2.2. Experimental Procedure

The animals (n = 6) per group were injected i.p. with saline solution (the control group) which were sacrificed immediately after injection to serve as time 0 control. Other group of animals (n = 6) was injected with single doses of 10 mg/kg KA [17]. Animals are treated with KA developing “have staring” spells; head nodding and several wet dog shakes during the first 20-30 min in the final phase present a recurrent limbic motor seizure evolving to a full motor limbic SE. The animals with extensive tonic-clonic seizures were included in the time course study and were sacrificed at 1, 6, 12, 24, and 48 h. For PTZ kindling, the animals were treated with a subconvulsant 3 doses of 25 mg/kg (body weight) of PTZ for i.p. each for 15 minutes and were sacrificed at 1, 12, 24, 48, and 72 h after the last injection [18]. Cerebral Antioxidant Enzymes Like SOD and CAT Essay. The PTZ injections induced generalized clonic or tonic seizures consistent with Racine scale, whose classification is as follows: 0—no reaction; 1—stereotypic mounting, eye blinking, and/or mild facial clonus; 2—head nodding and/or multiple facial clonus; 3—myoclonic jerks in the forelimbs; 4—clonic convulsions in the forelimbs with rearing; and 5—generalized clonic convulsions and loss of balance. The latency and duration of seizure were observed behaviorally [19, 20].

2.3. Histopathological and Immunohistochemical Study

The animals were deeply anesthetized with overdoses of pentobarbital (100 mg/kg i.p.) and perfused via the ascending aorta with phosphate-buffered saline (PBS), followed by 10% w/v buffered formalin solution at 4°C for histopathological examination. The whole brains were removed from the skull, were immersed in the same fixative, and after were cut with a matrix (coronal rodent brain matrix, EMS) at 2 mm thick [21]. The sections between −3.14 and −4.16 mm posterior to the bregma suture containing the dorsal hippocampus were selected using a Paxinos and Watson stereotaxic atlas [22]. Brain tissue samples were embedded in paraffin, were cut at 5 μ, and stained with cresyl violet. Pathological analysis was performed for all rats with a light microscope Axio Lab A1 Zeiss with Axiocam ICC5 camera at a magnification of 1000x.

For immunohistochemical studies, a LSAB System HRP (Dako, Carpinteria, CA), antisuperoxide dismutase polyclonal antibody (Abcam International, USA), antiglutathione peroxidase (Abcam International, USA), and catalase (Abcam International, USA) were used. In brief, according to Juárez-Rebollar et al. [21], the sections were deparaffinized, after hydrated with decreasing alcohol concentrations and washed three times for 3 min each time in 0.01 M phosphate-buffered saline (PBS, pH 7.4) for heat-induced epitope retrieval; the sections were boiled in citrate buffer (pH 6 or 9) in a microwave oven for 2 × 10 min. The sections were preincubated with 0.3% hydrogen peroxide in PBS and later incubated with SOD antibody (1 : 100), GPx (1 : 100), and CAT (1 : 100) by 90 min at room temperature. Slices were washed two times with PBS for 2 min followed by incubation with a secondary biotinylated antisera and then immersed in avidin–biotin peroxidase complex (LSAB System HRP, Dako, Carpinteria, CA) for 20 min at room temperature. The negative control sections were treated in the same way as described above except for each primary antibody was omitted. The immune reaction resulted in the oxidation of the 3,3′-diaminobenzidine by peroxidase (Liquid DAB, Dako, Carpinteria, CA) into an insoluble brown precipitate. Counterstaining with hematoxylin was performed after immunostaining. For immunostainings, changes in the number of these immunopositive cells were counted under a light microscope Axio Lab A1 Zeiss with Axiocam ICC5 camera at a magnification of ×1000. Cerebral Antioxidant Enzymes Like SOD and CAT Essay.

2.4. Morphometric Analysis

Three slides per rat were prepared and analyzed under light microscopy. Images from ten immersion oil fields were selected of the hippocampus area. Normal and damaged cells were counted in those images. The damaged cells were identified according to the criteria followed by Chang [23]. Percentages of damaged cells were obtained by dividing the number of damaged cells between total cells in the ten fields counted in each slide, and the total number of cells was expressed as the average of cells per field in each slide per rat. Similar analysis was performed to evaluate the immunohistochemical positive cells.

3.2. Histological and Immunohistochemical Analysis

The histopathological analysis shows the pyramidal cells with the nucleus, nucleus and cytoplasm, and glial cells and a neuropil with a normal appearance in the hippocampal region (Figures 1(a)–1(d) and 2(a)–2(d)). In the group treated with KA, no evident cell damage was observed at 1 h (Figure 1(e)); while at 6 and 12 h, the damage to the pyramidal cells is observed with an increase with the presence of hyperchromatic nuclei, loss of pyramidal cells, and interstitial edema (Figures 1(i) and 1(m)). At 24 and 48 h, the damage is observed as severe damage in the pyramidal cells of the hippocampus; there are numerous pyknotic cells, cellular atrophy, loss of cells, presence of intense interstitial edema, and neuronal necrosis in the hippocampal subfields CA1 and CA3 (Figures 1(q) and 1(u)). On the other hand, in the group treated with PTZ, no damage was observed in the pyramidal cells at 1 h of treatment (Figure 2(e)). At 12 and 24 h, the damage was evident of nuclear changes, presence of pyknotic pyramidal cells, and numerous nuclei with karyolysis (Figures 2(i) and 2(m)); at 48 and 72 h (Figures 2(q)and 2(u)), there is an increase in the loss of pyramidal cells, severe karyolysis with lack of nuclei, and intense interstitial edema. Cerebral Antioxidant Enzymes Like SOD and CAT Essay.