João Pereira Leite

Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy

Objective: Sodium-coupled transporters remove extracellular neurotransmitters and alterations in their function could enhance or suppress synaptic transmission and seizures. This study determined hippocampal gamma-aminobutyric acid (GABA) and glutamate transporter immunoreactivity (IR) in temporal lobe epilepsy (TLE) patients. Methods: Hippocampal sclerosis (HS) patients (n = 25) and non-HS cases (mass lesion and cryptogenic; n = 20) were compared with nonseizure autopsies (n = 8). Hippocampal sections were studied for neuron densities along with IR for glutamate decarboxylase (GAD; presynaptic GABA terminals), GABA transporter-1 (GAT-1; presynaptic GABA transporter), GAT-3 (astrocytic GABA transporter), excitatory amino acid transporter 3 (EAAT3; postsynaptic glutamate transporter), and EAAT2-1 (glial glutamate transporters). Results: Compared with autopsies, non-HS cases with similar neuron counts showed: 1) increased GAD IR gray values (GV) in the fascia dentata outer molecular layer (OML), hilus, and stratum radiatum; 2) increased GAT-1 OML GVs; 3) increased astrocytic GAT-3 GVs in the hilus and Ammon’s horn; and 4) no IR differences for EAAT3-1. HS patients with decreased neuron densities demonstrated: 1) increased OML and inner molecular layer GAD puncta; 2) decreased GAT-1 puncta relative to GAD in the stratum granulosum and pyramidale; 3) increased GAT-1 OML GVs; 4) decreased GAT-3 GVs; 5) increased EAAT3 IR on remaining granule cells and pyramids; 6) decreased glial EAAT2 GVs in the hilus and CA1 stratum radiatum associated with neuron loss; and 7) increased glial EAAT1 GVs in CA2/3 stratum radiatum. Conclusions: Hippocampal GABA and glutamate transporter IR differ in TLE patients compared with autopsies. These data support the hypothesis that excitatory and inhibitory neurotransmission and seizure susceptibility could be altered by neuronal and glial transporters in TLE patients.

The pathogenic and progressive features of chronic human hippocampal epilepsy

To design useful experimental models of epilepsy, it is necessary to clearly understand the known clinical-pathologic features of the disease process. Studies of mesial temporal lobe epilepsy (MTLE) patients have identified several distinctive clinical and pathophysiologic characteristics and many of these can be analyzed in experimental models. For example, patients with typical MTLE have medical histories that often contain an initial precipitating injury (IPI), are likely to have hippocampal sclerosis in the surgical specimen, and have better seizure outcomes than patients with typical idiopathic temporal seizures (i.e. cryptogenic). Hippocampal from children as young as age 1 year with IPI histories also demonstrate neuron damage similar to adults with hippocampal sclerosis. Compared to IPI patients without seizures (i.e. trauma, hypoxia, etc.), IPI cases with severe seizures showed younger ages at the IPI, shorter latent periods, and longer durations of habitual MTLE. Hippocampal damage is often bilateral, however, the epileptogenic side shows hippocampal sclerosis and the opposite side usually shows only mild neuron losses. Moreover, MTLE patients show declines in hippocampal neuron densities with very long histories of habitual seizures (15 to 20 years), however, the additional neuron loss adds to the template of hippocampal sclerosis and occurs in limited subfields (granule cells, CA1 and prosubiculum). Hippocampal axon and synaptic reorganization is another pathologic feature of MTLE, and involves granule cell mossy fibers and axons immunoreactive for neuropeptide upsilon, somatostatin, and glutamate decarboxylase (which synthesizes GABA). Finally, MTLE patients with hippocampal sclerosis show increased granule cell mRNA levels for brain derived neurotropic factor, nerve growth factor, and neurotrophin-3 that correlate with mossy fiber sprouting or with declines in Ammons horn neuron densities. Taken together, our data support the following concepts: (1) The pathogenesis of MTLE is associated with IPI histories that probably injure the hippocampus at some time prior to habitual seizure onsets, (2) most of the damage seems to occur with the IPI, (3) there can be additional neuron loss associated with long histories, (4) another pathologic feature of MTLE is axon reorganization of surviving fascia dentata and hippocampal neurons, and (5) reorganized axon circuits probably contribute to seizure or propagation.

New insights from the use of pilocarpine and kainate models

Local or systemic administration of pilocarpine and kainate in rodents leads to a pattern of repetitive limbic seizures and status epilepticus, which can last for several hours. A latent period follows status epilepticus and precedes a chronic phase, which is characterized by the occurrence of spontaneous limbic seizures. These distinct features, in a single animal preparation, of an acute damage induced by status epilepticus, a silent interval between injury and the onset of spontaneous seizures, and a chronic epileptic state have allowed antiepileptic drug (AED) studies with different purposes, (a) in the acute phase, identification of compounds with efficacy against refractory status epilepticus and/or neuroprotection against damage induced by sustained seizures; (b) in the latent period, identification of agents with a potential for preventing epileptogenesis and/or against seizure-induced long-term behavioral deficits and (c) in the chronic phase, testing drugs effective against partial and secondarily generalized seizures. Studies on pilocarpine and kainate models have pointed out that some AEDs or other compounds exert an antiepileptogenic effect. The analogy of the latent phase of pilocarpine and kainate models with the acquisition of amygdala kindling should encourage testing of drugs that have proved to suppress the evolution of amygdala kindling. Drug testing in the chronic phase should not address only the suppression of secondarily generalized motor seizures. Most of current tools used to quantify spontaneous seizure events need to be coupled to electrophysiology and more sophisticated systems for recording and analyzing behavior.

Hippocampal EEG excitability and chronic spontaneous seizures are associated with aberrant synaptic reorganization in the rat intrahippocampal kainate model

Previously, Mathern et al. (1992) demonstrated progressive mossy fiber (MF) sprouting in the intrahippocampal rat kainate seizure model. This study looked at the time course of EEG hyperexcitability and spontaneous seizure activity in the same in vivo model to determine if seizures were associated with MF sprouting. Results showed that animals progressed through 4 distinct EEG and behavioral phases and that in the chronic phase (greater than 90 days post kainate) MF sprouting was strongly associated with hippocampal epileptogenesis. Progressive MF sprouting into the inner molecular layer (IML) of the fascia dentata paralleled the EEG and behavioral appearance of independent hippocampal interictal epileptiform transients and chronic seizures. Hippocampi from chronic animals that demonstrated unilateral MF IML sprouting were observed to have interictal epileptiform transients and spontaneous seizures that lateralized to the hippocampus with synaptically reorganized MFs. Chronic animals with bilateral MF sprouting were observed to have bilateral independent EEG and behavioral hyperexcitability. Control animals and kainate treated animals that lacked hippocampal cell loss and MF sprouting did not show signs of chronic hippocampal EEG hyperexcitability or chronic seizures. These data support the idea that MF sprouting contributes to chronic hippocampal seizures by feedback excitation which leads to the excitability and synchronization required for a damaged hippocampus to become an epileptic focus.

Validation of the National Institutes of Health Stroke Scale, Modified Rankin Scale and Barthel Index in Brazil: The Role of Cultural Adaptation and Structured Interviewing

Background: We aimed to validate three widely used scales in stroke research in a multiethnic Brazilian population. Methods: The National Institutes of Health Stroke Scale (NIHSS), modified Rankin Scale (mRS) and Barthel Index (BI) were translated, culturally adapted and applied by two independent investigators. The mRS was applied with or without a previously validated structured interview. Interobserver agreement (kappa statistics) and intraclass correlation coefficients were calculated. Results: 84 patients underwent mRS (56 with and 28 without a structured interview), 57 BI and 62 NIHSS scoring. Intraclass correlation coefficient was 0.902 for NIHSS and 0.967 for BI. For BI, interobserver agreement was good (kappa = 0.70). For mRS, the structured interview improved interobserver agreement (kappa = 0.34 without a structured interview; 0.75 with a structured interview). Conclusion: The NIHSS, BI and mRS show good validity when translated and culturally adapted. Using a structured interview for the mRS improves interobserver concordance rates.

Children with severe epilepsy: evidence of hippocampal neuron losses and aberrant mossy fiber sprouting during postnatal granule cell migration and differentiation

Surgically resected hippocampi from children with extrahippocampal seizures and structurally non-atrophic brains were examined to determine the relationship of neuron losses and aberrant mossy fiber (MF) sprouting to the postnatal migration and differentiation of the fascia dentata (FD) granule cells (GC). Percent neuron loss compared to age-matched autopsy controls was determined by quantitative cell densities, and aberrant MF sprouting by neo-Timm histochemistry. Postnatal immature GC migration and differentiation was demonstrated by the transient but GC-specific expression of the immature form of neural cell adhesion molecule (NCAM-H). Results showed that the hippocampi from children with seizures appeared microanatomically intact without focal areas of damage. However, significant neuron losses were found by neuron counts in the fascia dentata (P < 0.01), CA4 (P < 0.01), and CA2 (P < 0.05). Aberrant supragranular inner molecular layer MF sprouting was found in hippocampi of children with seizures, and the MFs showed smaller puncta in specimens resected under 2 years of age (n = 3) compared to the larger puncta in older children (n = 5). Hippocampi from children under 2 years of age also demonstrated NCAM-H positive primitive cells in the infragranular and stratum granulosum of the fascia dentata consistent with the postnatal migration and differentiation of GCs, the parent neurons of the MFs. These results indicate that seizures in the immature but structurally intact human hippocampus are associated with decreased neuron densities and aberrant MF sprouting very early in postnatal development. The data also show that aberrant MF sprouting is found during postnatal migration, differentiation and axogenesis of GCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Seizures Decrease Postnatal Neurogenesis and Granule Cell Development in the Human Fascia tDentata

Summary:  Purpose: There is considerable controversy whether childhood seizures damage existing neurons and/or adversely affect neurogenesis and synaptogenesis. This study addressed this question by examining fascia dentata neurogenesis, cell death, and aberrant axon connections in hippocampi from children with extratemporal seizure foci.

Neuron loss, mossy fiber sprouting, and interictal spikes after intrahippocampal kainate in developing rats

This study determined neuron losses, mossy fiber sprouting, and interictal spike frequencies in adult rats following intrahippocampal kainic acid (KA) injections during postnatal (PN) development. KA (0.4 micrograms/0.2 microliters; n = 64) was injected into one hippocampus and saline into the contralateral side between PN 7 to 30 days. Animals were sacrificed 28 to 256 days later, along with age-matched naive animals (controls; n = 20). Hippocampi were studied for: (1) Fascia dentata granule cell, hilar, and CA3c neuron counts; (2) neo-Timms stained supragranular mossy fiber sprouting; and (3) hippocampal and intracerebral interictal spike densities (n = 13). Mossy fiber sprouting was quantified as the gray value differences between the inner and outer molecular layer. Statistically significant results (p < 0.05) showed the following: (1) Compared to controls, CA3c and hilar neuron counts were reduced in KA-hippocampi with injections at PN 7-10 and PN 12-14 respectively and counts decreased with older PN injections. Granule cell densities on the KA-side and saline injected hippocampi were not reduced compared to controls. (2) In adult rats, supragranular mossy fiber sprouting was observed in 2 of 7 PN 7 injected animals. Compared to controls, increased gray value differences, indicating mossy fiber sprouting, were found on the KA-side beginning with injuries at PN 12-14 and increasing with older PN injections. On the saline-side only PN 30 animals showed minimal sprouting. (3) Mossy fiber sprouting progressively increased on the KA-side with longer survivals in rats injured after PN 15. Sprouting correlated positively with later PN injections and longer post-injection survival intervals, and not with reduced hilar or CA3c neuron counts. (4) On the KA-side, mossy fiber gray value differences correlated positively with in vivo intrahippocampal interictal spike densities. These results indicate that during postnatal rat development intrahippocampal kainate excitotoxicity can occur as early as PN 7 and increases with older ages at injection. This rat model reproduces many of the pathologic, behavioral, and electrophysiologic features of human mesial temporal lobe epilepsy, and supports the hypothesis that hippocampal sclerosis can be the consequence of focal injury during early postnatal development that progressively evolves into a pathologic and epileptic focus.

Animal models of epilepsy: use and limitations.

Epilepsy is a chronic neurological condition characterized by recurrent seizures that affects millions of people worldwide. Comprehension of the complex mechanisms underlying epileptogenesis and seizure generation in temporal lobe epilepsy and other forms of epilepsy cannot be fully acquired in clinical studies with humans. As a result, the use of appropriate animal models is essential. Some of these models replicate the natural history of symptomatic focal epilepsy with an initial epileptogenic insult, which is followed by an apparent latent period and by a subsequent period of chronic spontaneous seizures. Seizures are a combination of electrical and behavioral events that are able to induce chemical, molecular, and anatomic alterations. In this review, we summarize the most frequently used models of chronic epilepsy and models of acute seizures induced by chemoconvulsants, traumatic brain injury, and electrical or sound stimuli. Genetic models of absence seizures and models of seizures and status epilepticus in the immature brain were also examined. Major uses and limitations were highlighted, and neuropathological, behavioral, and neurophysiological similarities and differences between the model and the human equivalent were considered. The quest for seizure mechanisms can provide insights into overall brain functions and consciousness, and animal models of epilepsy will continue to promote the progress of both epilepsy and neurophysiology research.

Plasticity‚ synaptic strength‚ and epilepsy: what can we learn from ultrastructural data?

Summary:  Central nervous system synapses have an intrinsic plastic capacity to adapt to new conditions with rapid changes in their structure. Such activity‐dependent refinement occurs during development and learning, and shares features with diseases such as epilepsy. Quantitative ultrastructural studies based on serial sectioning and reconstructions have shown various structural changes associated with synaptic strength involving both dendritic spines and postsynaptic densities (PSDs) during long‐term potentiation (LTP). In this review, we focus on experimental studies that have analyzed at the ultrastructural level the consequences of LTP in rodents, and plastic changes in the hippocampus of experimental models of epilepsy and human tissue obtained during surgeries for intractable temporal lobe epilepsy (TLE). Modifications in spine morphology, increases in the proportion of synapses with perforated PSDs, and formation of multiple spine boutons arising from the same dendrite are the possible sequence of events that accompany hippocampal LTP. Structural remodeling of mossy fiber synapses and formation of aberrant synaptic contacts in the dentate gyrus are common features in experimental models of epilepsy and in human TLE. Combined electrophysiological and ultrastructural studies in kindled rats and chronic epileptic animals have indicated the occurrence of seizure‐ and neuron loss‐induced changes in the hippocampal network. In these experiments, the synaptic contacts on granule cells are similar to those described for LTP. Such changes could be associated with enhancement of synaptic efficiency and may be important in epileptogenesis.