Cases were selected from the pathology archives at the Departments of Neurology and Neurosurgery, Seoul National University Hospital, Seoul, Korea. The research was approved by the Hospital Committee on Human Ethics. The hippocampal specimens were obtained from 26 patients who had undergone temporal lobectomy between 2000 and 2006, including hippocampal resection, for the treatment of medically intractable mTLE with HS. Medically intractable patients were defined as those whose seizures were poorly controlled with two or more anticonvulsant drugs prescribed by an epileptologist, including at least one of the following: phenytoin, carbamazepine, and valproic acid.17 All patients went through a comprehensive clinical, electrophysiological, neuropsychological, and imaging evaluation before the epilepsy surgery.

Clinical data were obtained for each patient by reviewing all records available in the electronic medical records at the hospital. These data included the patient's sex, age at epilepsy onset, age at surgery, duration of epilepsy, average frequency of preoperative complex partial and generalized seizures, history of childhood febrile seizure (FS) or other significant IPI,18 age at FS or IPI, history of SE, and surgical outcomes.

The brain specimens were cryopreserved as tissue blocks for cryostat sectioning at a thickness of 7 µm. Coronal sections were taken from the right or left hippocampus, and every seventh section of the hippocampus (6 sections per specimen) was subjected to semiquantitative immunohistochemical analysis. All hippocampal specimens were processed identically to minimize the effect of differential shrinkage on neuronal density measurements. Histological evaluations were performed as described previously.19-21 Paraffin-embedded sections from selected HS patients were dewaxed, rehydrated through graded alcohols, and taken to water. Sections were microwaved for 15 minutes in 0.05 M EDTA (pH 7.5) and then allowed to cool for 20 minutes.

Sections were stained using the Nissl method and labeled with antinestin (1 : 100, Chemicon, Temecula, CA, USA) and anti-Prox1 antibodies (1 : 60, Abcam, Cambridge, UK). Nestin, which is a protein of the intermediate filament family, is typical of undifferentiated neural stem and progenitor cells.22 Prox1 is expressed in postmitotic dentate granule cells, is specific for this cell type in the adult rat dentate gyrus,23-25 and is used to identify ectopic granule cells following pilocarpine-induced SE.26,27 Biotinylated goat antimouse IgG (ABC, Sigma, Poole, UK) was used as the secondary antibody. The biotin signal was detected using 3,3'-diaminobenzidine (brown) and Vector VIP (purple). Prox1 expression was detected by nickel-enhanced diaminobenzidine peroxidase immunohistochemistry, as described previously.26,27 We also examined the extrahippocampal pathological findings. The surgically excised temporal lobe specimens were fixed in formaldehyde after extirpation and then stained with hematoxylin and eosin.

Histological analysis was performed in the predefined areas of consecutive coronal blocks of 6 serial sections. The cells of interest were evaluated in the CA3 area for hippocampal neuronal damage, the hilus for nestin immunoreactivities, and the granule cell layer for GCD. The region of interest was set to include the proximal, middle, and distal subfields of area CA3 stratum pyramidale (magnification ×400), the whole hilar area (magnification ×400), and the superior and inferior portions of the granule cell layer (magnification ×600). It is not possible to perform unbiased stereology in human surgical tissue samples where the entire region of interest is not present,28 and so we adapted the semiquantitative scoring system of histopathological results as follows. Neuronal loss was measured on a scale of 0-3, where grades 0, 1, 2, and 3 corresponded to 0%, 20%, 20-50%, and >50% of the neurons lost in the particular subfield, respectively. The mean and SD values of the score were calculated from the scored sections.28 Cell genesis was measured on a scale of 1-4, where grade 1 was "few or no nestin-positive cells present (<3 cells/high-power field, HPF)", grade 2 was "a few nestin-positive cells present (3-10 cells/HPF)", grade 3 was "a moderate number of nestin-positive cells present (10-100 cells/HPF)", and grade 4 was "a high number of nestin-positive cells present (>100 cells/HPF)." The mean and SD values were calculated from the scored sections for each area separately (modified from Pirttilä et al.28). GCD was also measured on a scale of 1-4, where grade 1 describes epileptic qualities and a nondispersed appearance, grade 2 describes granule cell bodies dispersed into the molecular layer forming an irregular outer border, grade 3 describes double-layer granule cell bodies organized into two layers, and grade 4 describes predominantly granule cell death (Fig. 1).3,28

The data are presented as mean±SD values. The Pearson correlation coefficient was determined to assess the associations between variables. Two groups were compared by chi-square analysis with arbitrary dichotomization, using SPSS for windows (version 15.0; SPSS Inc, Chicago, IL, USA). A value of p<0.05 was considered significant.

The clinical characteristics of the 26 patients, who comprised 15 men and 11 women who were aged 32.07±9.29 years (range, 16-43 years) at the time of surgery, are listed in Table 1. The age at the onset of epilepsy was 11.11±6.98 years (range, 1-27 years), and the duration of epilepsy at the time of collection was 17.76±10.58 years (range, 1-42 years). All patients had complex partial seizures, and the frequency of seizures per month was 4.25±5.98 (range, 0.5-30). In 16 patients (61.5%), secondary generalization frequently occurred more than once per month. A history of childhood FS was reported in 13 patients (50%), and 7 patients reported other relevant causative childhood events (IPI), for example head trauma, meningitis, encephalitis, and Reye syndrome. No patient reported a history of SE. Most of the patients (80.7%) became seizure-free after their epilepsy surgery.

The pathological grading scores of Nissl (for neuronal loss), nestin (for cell genesis), and Prox1 (for GCD) are listed in Table 2, based on the grading system presented in Fig. 1. Most patients showed a moderate-to-severe amount of neuronal loss: grade 3 in 12 patients (46.2%), grade 2 in 10 patients (38.5%), and grade 1 in 1 patient (3.8%). Most patients showed a mild-to-moderate amount of cell genesis: grade 1 in 8 patients (30.8%), grade 2 in 6 patients (23.1%), grade 3 in 11 patients (42.3%), and grade 4 in 1 patient (3.8%). All patients exhibited some degree of GCD: grade 2 in 13 patients (50%), grade 3 in 7 patients (26.9%), and grade 4 in 6 patients (23.1%). With regard to the extrahippocampal pathological findings, only one patient exhibited apparent cortical dysplasia on magnetic resonance imaging (MRI) and macroscopic examination. However, 11 patients (42.3%) exhibited MRI-negative microscopic dysplasia in the resected temporal lobe specimens on histological examination.

There were significant correlations between cell genesis and GCD (r=0.43, p=0.03) and between neuronal loss and GCD (r=0.73, p<0.01). Clinical variables, including age at onset, age at surgery, duration of epilepsy, and mean frequency of preoperative seizures, were not correlated with the pathological variables, including neuronal loss, cell genesis, and GCD (Table 3). We dichotomized the results of nestin immunoreactivity into a mild degree of cell genesis (grades 1 and 2) and a moderate-to-severe degree (grades 3 and 4). Moderate-to-severe cell genesis occurred more frequently in patients with a history of FS (p=0.03)(Fig. 2) and in those with frequent generalized seizures (p=0.026, both by chi-square test)(Fig. 2). Neuronal loss and GCD were not significantly correlated with FS or with the frequency of generalized seizures. The presence of temporal microscopic dysplasia was not significantly correlated with the other pathological findings, including neuronal loss, cell genesis, or GCD, or with any clinical variable including the history of FS and frequent generalized seizures.