Neuroprotective Effects of Diazepam, Carbamazepine, Phenytoin and Ketamine after Pilocarpine-induced Status Epilepticus

Cell damage and spatial localization deficits are often reported as long-term consequences of pilocarpine-induced status epilepticus. In this study, we investigated the neuroprotective effects of repeated drug administration after long-lasting status epilepticus. Groups of six to eight Wistar rats received microinjections of pilocarpine (2.4 mg/mu l, 1 mu l) in the right dorsal hippocampus to induce a status epilepticus, which was attenuated by thiopental injection (35 mg/kg, i.p.) 3 hrs after onset. Treatments consisted of i.p. administration of diazepam, ketamine, carbamazepine, or phenytoin at 4, 28, 52, and 76 hr after the onset of status epilepticus. Two days after the treatments, rats were tested in the Morris water maze and 1 week after the cognitive tests, their brains were submitted to histology to perform haematoxylin and eosin staining and glial fibrillary acidic protein (GFAP) immunofluorescence detection. Post-status epilepticus rats exhibited extensive gliosis and cell loss in the hippocampal CA1, CA3 (70% cell loss for both areas) and dentate gyrus (60%). Administration of all drugs reduced cell loss in the hippocampus, with best effects observed in brains slices of diazepam-treated animals, which showed less than 30% of loss in the three areas and decreased GFAP immunolabelling. Treatments improved spatial navigation during training trials and probe trial, with exception of ketamine. Interestingly, in the probe trial, only diazepam-treated animals showed preference for the goal quadrant. Our data point to significant neuroprotective effects of repeated administration of diazepam against status epilepticus-induced cell damage and cognitive disturbances.

Absence-like seizures in adult rats following pilocarpine-induced status epilepticus early in life

Administration of pilocarpine causes epilepsy in rats if status epilepticus (SE) is induced at an early age. To determine in detail the electrophysiological patterns of the epileptogenic activity in these animals, 46 Wistar rats, 7-17 days old, were subjected to SE induced by pilocarpine and electro-oscillograms from the cortex, hippocampus, amygdala, thalamus and hypothalamus, as well as head, rostrum and vibrissa, eye, ear and forelimb movements, were recorded 120 days later. Six control animals of the same age range did not show any signs of epilepsy. In all the rats subjected to SE, iterative spike-wave complexes (8.1 ± 0.5 Hz in frequency, 18.9 ± 9.1 s in duration) were recorded from the frontal cortex during absence fits. However, similar spike-wave discharges were always found also in the hippocampus and, less frequently, in the amygdala and in thalamic nuclei. Repetitive or single spikes were also detected in these same central structures. Clonic movements and single jerks were recorded from all the rats, either concomitantly with or independently of the spike-wave complexes and spikes. We conclude that rats made epileptic with pilocarpine develop absence seizures also occurring during paradoxical sleep, showing the characteristic spike-wave bursts in neocortical areas and also in the hippocampus. This is in contrast to the well-accepted statement that one of the main characteristics of absence-like fits in the rat is that spike-wave discharges are never recorded from the hippocampal fields.

Consequences of pilocarpine-induced status epilepticus in immunodeficient mice

Systemic injection of pilocarpine in rodents induces status epilepticus (SE) and reproduces the main characteristics of temporal lobe epilepsy (TLE). Different mechanisms are activated by SE contributing to cell death and immune system activation. We used BALB/c nude mice, a mutant that is severely immunocompromised, to characterize seizure pattern, neurochemical changes, cell death and c-Fos activation secondarily to pilocarpine-induced SE. The behavioral seizures were less severe in BALB/c nude than in BALB/c wild type mice. However, nude mice presented more tonic clonic episodes and higher mortality rate during SE. The c-Fos expression was most prominent in the caudate-putamen, CA3 (p < 0.05), dentate gyrus, entorhinal cortex (p < 0.001), basolateral nucleus of amygdala (p < 0.01) and piriform cortex (p < 0.05) of BALB/c nude mice than of BALB/c. Besides, nude mice subjected to SE presented high number of Fluorojade-B (FJB) stained cells in the piriform cortex, amygdala (p < 0.05) and hilus (p < 0.05) in comparison with BALB/c mice. A significant increase in the level of glutamate and GABA was found in the hippocampus and cortex of BALB/c mice presenting SE in comparison to controls. However, the level of glutamate was higher in the brains of BALB nude mice than in the brains of BALB/c wild type mice, while the levels of GABA were unchanged. These results indicate that the brains of immunodeficient nude mice are more vulnerable to the deleterious effects of pilocarpine-induced SE as they present intense activation, increased glutamate levels and more cell death. Published by Elsevier B.V.

Effects of voluntary running on spatial memory and mature brain-derived neurotrophic factor expression in mice hippocampus after status epilepticus

Voluntary physical activity improves memory and learning ability in rodents, whereas status epilepticus has been associated with memory impairment. Physical activity and seizures have been associated with enhanced hippocampal expression of BDNF, indicating that this protein may have a dual role in epilepsy. The influence of voluntary physical activity on memory and BDNF expression has been poorly studied in experimental models of epilepsy. In this paper, we have investigated the effect of voluntary physical activity on memory and BDNF expression in mice with pilocarpine-incluced epilepsy. Male Swiss mice were assigned to four experimental groups: pilocarpine sedentary (PS), pilocarpine runners (PRs), saline sedentary (SS) and saline runners (SRs). Two days after pilocarpine-induced status epilepticus, the affected mice (PR) and their running controls (SR) were housed with access to a running wheel for 28 days. After that, the spatial memory and the expression of the precursor and mature forms of hippocampal BDNF were assessed. PR mice performed better than PS mice in the water maze test. In addition, PR mice had a higher amount of mature BDNF (14 kDa) relative to the total BDNF (14 kDa + 28 kDa + 32 kDa forms) content when compared with PS mice. These results show that voluntary physical activity improved the spatial memory and increased the hippocampal content of mature BDNF of mice with pilocarpine-induced status epilepticus. (C) 2009 Elsevier B.V. All rights reserved.

Spastin in the human and mouse central nervous system with special reference to its expression in the hippocampus of mouse pilocarpine model of status epilepticus and temporal lobe epilepsy

In the present in situ hybridization and immunocytochemical studies in the mouse central nervous system (CNS), a strong expression of spastin mRNA and protein was found in Purkinje cells and dentate nucleus in the cerebellum, in hippocampal principal cells and hilar neurons, in amygdala, substantia nigra, striatum, in the motor nuclei of the cranial nerves and in different layers of the cerebral cortex except piriform and entorhinal cortices where only neurons in layer II were strongly stained. Spastin protein and mRNA were weakly expressed in most of the thalamic nuclei. In selected human brain regions such as the cerebral cortex, cerebellum, hippocampus, amygdala, substania nigra and striatum, similar results were obtained. Electron microscopy showed spastin immunopositive staining in the cytoplasma, dendrites, axon terminals and nucleus. In the mouse pilocarpine model of status epilepticus and subsequent temporal lobe epilepsy, spastin expression disappeared in hilar neurons as early as at 2h during pilocarpine induced status epilepticus, and never recovered. At 7 days and 2 months after pilocarpine induced status epilepticus, spastin expression was down-regulated in granule cells in the dentate gyrus, but induced expression was found in reactive astrocytes. The demonstration of widespread distribution of spastin in functionally different brain regions in the present study may provide neuroanatomical basis to explain why different neurological, psychological disorders and cognitive impairment occur in patients with spastin mutation. Down-regulation or loss of spastin expression in hilar neurons may be related to their degeneration and may therefore initiate epileptogenetic events, leading to temporal lobe epilepsy.

Comparative neuroanatomical and temporal characterization of FluoroJade-positive neurodegeneration after status epilepticus induced by systemic and intrahippocampal pilocarpine in Wistar rats

The aims of this study were to characterize the spatial distribution of neurodegeneration after status epilepticus (SE) induced by either systemic (S) or intrahippocampal (H) injection of pilocarpine (PILO), two models of temporal lobe epilepsy (TLE), using FluoroJade (FJ) histochemistry, and to evaluate the kinetics of FJ staining in the H-PILO model. Therefore, we measured the severity of behavioral seizures during both types of SE and also evaluated the FJ staining pattern at 12, 24, and 168 h (7 days) after the H-PILO insult. We found that the amount of FJ-positive (FJ+) area was greater in SE induced by S-PILO as compared to SE induced by H-PILO. After SE induced by H-PILO, we found more FJ+ cells in the hilus of the dentate gyrus (DG) at 12 h, in CA3 at 24 h, and in CA1 at 168 h. We found also no correlation between seizure severity and the number of FJ+ cells in the hippocampus. Co-localization studies of FJ+ cells with either neuronal-specific nuclear protein (NeuN) or glial fibrillary acidic protein (GFAP) labeling 24 h after H-PILO demonstrated spatially selective neurodegeneration. Double labeling with FJ and parvalbumin (PV) showed both FJ+/PV+ and FJ+/PV- cells in hippocampus and entorhinal cortex, among other areas. The current data indicate that FJ+ areas are differentially distributed in the two TLE models and that these areas are greater in the S-PILO than in the H-PILO model. There is also a selective kinetics of FJ+ cells in the hippocampus after SE induced by H-PILO, with no association with the severity of seizures, probably as a consequence of the extra-hippocampal damage. These data point to SE induced by H-PILO as a low-mortality model of TLE, with regional spatial and temporal patterns of FJ staining. (C) 2010 Elsevier B.V. All rights reserved.

Study of spontaneous recurrent seizures and morphological alterations after status epilepticus induced by intrahippocampal injection of pilocarpine

Epileptic seizures are clinical manifestations of neuronal discharges characterized by hyperexcitability and/or hypersynchrony in the cortex and other subcortical regions. The pilocarpine (PILO) model of epilepsy mimics temporal lobe epilepsy (TLE) in humans. In the present study, we used a more selective approach: microinjection of PILO into the hilus of the dentate gyrus (H-PILO). Our main goal was to evaluate the behavioral and morphological alterations present in this model of TLE. Seventy-six percent of all animals receiving H-PILO injections had continuous seizures called status epilepticus (SE). A typical pattern of evolution of limbic seizures during the SE with a latency of 29.3 +/- 16.3 minutes was observed using an analysis of behavioral sequences. During the subsequent 30 days, 71% of all animals exhibited spontaneous recurrent seizures (SRSs) during a daily 8-hour videotaping session. These SRSs had a very conspicuous and characteristic pattern detected by behavioral sequences or neuroethological analysis. Only the animals that had SE showed positive Neo-Timm staining in the inner molecular layer of the dentate gyrus (sprouting) and reduced cell density in Ammon`s horn pyramidal cell subfield CA1. However, no correlation between the intensity of sprouting and the mean number and total number of SRSs was found. Additionally, using Fluoro-Jade staining, we observed neurodegeration in the hilus and pyramidal cell subfields CA3 and CM 24 hours after SE. These data indicate that H-PILO is a reliable, selective, efficient, low-mortality model that mimics the acute and chronic behavioral and morphological aspects of TLE. (C) 2010 Elsevier Inc. All rights reserved.

Changes in lipid composition in hippocampus early and late after status epilepticus induced by kainic acid in wistar rats

Oxidative damage to biological membranes has been reported as a cause of alterations in many different diseases. We had previously reported lipid peroxidation in the kainic acid model of temporal epilepsy. In this study we evaluated earlier and later modifications in the lipid composition after status epileticus induced by kainic acid. Lipid composition was determined by thin-layer chromatography, in the cortex and hippocampus 12-14 h, 7-8, 75-80, or 140-150 days after the end of status epileticus. In the hippocampus there was a significant change in the lipid protein ratio after status epileticus and this was accompanied by an alteration in lipid composition in all tested times. These results suggested that lipid peroxidation induced by kainic acid could be accompanied by chronic changes in the lipid composition that could be related to the development of seizures.

Role of morphological changes in newly born granule cells of hippocampus after status epilepticus induced by pilocarpine in hyperexcitability

JT is the recipient of a Post-Doctoral Fellowship from CNPq, Brazil. NGC and ACR are recipients of grants from CNPq, FAPESP, FAPESP-Cinapce, CAPES-PROEX, CNPq-Research Fellowships, Brazil.

What is the value of hypothermia in acute neurologic diseases and status epilepticus?

Patients with status epilepticus that proves refractory to anesthetic agents represent a daunting challenge for treating clinicians. Animal data support the neuroprotective action of brain hypothermia, and its efficacy in status epilepticus models. This approach, targeting a core temperature of about 33°C for at least 24 hours together with pharmacological sedation, has been described in adults and children. However, although relatively safe if concomitant barbiturates are avoided, it seems that mild hypothermia rarely allows a sustained control of ongoing status epilepticus, since seizures tend to recur in normothermia. Conversely, mild hypothermia has a high-evidence level and is increasingly used in postanoxic encephalopathy, both in newborns and adults. Due to the paucity of available clinical data, prospective studies are needed to assess the value of hypothermia in status epilepticus.

Predictors of awakening from postanoxic status epilepticus after therapeutic hypothermia.

BACKGROUND: Postanoxic status epilepticus (PSE) is considered a predictor of fatal outcome and therefore not intensively treated; however, some patients have had favorable outcomes. The aim of this study was to identify favorable predictors for awakening beyond vegetative state in PSE. METHODS: We studied six subjects treated with hypothermia improving beyond vegetative state after cerebral anoxia, despite PSE. They were among a cohort of patients treated for anoxic encephalopathy with therapeutic hypothermia in our institution between October 1999 and May 2006 (retrospectively, 3/107 patients) and June 2006 and May 2008 (prospectively, 3/74 patients). PSE was defined by clinical and EEG criteria. Outcome was assessed according to the Glasgow-Pittsburgh Cerebral Performance Categories (CPC). RESULTS: All improving patients had preserved brainstem reflexes, cortical somatosensory evoked potentials, and reactive EEG background during PSE. Half of them had myoclonic PSE, while three had nonconvulsive PSE. In the prospective arm, 3/28 patients with PSE showed this clinical-electrophysiologic profile; all awoke. Treatments consisted of benzodiazepines, various antiepileptic drugs, and propofol. One subject died of pneumonia in a minimally conscious state, one patient returned to baseline (CPC1), three had moderate impairment (CPC2), and one remained dependent (CPC3). Patients with nonconvulsive PSE showed a better prognosis than subjects with myoclonic PSE (p = 0.042). CONCLUSION: Patients with postanoxic status epilepticus and preserved brainstem reactions, somatosensory evoked potentials, and EEG reactivity may have a favorable outcome if their condition is treated as status epilepticus.

Bowel ischemia: a rare complication of thiopental treatment for status epilepticus.

BACKGROUND: Refractory status epilepticus (RSE) treatment is usually performed with coma induction using an appropriate general anesthetic. Most frequent complications are represented by hypotension and infection. Other side-effects may however be encountered. OBSERVATIONS: We describe two patients suffering from acute bowel ischemia after thiopental (THP) treatment for RSE. A 73-year-old man with a complex-patial RSE following an acute stroke received THP (303 mg/kg over 48 h); 36 h after THP discontinuation, he presented abdominal tenderness and lactate elevation. Necrosis of the terminal ileum and colon was seen during surgical exploration; he deceased shortly thereafter. A 21 year-old woman had a cryptogenic de novo generalized-convulsive RSE resistant to 5 attempts of EEG burst-suppression. During the 6th attempt, after THP (840 mg/kg over 150 h) together with mild hypothermia, she developed an ileus with elevated serum lactate; caecum necrosis was observed during surgery. Hypernatremia, acidosis and hyperlactatemia heralded this complication in both patients. CONCLUSION: In these two patients, mechanical vascular ischemia may have resulted from drug-induced paralytic ileus. To our knowledge, this is the first report describing this potential fatal side effect in adults with RSE.

Morphological Alterations in Newly Born Dentate Gyrus Granule Cells That Emerge after Status Epilepticus Contribute to Make Them Less Excitable

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.

Status epilepticus and lymphocytic pneumonitis following hepatitis B vaccination

The case reported refers to a patient who developed status epilepticus in the day of her third dose of hepatitis B vaccination and we review the literature on this subject. A 12 year-old girl, without a relevant previous history, taking no drugs, developed a seizure attack followed by unconsciousness, and eventually died after three days of her third dose of hepatitis B (HB) vaccination. Autopsy study revealed cerebral edema with congestion and herniation and diffuse interstitial type pneumonitis. There seem to be a straight forward time relationship between the third HB vaccine, the event of convulsion and the sudden death of the patient. We suggest that, in some cases, vaccination may be the triggering factor for autoimmune and neurological disturbances in genetically predisposed individuals and physicians should be aware of this possible association. (c) 2007 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.