons exhibited some changes following SE. Taken together, our findings suggest that altered expressions of HCN1 following SE may be mainly involved in the imbalances of neurotransmissions to hippocampal circuits; thus, it is proposed that HCN1 may play an important role in the epileptogenic period as a compensatory response. [BMB Reports 2012; 45(11): 635-640]     

CCL28 in the mouse hippocampal CA1 area and the dentate gyrus during and after pilocarpine-induced status epilepticus

The present study showed a wide presence of CCL28 in mouse CNS, including cerebral, cerebellum, brain stem and spinal cord. In hippocampus, the expression of CCL28 at both mRNA and protein level was clarified. The CCL28 expression was mainly localized in pyramidal cells of CA area, granular cells of dentate gyrus and some interneurons in CA area and hilus. Double-labelling immunocytochemistry revealed that most of calbindin, calretinin and parvalbumin immunopositive neurons expressed CCL28. During and after pilocarpine induced status epilepticus (SE), a down-regulated expression of CCL28 in hippocampal interneurons in the CA1 area and in the hilus of the dentate gyrus was demonstrated. The present study may, therefore provide evidence that CCL28 may have a novel role in CNS and may be involved in the loss of hippocampal interneurons, and subsequent disinhibition of pyramidal neurons.     

Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus

Long non‐coding RNAs (lncRNAs) have been implicated in the regulation of gene expression at various levels. However, to date, the expression profile of lncRNAs in status epilepticus (SE) was unclear. In our study, the expression profile of lncRNAs was investigated by high‐throughput sequencing based on a lithium/pilocarpine‐induced SE model in immature rats. Furthermore, weighted correlation network analysis (WGCNA), gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to construct co‐expression networks and establish functions of the identified hub lncRNAs in SE. The functional role of a hub lncRNA (NONRATT010788.2) in SE was investigated in an in vitro model. Our results indicated that 7082 lncRNAs (3522 up‐regulated and 3560 down‐regulated), which are involved in cell proliferation, inflammatory responses, angiogenesis and autophagy, were dysregulated in the hippocampus of immature rats with SE. Additionally, WGCNA identified 667 up‐regulated hub lncRNAs in turquoise module that were involved in apoptosis, inflammatory responses and angiogenesis via regulation of HIF‐1, p53 and chemokine signalling pathways and via inflammatory mediator regulation of TRP channels. Knockdown of an identified hub lncRNA (NONRATT010788.2) inhibited neuronal apoptosis in vitro. Taken together, our study is the first to demonstrate the expression profile and potential function of lncRNAs in the hippocampus of immature rats with SE. The defined hub lncRNAs may participate in the pathogenesis of SE via lncRNA‐miRNA‐mRNA network.     

Genetic deletion of the neuronal glutamate transporter,EAAC1, results in decreased neuronal death after pilocarpine-induced status epilepticus

Excitatory amino acid carrier 1 (EAAC1 also called EAAT3) is a Na⁺-dependent glutamate transporter expressed by both glutamatergic and GABAergic neurons. It provides precursors for the syntheses of glutathione and GABA and contributes to the clearance of synaptically released glutamate. Mice deleted of EAAC1 are more susceptible to neurodegeneration in models of ischemia, Parkinson’s disease, and aging. Antisense knock-down of EAAC1 causes an absence seizure-like phenotype. Additionally, EAAC1 expression increases after chemonvulsant-induced seizures in rodent models and in tissue specimens from patients with refractory epilepsy. The goal of the present study was to determine if the absence of EAAC1 affects the sensitivity of mice to seizure-induced cell death. A chemoconvulsant dose of pilocarpine was administered to EAAC1^−/− mice and to wild-type controls. Although EAAC1^−/− mice experienced increased latency to seizure onset, no significant differences in behavioral seizure severity or mortality were observed. We examined EAAC1 immunofluorescence 24 h after pilocarpine administration and confirmed that pilocarpine causes an increase in EAAC1 protein. Forty-eight hours after induction of seizures, cell death was measured in hippocampus and in cortex using Fluoro-Jade C. Surprisingly, there was ∼2-fold more cell death in area CA1 of wild-type mice than in the corresponding regions of the EAAC1^−/− mice. Together, these studies indicate that absence of EAAC1 results in either a decrease in pilocarpine-induced seizures that is not detectable by behavioral criteria (surprising, since EAAC1 provides glutamate for GABA synthesis), or that the absence of EAAC1 results in less pilocarpine/seizure-induced cell death, possible explanations as discussed.     

Brain-derived neurotrophic factor facilitates TrkB down-regulation and neuronal injury after status epilepticus in the rat hippocampus

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of neuronal biology and hippocampal physiology. Status epilepticus (SE) is a condition in which prolonged seizures lead to neuronal degeneration. SE-induced in rodents serves as a model of Temporal Lobe Epilepsy with hippocampal sclerosis, the most frequent epilepsy in humans. We have recently described a strong correlation between TrkB decrease and p75ntr increase with neuronal degeneration ( Neuroscience 154:978, 2008). In this report, we report that local, acute intra-hippocampal infusion of function-blocking antibodies against BDNF prevented both early TrkB down-regulation and neuronal degeneration after SE. Conversely, the infusion of recombinant human BDNF protein after SE greatly increased neuronal degeneration. The inhibition of BDNF mRNA translation by the infusion of antisense oligonucleotides induced a rapid decrease of BDNF protein levels, and a delayed increase. If seizures were induced at the time endogenous BDNF was decreased, SE-induced neuronal damage was prevented. On the other hand, if seizures were induced at the time endogenous BDNF was increased, SE-induced neuronal damage was exacerbated. These results indicate that under a pathological condition BDNF exacerbates neuronal injury.     

Proteome changes associated with hippocampal MRI abnormalities in the lithium pilocarpine-induced model of convulsive status epilepticus

Convulsive status epilepticus is associated with subsequent hippocampal damage and development of mesial temporal sclerosis in a subset of individuals. The lithium pilocarpine model of status epilepticus (SE) in the rat provides a model in which to investigate the molecular and pathogenic process leading to hippocampal damage. In this study, a 2-DE-based approach was used to detect proteome changes in the hippocampus, at an early stage (2 days) after SE, when increased T2 values were detectable by magnetic resonance imaging. Gel image analysis was followed by LC-MS/MS identification of protein species that differed in abundance between pilocarpine-treated and control rats. The most significantly up-regulated species in the experimental animals was identified as heat shock 27-kDa protein, in line with findings in humans and in other experimental models of epilepsy. Additional up-regulated species included dihydropyrimidinase-related protein-2, cytoskeletal proteins (alpha-tubulin and ezrin) and dihydropteridine reductase. In summary, the hippocampus of rats subject to pilocarpine-induced SE exhibits specific changes in protein abundance, which likely relate to pathogenic, neuroprotective and neurogenic responses.     

Diazepam administration after prolonged status epilepticus reduces neurodegeneration in the amygdala but not in the hippocampus during epileptogenesis

An episode of status epilepticus (SE), if left untreated, can lead to death, or brain damage with long-term neurological consequences, including the development of epilepsy. The most common first-line treatment of SE is administration of benzodiazepines (BZs). However, the efficacy of BZs in terminating seizures is reduced with time after the onset of SE; this is accompanied by a reduced efficacy in protecting the hippocampus against neuronal damage, and is associated with impaired function and internalization of hippocampal GABA_A receptors. In the present study, using Fluoro-Jade C staining, we found that administration of diazepam to rats at 3 h after the onset of kainic acid-induced SE, at a dose sufficient to terminate SE, had no protective effect on the hippocampus, but produced a significant reduction in neuronal degeneration in the amygdala, piriform cortex, and endopiriform nucleus, examined on days 7–9 after SE. Thus, in contrast to the hippocampus, the amygdala and other limbic structures are responsive to neuroprotection by BZs after prolonged SE, suggesting that GABA_A receptors are not significantly altered in these structures during SE.     

Oxidative stress and modification of synaptic proteins in hippocampus after traumatic brain injury

Oxidative stress, an imbalance between oxidants and antioxidants, contributes to the pathogenesis of traumatic brain injury (TBI). Oxidative neurodegeneration is a key mediator of exacerbated morphological responses and deficits in behavioral recoveries. The present study assessed early hippocampal sequential imbalance to possibly enhance antioxidant therapy. Young adult male Sprague-Dawley rats were subjected to a unilateral moderate cortical contusion. At various times post-TBI, animals were killed and the hippocampus was analyzed for antioxidants (GSH, GSSG, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, superoxide dismutase, and catalase) and oxidants (acrolein, 4-hydroxynonenal, protein carbonyl, and 3-nitrotyrosine). Synaptic markers (synapsin I, postsynaptic density protein 95, synapse-associated protein 97, growth-associated protein 43) were also analyzed. All values were compared with those for sham-operated animals. Significant time-dependent changes in antioxidants were observed as early as 3 h posttrauma and paralleled increases in oxidants (4-hydroxynonenal, acrolein, and protein carbonyl), with peak values obtained at 24-48 h. Time-dependent changes in synaptic proteins (synapsin I, postsynaptic density protein 95, and synapse-associated protein 97) occurred well after levels of oxidants peaked. These results indicate that depletion of antioxidant systems following trauma could adversely affect synaptic function and plasticity. Early onset of oxidative stress suggests that the initial therapeutic window following TBI appears to be relatively short, and it may be necessary to stagger selective types of antioxidant therapy to target specific oxidative components.     

Reexpression of the embryonic form of NCAM in the rat hippocampus after status epilepticus induced by neurotoxic agent

The neural cell adhesion molecule (NCAM) is known to take part in the cohesion of cellular interactions through a homophilic binding mechanism. During development, NCAM shifts from an embryonic polysialic acid-rich form to a poorer adult one. This conversion reflects a loss of plasticity to the benefit of more stability. We have shown here an inverse process, namely the reexpression of the embryonic form of NCAM in adult rats following a status epilepticus induced through systemic administration of kainic acid.     

Calpain activation is involved in early caspase-independent neurodegeneration in the hippocampus following status epilepticus

Evidence for increased calpain activity has been described in the hippocampus of rodent models of temporal lobe epilepsy. However, it is not known whether calpains are involved in the cell death that accompanies seizures. In this work, we characterized calpain activation by examining the proteolysis of calpain substrates and in parallel we followed cell death in the hippocampus of epileptic rats. Male Wistar rats were injected with kainic acid (10 mg/kg) intraperitoneally and killed 24 h later, after development of grade 5 seizures. We observed a strong Fluoro-Jade labeling in the CA1 and CA3 areas of the hippocampus in the rats that received kainic acid, when compared with saline-treated rats. Immunohistochemistry and western blot analysis for the calpain-derived breakdown products of spectrin showed evidence of increased calpain activity in the same regions of the hippocampus where cell death is observed. No evidence was found for caspase activation, in the same conditions. Treatment with the calpain inhibitor MDL 28170 significantly prevented the neurodegeneration observed in CA1. Taken together, our data suggest that early calpain activation, but not caspase activation, is involved in neurotoxicity in the hippocampus after status epilepticus .     

Depression and/or potentiation of cortical responses after status epilepticus in immature rats

Lithium-pilocarpine status epilepticus (SE) resulted in delayed changes of single cortical interhemisperic (transcallosal) responses in immature rats. Low-frequency stimulation inducing depression and/or potentiation was studied to analyze possible dynamic changes in cortical responses. Status was elicited in 12-day-old (SE12) or 25-day-old (SE25) rats. Control siblings received saline instead of pilocarpine. Interhemispheric responses were elicited by stimulation of the sensorimotor region of the cerebral cortex 3, 6, 9, 13, or 26 days after status. A series of 5 biphasic pulses with intensity equal to twofold threshold were used for stimulation. The interval between pulses was 100, 125, 160, 200 or 300 ms, eight responses were always averaged. Peak amplitude of the first positive, first negative and second positive waves was measured and responses to the second, third, fourth and fifth pulse were compared with the first one. Animals after status epilepticus as well as lithium-paraldehyde controls exhibit a frequency depression at nearly all the intervals studied. An outlined increase of responses in SE rats in comparison with the controls three days after SE stayed just below the level of statistical significance. In addition, animals in the SE12 group exhibited potentiation of responses at this interval after SE. With longer intervals after SE, the relation between SE and control animals changed twice resulting in a tendency to lower amplitude of responses in SE than in control rats 26 days after SE. Rats in the SE25 group exhibited higher responses than controls 13 days after status, but this difference was not present at the longest interval after SE. Low-frequency stimulation did not reveal increased cortical excitability as a long-lasting consequence of status epilepticus induced in immature rats. In addition, the outlined differences between SE and control rats changed with the time after SE.     

Changes in phosphorylation of the NMDA receptor in the rat hippocampus induced by status epilepticus

Systemic administration of pilocarpine preceded by lithium induces status epilepticus (SE) that results in neurodegeneration and may lead to the development of spontaneous recurrent seizures. We investigated the effect of Li/pilocarpine-induced SE on phosphorylation of the NMDA receptor in rat hippocampus. Phosphorylation of NR1 by PKC on Ser890 was decreased to 45% of control values immediately following 1 h of SE. During the first 3 h following the termination of SE, phosphorylation of Ser890 increased 4-fold before declining to control values by 24 h. Phosphorylation of NR1 by PKA was also depressed relative to controls immediately following SE and transiently increased above control values upon the termination of SE. SE was accompanied by a general increase in tyrosine phosphorylation of hippocampal proteins that lasted for several hours following the termination of seizures. Tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDAR increased 3-4-fold over control values during SE, continued to increase during the first hour following SE and then declined to control levels by 24 h. SE resulted in the activation of Src and Pyk2 associated with the postsynaptic apparatus, suggesting a role for these enzymes in the SE-induced increase in tyrosine phosphorylation. Changes in phosphorylation of the NMDA receptor may play a role in the pathophysiological consequences of SE.     

GABA metabolism in the substantia nigra,cortex, and hippocampus during status epilepticus

The metabolism of GABA and other amino acids was studied in the substantia nigra, the hippocampus and the parietal cortex of rats following microinjections of GAMMA-vinyl-GABA during status epilepticus induced by lithium and pilocarpine. GABA metabolism showed striking regional variations. In controls, both GABA concentration and rate of GABA synthesis were highest in the substantia nigra and lowest in cortex, as expected. In substantia nigra, status epilepticus resulted in a 2 1/2 fold decline in the rate of GABA synthesis and in a 307% increase in the turnover time of the GABA pool. In hippocampus, the rate of GABA synthesis was not altered significantly, but the turnover time of the GABA pool was 284% of controls, and the size of that pool increased to 208% of controls. By contrast, in cortex, where seizure activity is limited in this model, the rate of GABA synthesis increased to 230% of controls while pool size and turnover time did not change. Aspartate concentration decreased in all three brain regions. These data suggest that the observed reduction of the rate of GABA synthesis in substantia nigra could play a key role in seizure spread in this model of status epilepticus.Special Issue dedicated to Claude Baxter.     

Decreased calmodulin kinase activity after status epilepticus

Status epilepticus was induced in paralyzed, ventilated rats using bicuculline and was maintained for 50 to 120 minutes. Cerebral cortex, hippocampus, and cerebellum were assayed for calmodulin kinase II activity in vitro using [-³²P]ATP and polyacrylamide gel electrophoresis. Seizures resulted in a 3.2 fold decrease in calmodulin kinase activity in crude synaptic membranes of cortex and in a 8.2 fold decrease in hippocampal membranes. Cytosolic calmodulin kinase activity was slightly increased in rats in status epilepticus but statistical significance was not reached. Status epilepticus did not affect calcium/calmodulin-dependent kinase activity in cerebellar membranes or cytosol. These data suggest that intense firing associated with continuous seizure activity decreases calmodulin kinase activity in cortical and hippocampal synaptic membranes, which may result in altered neuronal excitability.     

Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.     

Oxidative stress and diabetes in pregnant rats

A considerable amount of clinical and experimental evidence now exists suggesting the involvement of free radical-mediated oxidative processes in the pathogenesis of diabetic complications. If the diabetic state is associated with a generalized increase in oxidative stress, it might well be reflected in the alterations in embryonic and fetal development during pregnancy. In the present study, incidence of the malformed fetuses, biochemical parameters and antioxidant system activity of streptozotocin (STZ)-induced diabetic pregnant rats was investigated and the results obtained were compared with those of the control group (non-diabetic). Virgin female Wistar rats were injected with 40 mg/kg streptozotocin (STZ) before mating. All the females were killed on Day 21 of pregnancy and the fetuses were analyzed. A maternal blood sample was collected by venous puncture and the maternal liver was removed for biochemical measurement. The diabetic dams presented hyperglycemia, hyperlipemia, hypertriglyceridemia, hypercholesterolemia, hyperuricemia, decreased reduced glutathione (GSH), hepatic glycogen and superoxide dismutase (SOD) determinations. There was an increased incidence of skeletal and visceral malformation in fetuses from diabetic rats. Our findings suggest that oxidative stress occurs in the diabetic pregnant state, which might promote maternal homeostasis alterations. These diabetic complications might be a contributory factor to conceptus damage causing embryonic death (abortion/miscarriage) or the appearance of malformations in the fetuses of diabetic dams. Antioxidant treatment of women with diabetes may be important in future attempts to prevent congenital malformations.     

Pilocarpine-induced status epilepticus in rats involves ischemic and excitotoxic mechanisms

The neuron loss characteristic of hippocampal sclerosis in temporal lobe epilepsy patients is thought to be the result of excitotoxic, rather than ischemic, injury. In this study, we assessed changes in vascular structure, gene expression, and the time course of neuronal degeneration in the cerebral cortex during the acute period after onset of pilocarpine-induced status epilepticus (SE). Immediately after 2 hr SE, the subgranular layers of somatosensory cortex exhibited a reduced vascular perfusion indicative of ischemia, whereas the immediately adjacent supragranular layers exhibited increased perfusion. Subgranular layers exhibited necrotic pathology, whereas the supergranular layers were characterized by a delayed (24 h after SE) degeneration apparently via programmed cell death. These results indicate that both excitotoxic and ischemic injuries occur during pilocarpine-induced SE. Both of these degenerative pathways, as well as the widespread and severe brain damage observed, should be considered when animal model-based data are compared to human pathology.     

Oxidative stress in the chronic phase after stroke

The spontaneous and the stimulated extracellular generation of reactive oxygen species (ROS) by peripheral phagocytes, the blood antioxidant capacity and the degree of oxidative damage were evaluated in patients with severe ischemic and hemorrhagic stroke in the chronic phase of disease. It was found in patients compared to the control group that: (i) the spontaneous phagocyte oxidative activity was enhanced independently of the type of stroke and the time elapsed after stroke onset; (ii) there was no difference in the extracellular ROS generation stimulated by opsonin-dependent and independent receptor mechanisms; (iii) there was no change in the indices of blood antioxidant capacity; (iv) the concentration of plasma lipid peroxides was enhanced regardless of the type of stroke, but it significantly increased over time; and (v) the concentration of blood thiobarbituric acid-reactive material was also enhanced. It was independent of the type of stroke and remained elevated during the whole period studied. We have demonstrated an enhanced spontaneous phagocyte oxidative activity and oxidative damage to lipids in patients in the chronic phase after stroke. The elimination of generated ROS and products of lipid peroxidation from the circulation could prevent the aggravation of chronic vascular injury in patients and could reduce the possibility of a subsequent stroke. This suggests the need for complex therapy, including antioxidant treatment directed to exclude the effects of free radicals, after the oxidative stress of stroke.