Immunopathology in Drug Resistant Mesial Temporal Lobe Epilepsy with Different Types of Hippocampal Sclerosis

PURPOSE
There is evidence that autoimmunity has a specific role in temporal lobe seizures of limbic encephalitis patients. Our aim in this study was to investigate any histopathological clues of autoimmune process in refractory temporal lobe epilepsy (TLE) patients with different pathologically proven hippocampal sclerosis (HS) types.


METHODS
22 patients who had undergone epilepsy surgery due to mesial TLE-HS were included. The sera of patients are tested for neuronal antibodies to N-methyl-D-aspartate receptors (NMDAR), leucine-rich, glioma inactivated 1 (LGI1), contactin-associated protein 2 (CASPR2), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), gamma-aminobutyric acid B receptor (GABABR) and glutamic acid decarboxylase (GAD). Pathological and immunohistochemical investigations including neuronal nuclei (NeuN), NMDAR, GAD, glial fibrillary acidic protein (GFAP), CD8+-CD3+ lymphocytes and immunoglobulin G (IgG) were done. Patients were grouped according to type of HS. Clinical features and immunohistochemical changes were defined in these groups.


RESULTS
Available sera of 15 patients did not have any neuronal antibodies. Thirteen of 22 patients had HS type 1, three had HS type 2 and two had HS type 3. According to immunohistochemical investigations CD3+ and CD8+ T cell infiltration was more prominent in the hippocampus of patients with classical HS (International League Against Epilepsy (ILAE) Type 1 HS) and there was a significant negative correlation between epilepsy duration and numbers of CD3+-CD8+ lymphocytes in temporal lobe parenchyma.


CONCLUSION
The role of T cell-mediated immunopathology and immunopathological difference in a variety of drug resistant TLE-H2S patients was suggested. These findings can be helpful in understanding the epileptogenicity of HS.

synaptophysin, GFAP) stains. The ILAE classification system was employed to identify the HS types in our study: HS ILAE type 1 = classical hippocampal sclerosis with pronounced neuronal cell loss in all hippocampal subfields (in particular CA1 >80% cell loss; CA4 >40% neuronal cell loss); HS ILAE type 2 = hippocampal sclerosis with predominant neuronal cell loss in CA1 (>78%) and less severe neuronal cell loss in all other subfields (<25%); and HS ILAE type 3 = hippocampal sclerosis with predominant neuronal cell loss in CA4 (>45%).
Hippocampal specimens with gliosis only (no-HS) and cortical lamination abnormalities in the temporal lobe associated with HS, called FCD IIIa, were also defined. All pathological specimens were examined semiquantitatively in accordance with the latest ILAE report by an experienced neuropathologist (FS).

Immunohistochemical Investigation:
Brain tissues obtained from surgical specimens were also investigated by immunohistochemical analysis for the presence of inflammatory infiltrates (CD3 + -CD8 + T cells), IgG deposits, NMDAR and GAD expressing neurons.
During the epilepsy surgery, a biopsy sample of a diameter of 0.5-1 cm brain tissue including the temporal lobe and hippocampus samples were taken. Tissue samples were frozen in liquid nitrogen (-196 0 C) in the surgery room. Samples were then stored at -80 0 C until sectioning. A total of 22 fresh frozen brain samples were collected. 5 μm sections were cut using a cryostat-microtome (Leica CM 1900 UV) then mounted on positive charged slides and stored at −20 0 C. All samples were stained with methylene azure II and with haematoxylin and eosin for routine histologic evaluation.
For immunohistochemical evaluation, sections were fixed with precooled (in -20 0 C) acetone for 10 minutes and dried at room temperature. After fixation 0.3% H 2 O 2 was applied for 10 minutes. Sections were washed for 15 minutes with phosphate buffered saline, which was used at every step of washing. Serum blocking solution was applied for 10 minutes. After these preparation steps primary antibodies with predetermined concentrations applied for NeuN, anti-GFAP, anti-CD3, anti-CD8, anti-NMDAR and anti-GAD65 were applied at +4 0 C overnight. Next day after washing appropriate biotinylated secondary antibody was applied for 20 minutes at room temperature followed by washing and then application of peroxidase solution for 20 minutes. As a chromogen, DAB (3-3' diaminobenzidine) was applied for 3-10 minutes in average. After washing sections were counterstained with hematoxylene for 40 seconds. These staining procedures were followed with serial alcohol and xylene applications. Sections were then mounted. For Anti-Ig G antibody staining we used a polyclonal florescent anti-human Ig G antibody. After washing 10 minutes with PBS, sections were put into alcohol (96%) for 20 minutes. Following another washing step polyclonal florescent IgG antibody was applied for 45 minutes in dark room, sections were washed and then mounted with a water based DAPI containing medium.
Hippocampal and temporal lobe tissue samples of a Rasmussen encephalitis patient who underwent hemispherotomy with ATL-AH during the study period was investigated as a positive control with the same procedures of pathologic investigation and immunohistochemical analysis. Hippocampus and temporal lobe samples were analyzed for each antigen marker semiquantitatively in different magnifications for both antigen positive cell count and positivity grading. NeuN positive regions were used to determine neuron dense areas. Images of CD3, CD8 were captured at x400 magnification. We focused around the vessels and counted lymphocytes in five areas for each. NMDAR immunoreactivity was determined by counting positive cells in 10 areas at x100 magnification. GFAP and GAD antigen immunoreactivities were scored at x100 magnification with a scoring system between 0-3 (0: none, 1: mild, 2: moderate, 3: severe). The results of the stainings for the antibodies were scored by experienced investigators (AF and FK).

Data analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS 17.0) for Mac OS X. Categorical variables were examined by the chi-square test, Fisher's exact test as appropriate. The Mann-Whitney U test, independent-dependent samples t-test and Pearson's chi-square test were used to determine potentially significant differences, and a p value less than 0.05 was considered significant.
The effect of any risk factors for epilepsy (febrile convulsion, history of trauma, family history and status epilepticus) on the numbers of NMDAR positive cells or CD8 + -CD3 + lymphocytes in hippocampal or other temporal lobe tissues were analyzed by Mann-Whitney U tests. Immunopathological findings of patients according to type of HS were also analyzed by independent samples, Mann Whitney U tests.