Oral glutamine supplementation increases seizure severity in a rodent model of mesial temporal lobe epilepsy.

Background: Glutamine synthetase (GS) is the only enzyme known to synthesize significant amounts of glutamine in mammals, and loss of GS in the hippocampus has been implicated in the pathophysiology of medication refractory mesial temporal lobe epilepsy (MTLE). Moreover, loss-of-function mutations of the GS gene causes severe epileptic encephalopathy, and supplementation with glutamine has been shown to normalize EEG and possibly improve the outcome in these patients. Here we examined whether oral glutamine supplementation is an effective treatment for MTLE by assessing the frequency and severity of seizures after supplementation in a translationally relevant model of the disease.Methods: Male Sprague Dawley rats (380-400 g) were allowed to drink unlimited amounts of glutamine in water (3.6% w/v; n = 8) or pure water (n = 8) for several weeks. Ten days after the start of glutamine supplementation, GS was chronically inhibited in the hippocampus to induce MTLE. Continuous video-intracranial EEG was collected for 21 days to determine the frequency and severity of seizures.Results: While there was no change in seizure frequency between the groups, the proportion of convulsive seizures was significantly higher in glutamine treated animals during the first three days of GS inhibition.Conclusion: The results suggest that oral glutamine supplementation transiently increases seizure severity in the initial stages of an epilepsy model, indicating a potential role of the amino acid in seizure propagation and epileptogenesis.

Gene expression in the epileptic (EL) mouse hippocampus.

The neuropathology of hippocampal seizure foci in human temporal lobe epilepsy (TLE) and several animal models of epilepsy reveal extensive neuronal loss along with astrocyte and microglial activation. Studies of these models have advanced hypotheses that propose both pathological changes are essential for seizure generation. However, some seizure foci in human TLE show an extreme loss of neurons in all hippocampal fields, giving weight to hypotheses that favor neuroglia as major players. The epileptic (EL) mouse is a seizure model in which there is no observable neuron loss but associated proliferation of microglia and astrocytes and provides a good model to study the role of activated neuroglia in the presence of an apparently normal population of neurons. While many studies have been carried out on the EL mouse, there is a paucity of studies on the molecular changes in the EL mouse hippocampus, which may provide insight on the role of neuroglia in epileptogenesis. In this paper we have applied high throughput gene expression analysis to identify the molecular changes in the hippocampus that may explain the pathological processes. We have observed several classes of genes whose expression levels are changed. It is hypothesized that the upregulation of heat shock proteins (HSP70, HSP72, FOSL2 (HSP40), and their molecular chaperones BAG3 and DNAJB5 along with the down regulated gene MALAT1 may contribute to the neuroprotection observed. The increased expression of BDNF along with immediate early gene expression (FosB, JunB, ERG4, NR4A1, NR4A2, FBXO3) and the down regulation of GABRD, DBP and MALAT1 it is hypothesized may contribute to the hyperexcitability of the hippocampal neurons in this model. Activated astrocytes and microglia may also contribute to excitability pathomechanisms. Activated astrocytes in the ELS mouse are deficient in glutamine synthetase and thus reduce the clearance of extracellular glutamate. Activated microglia which may be associated with C1Q and MHC class I molecules we propose may mediate a process of selective removal of defective GABAergic synapses through a process akin to trogocytosis that may reduce neuronal inhibition and favor hyperexcitability.

Metabolic injury in a variable rat model of post-status epilepticus.

OBJECTIVE: In vivo studies of epilepsy typically use prolonged status epilepticus to generate recurrent seizures. However, reports on variable status duration have found discrete differences in injury after 40-50 min of seizures, suggesting a pathophysiologic sensitivity to seizure duration. In this report we take a multivariate cluster analysis to study a short duration status epilepticus model using in vivo 7T magnetic resonance spectroscopy (MRS) and histologic evaluation. METHODS: The Hellier Dudek model was applied with 45 min of status epilepticus after which the animals were imaged twice, at 3 days and 3 weeks post-status epilepticus. Single voxel point resolved spectroscopy (PRESS) MRS was used to acquire data from the dentate gyrus and CA3 region of the hippocampus, assessing metabolite ratios to total creatine (tCr). In a subset of animals after the second imaging study, brains were analyzed histologically by Nissl staining. RESULTS: A hierarchical cluster analysis performed on the 3-day data from 21 kainate-treated animals (dentate gyrus voxel) segregated into two clusters, denoted by KM (more injured, n = 6) and KL (less injured, n = 15). Although there was no difference in kainate dosing or seizure count between them, the metabolic pattern of injury was different. The KM group displayed the largest significant changes in neuronal and glial parameters; the KL group displayed milder but significant changes. At 3 weeks, the KL group returned to normal compared to controls, whereas the KM group persisted with depressed N-acetyl aspartate (NAA)/tCr, glutamate/tCr, and increased inositol/tCr and glutamine/tCr. The classification was also consistent with subsequent histologic patterns at 3 weeks. SIGNIFICANCE: Although a short status period might be expected to generate a continuous distribution of metabolic injury, these data show that the short Hellier Dudek model appears to generate two levels of injury. The changes seen in segregated groups persisted into 3 weeks, and can be interpreted according to neuronal and glial biomarkers consistent with histology results.

Periventricular White Matter Alterations From Explosive Blast in a Large Animal Model: Mild Traumatic Brain Injury or "Subconcussive" Injury?

The neuropathology of mild traumatic brain injury in humans resulting from exposure to explosive blast is poorly understood as this condition is rarely fatal. A large animal model may better reflect the injury patterns in humans. We investigated the effect of explosive blasts on the constrained head minimizing the effects of whole head motion. Anesthetized Yucatan minipigs, with body and head restrained, were placed in a 3-walled test structure and exposed to 1, 2, or 3 explosive blast shock waves of the same intensity. Axonal injury was studied 3 weeks to 8 months postblast using ß-amyloid precursor protein immunohistochemistry. Injury was confined to the periventricular white matter as early as 3-5 weeks after exposure to a single blast. The pattern was also present at 8 months postblast. Animals exposed to 2 and 3 blasts had more axonal injury than those exposed to a single blast. Although such increases in axonal injury may relate to the longer postblast survival time, it may also be due to the increased number of blast exposures. It is possible that the injury observed is due to a condition akin to mild traumatic brain injury or subconcussive injury in humans, and that periventricular injury may have neuropsychiatric implications.

Effects of Branched-Chain Amino Acid Supplementation on Spontaneous Seizures and Neuronal Viability in a Model of Mesial Temporal Lobe Epilepsy.

BACKGROUND: The essential branched-chain amino acids (BCAAs) leucine, isoleucine, and valine have recently emerged as a potential novel treatment for medically refractory epilepsy. Blood-derived BCAAs can readily enter the brain, where they contribute to glutamate biosynthesis and may either suppress or trigger acute seizures. However, the effects of BCAAs on chronic (ie, spontaneous recurrent) seizures and epilepsy-associated neuron loss are incompletely understood. MATERIALS AND METHODS: Sixteen rats with mesial temporal lobe epilepsy were randomized into 2 groups that could drink, ad libitum, either a 4% solution of BCAAs in water (n=8) or pure water (n=8). The frequency and relative percent of convulsive and nonconvulsive spontaneous seizures were monitored for a period of 21 days, and the brains were then harvested for immunohistochemical analysis. RESULTS: Although the frequency of convulsive and nonconvulsive spontaneous recurrent seizures over a 3-week drinking/monitoring period were not different between the groups, there were differences in the relative percent of convulsive seizures in the first and third week of treatment. Moreover, the BCAA-treated rats had over 25% fewer neurons in the dentate hilus of the hippocampus compared with water-treated controls. CONCLUSIONS: Acute BCAA supplementation reduces seizure propagation, whereas chronic oral supplementation with BCAAs worsens seizure propagation and causes neuron loss in rodents with mesial temporal lobe epilepsy. These findings raise the question of whether such supplementation has a similar effect in humans.

Neuronal and glial changes in the brain resulting from explosive blast in an experimental model.

Mild traumatic brain injury (mTBI) is the signature injury in warfighters exposed to explosive blasts. The pathology underlying mTBI is poorly understood, as this condition is rarely fatal and thus postmortem brains are difficult to obtain for neuropathological studies. Here we report on studies of an experimental model with a gyrencephalic brain that is exposed to single and multiple explosive blast pressure waves. To determine injuries to the brain resulting from the primary blast, experimental conditions were controlled to eliminate any secondary or tertiary injury from blasts. We found small but significant levels of neuronal loss in the hippocampus, a brain area that is important for cognitive functions. Furthermore, neuronal loss increased with multiple blasts and the degree of neuronal injury worsened with time post-blast. This is consistent with our findings in the blast-exposed human brain based on magnetic resonance spectroscopic imaging. The studies on this experimental model thus confirm what has been presumed to be the case with the warfighter, namely that exposure to multiple blasts causes increased brain injury. Additionally, as in other studies of both explosive blast as well as closed head mTBI, we found astrocyte activation. Activated microglia were also prominent in white matter tracts, particularly in animals exposed to multiple blasts and at long post-blast intervals, even though injured axons (i.e. ß-APP positive) were not found in these areas. Microglial activation appears to be a delayed response, though whether they may contribute to inflammation related injury mechanism at even longer post-blast times than we tested here, remains to be explored. Petechial hemorrhages or other gross signs of vascular injury were not observed in our study. These findings confirm the development of neuropathological changes due to blast exposure. The activation of astrocytes and microglia, cell types potentially involved in inflammatory processes, suggest an important area for future study.

Metabolic changes in early poststatus epilepticus measured by MR spectroscopy in rats.

There is little experimental in vivo data on how differences in seizure duration in experimental status epilepticus influence metabolic injury. This is of interest given that in humans, status duration is a factor that influences the probability of subsequent development of epilepsy. This question is studied using 7-T magnetic resonance (MR) spectroscopy, T2 relaxometry in the incremented kainate rodent model of temporal lobe epilepsy, using two durations of status epilepticus, 1.5 and 3 hours. Histologic evaluation was performed in a subset of animals. Three days after status, single-voxel (8 mm(3)) point resolved spectroscopy (PRESS) MR spectroscopic measurements were acquired at 7 T to assess the cerebral metabolites measured as a ratio to total creatine (tCr). The status injury resulted in decreased N-acetylaspartate NAA/tCr, increased myo-inositol/tCr and glutamine/tCr, increased T2, and significant declines in NeuN-stained neuronal counts in both status groups. Regressions were identified in the status groups that provide evidence for neuronal injury and astrocytic reaction after status in both the short and long status duration groups. The long status group displays changes in glutathione/tCr that are not identified in the short status group, this difference possibly representing a maturation of injury and antioxidant response that occurs in synchrony with glutamatergic injury and glial activation.