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Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Abstract:  Cell damage and spatial localization deficits are often reported as long-term consequences of pilocarpine-induced status epilepticus. In this study, we investigated the neuroprotective effects of repeated drug administration after long-lasting status epilepticus. Groups of six to eight Wistar rats received microinjections of pilocarpine (2.4 mg/µl, 1 µl) in the right dorsal hippocampus to induce a status epilepticus, which was attenuated by thiopental injection (35 mg/kg, i.p.) 3 hrs after onset. Treatments consisted of i.p. administration of diazepam, ketamine, carbamazepine, or phenytoin at 4, 28, 52, and 76 hr after the onset of status epilepticus. Two days after the treatments, rats were tested in the Morris water maze and 1 week after the cognitive tests, their brains were submitted to histology to perform haematoxylin and eosin staining and glial fibrillary acidic protein (GFAP) immunofluorescence detection. Post-status epilepticus rats exhibited extensive gliosis and cell loss in the hippocampal CA1, CA3 (70% cell loss for both areas) and dentate gyrus (60%). Administration of all drugs reduced cell loss in the hippocampus, with best effects observed in brains slices of diazepam-treated animals, which showed less than 30% of loss in the three areas and decreased GFAP immunolabelling. Treatments improved spatial navigation during training trials and probe trial, with exception of ketamine. Interestingly, in the probe trial, only diazepam-treated animals showed preference for the goal quadrant. Our data point to significant neuroprotective effects of repeated administration of diazepam against status epilepticus-induced cell damage and cognitive disturbances.

Epilepsy is a group of brain disorders mostly characterized by unpredicted, recurrent and spontaneous seizures that often have an impact on patients’ quality of life [1–3]. In most cases, treatments consist of keeping patients seizure-free with chronic use of anti-epileptic drugs. However, none of the currently used anti-epileptic drugs were developed to alter the natural course of disease, that is, the epileptogenic process through which the brain undergoes as seizures occur [4]. Indeed, the examination of the neuronal tissue of experimental animals as well as epileptic patients has pointed to irreversible morphological, physiological, and biochemical changes [5–7]. In this respect, the most commonly observed alterations are: extensive neuronal loss, hippocampal mossy fiber sprouting, tissue hyperexcitability, and changes in receptor subunit composition [8]. Whether these alterations occur during the epileptogenic process or due to repetitive seizures is still a matter of discussion [6,9].

A number of studies using animal models in experimental research have gathered information on the neuroprotective properties of established and novel anti-epileptic drugs, with special reference to the self-sustained status epilepticus and the amygdala-kindling models, which mimic the most common type of epilepsy; the temporal lobe epilepsy [1,4]. In most of these studies, the administration of a single dose of the drug after the insult diminishes the frequency and severity of recurrent seizures but does not prevent the occurrence of epilepsy [1,4,9].

Chronic models of temporal lobe epilepsy consist of applying a chemical or electrical stimulus, which triggers a long-lasting ictal activity [10]. The administration of the cholinergic agonist, pilocarpine, systemically or centrally in rats, elicits a status epilepticus that may last for several hours and in some cases lead to the death of the animal [11]. Therefore, in order to diminish the incidence of death among experimental animals, most researchers stop behavioural status epilepticus with the administration of anaesthetics [12,13]. After status epilepticus, animals go through a highly variable silent or latent period in which epileptogenesis is considered to occur, and at the end, spontaneous and recurrent seizures start in a period commonly named as chronic phase [11]. Post-status epilepticus recurrent seizures differ from the initial status epilepticus in behaviour and pharmacology, as they result from the hyperexcitation of different neural sites [4,14].

Most studies apply single doses of neuroprotective drugs before and after status epilepticus or focus on the treatment of recurrent seizures. In contrast, our work aims at the examination of the efficacy of sub-chronic treatment immediately after the pilocarpine-induced status epilepticus using four commonly used drugs with distinct modes of action. As parameters, we chose to analyse the status epilepticus-induced neuronal damage as well as, rodents’ spatial learning and memory deficits in the Morris water maze.

Experimental procedures.

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

This work was approved by the Ethics Committee for Experimental Animals at the University Campus (CEUA-RP) that follows the Guidelines of the Brazilian College of Animal Experimentation; Guiding Principles for Research involving Animals and Human Beings; American Physiological Society and Ethical Guidelines for investigations of Experimental Pain in Conscious Animals. Also, every effort was made to avoid unnecessary stress and pain to the experimental animals.

Animals and surgery.

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Adult male Wistar rats (220–250 g) from the animal housing of the University Campus of Ribeirão Preto were used in the assays. The animals were kept in wire-mesh cages in a room with a 12-hr dark/light cycle (lights on at 7:00 a.m.) with standard laboratory rat food and water ad libitum. Also, conditions of luminosity and temperature (22°) were kept constant in the housing and experimental rooms.

Two days after arrival, animals were anaesthetized with sodium thiopental 40 mg/kg (Cristalia, Brazil) for stereotaxic implantation of stainless steel guide cannulae in the dorsal hippocampus following coordinates: AP: −3.8 mm, ML: −2.6 mm, DV: −2.8 mm, from bregma [15]. Cannulae were attached to the skull with acrylic resin, anchored with stainless steel screws and temporarily sealed with a stainless steel wire to protect it from obstruction. Animals were allowed to rest for 5–7 days to recover from surgery.

Pilocarpine-induced status epilepticus and drug treatment.

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

After recovering from surgery, some rats were injected with pilocarpine (2.4 mg/µl; l µl/1 min., Sigma-Aldrich, USA) in the right dorsal hippocampus and observed for the onset of status epilepticus, which consisted of one non-interrupted generalized seizure or seizures without recovery [16,17]. Seizures were behaviourally assessed using Racine scale for limbic seizures [18]. Three hours after the onset of status epilepticus, thiopental was administered (30 mg/kg, via i.p., Cristalia, Brazil) in order to attenuate behavioural seizures. One hour after thiopental administration, groups of animals (n = 6, each) were then submitted to different treatments. Treatments consisted of daily injections of ketamine (50 mg/kg, i.p., Ketalar®, Parke Davis Warner Lambert, Brazil), diazepam (2 mg/kg, i.p., Sanofi-Synthelabo, Brazil), carbamazepine (120 mg/kg, i.p., Tegretol®, Novartis Biosciences, Brazil) and phenytoin (Hidantal® 60 mg/kg, i.p., Hoechst Marion Roussel, Brazil), whereas status epilepticus-untreated animals were given saline injections (i.p.). Drugs and saline were injected in the same volume (0.2 ml) at 4, 28, 52, and 76 hrs after status epilepticus onset. All doses were chosen from previous data on anticonvulsant screening [19–21].

Treatment lasted for 4 days after which, 2 days were allowed before cognitive testing started in order to avoid drug-induced impairments. Control groups of animals (n = 5, each group), not injected with pilocarpine, where injected with all other drugs in order to assess the effects of treatments over spatial memory.

Morris water maze.

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

In these experiments, rats were submitted to a training period of 4 days in the Morris water maze in order to check cognitive impairments in spatial learning and memory [22]. The water maze apparatus consisted of a white polyethylene circular pool (diameter 140 cm, depth 50 cm), filled with water and milk up to 25 cm to the boards and was divided in four quadrants. An invisible platform was placed in the centre of a fixed quadrant (goal quadrant) and was not removed until the completion of the test.

Each training trial consisted of putting the animals on randomly chosen quadrants of the pool, except the platform quadrant. Animals were allowed to explore the pool for 90 sec. after which they were gently placed over the platform in order to rest for 30 sec. Animals were submitted to six attempts and the latencies to find the platform in the training periods were recorded. After the last training attempt, the platform was removed from the pool for the probe trial session when the occupancy in each quadrant of the pool was quantified.

Histological procedures.

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

At the end of the experimental period, animals were deeply anaesthetized with an overdose of thiopental and perfused in the left ventricle with saline 0.9% solution (40 ml at 4°) followed by paraformaldehyde 4% (200 ml, phosphate-buffered saline 0.5 M, pH 7.4, cooled) for 15 min. in a constant pressure of 50 mmHg. Next, brains were removed, disconnected from cerebellum and olfactory bulb and fixed in a fresh fixative paraformaldehyde solution (4%) for 12 hrs. After that, routine paraffin embedding was performed and all brains were identically processed in order to minimize the effects of differential shrinkage. Next, tissues were cut in 10 µm-sections using an 820″ Spencer microtome (American Optical Corporation, USA). Some sections were stained with haematoxylin and eosin, whereas other sections were submitted to immunofluorescence protocol for GFAP.

GFAP immunofluorescence detection.

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Before immunolabelling, slides were deparaffinized (at 58°), hydrated and washed in phosphate-buffered saline (0.05 M, pH 7.6). Next, slides were immersed in the first serum blocking solution (phosphate-buffered saline, BSA 2%, Triton X 0.5%) overnight at 4°. In the morning after, slides were immersed in the second serum blocking solution (phosphate-buffered saline, BSA 1%, Triton X 0.1%) at 37°, for 30 min. After blockade, sections were incubated with mouse primary antibody anti-GFAP (anti-human, Dakocytomation, Denmark) diluted in the first serum blocking solution (1 : 500 µl) at 37% for 2 hrs and then washed three times in phosphate-buffered saline (10 min. each). Next, slides were incubated with fluorescein isothiocyanate (FITC)-conjugated goat secondary antibody (anti-mouse IgG, Dakocytomation, Denmark) diluted in second serum blocking solution (1 : 200 µl) for 1 hr, after which the slides were washed three times in phosphate-buffered saline (10 min. each) and further mounted with coverslips using a solution containing phosphate-buffered saline and glycerol (1 : 1, v/v) and examined in a fluorescence microscope (Leica DM 4500 B microscope, Leica Microsystems, Germany). Immunofluorescence was evaluated qualitatively. All used reagents were purchased from Sigma-Aldrich.

Quantification of neuronal damage.

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Cell density estimates were performed in the sections (5 sections per each animal) selected at the same stereotaxic coordinates for all animals (AP: −2.8), according to the Atlas of Paxinos and Watson [15] in the hippocampal CA1, CA3 and dentate gyrus. The estimated number of neurons, diameter of the nucleus of each neuron and the size of histological areas were obtained using the software Q-Win (Leica Microsystems). The images were acquired with a Leica DM 4500 B microscope (Leica Microsystems) in 400-fold enlargements in 3 adjacent regions for each slice and were digitized by the Leica DFC 300FX camera (Leica Microsystems).

Data from these experiments were normalized using the Abercrombie correction method [23] according to the following formula:

  • image

In this formula, N stands for the real number of cells in each mm2, n the observed number of neurons, T section thickness, D mean diameter of neuronal nucleus and A the area of cell counts in mm2.

Statistical analysis.

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Data from cell counts for each hippocampal area were submitted to one-way anova followed by Student–Newman–Keuls post-test. Data from Morris water maze were analysed by anova with repeated measures (RM-anova), followed by Student–Newman–Keuls post-test. All data are demonstrated by means ± S.E.M. These analyses were performed using spss software, version 13.0 (USA, 2004).

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Pilocarpine-induced status epilepticus and drug treatment.

Within approximately 30 min. after hippocampal injection, 70% of rats developed behavioural seizures. The first signs consisted of orofacial automatisms, hypersalivation, body tremors that evolved to rearing, falling and generalized clonus. In most animals, a single injection of thiopental (35 mg/kg, i.p.) attenuated behavioural seizures, whereas in a small percentage of animals (5%) one additional injection of thiopental (10 mg/kg, i.p.) was required. One hour after the interruption of behavioural status epilepticus, the treatment was initiated, at which time animals were unresponsive to environmental stimulation and partially anaesthetized. We did not observe any strong side-effects due to daily drug treatment, except for ketamine that induced strong depressant effects over respiration and nervous system.

Morris water maze.

Data from all training trials are summarized in fig. 1 (A and B). Saline-injected control animals rapidly learned to find the hidden platform, as demonstrated by the progressive decrease in escape latencies, whereas it took longer for untreated status epilepticus animals reach the platform. RM-anova pointed to significant effects of training trials (F[3,27] = 24.094, P < 0.0001) and drugs (F[5,29] = 20.407, P < 0.0001), whereas training versus drugs interaction did not reach statistical significance (F[15,77] = 1.759, P = 0.057) (fig. 1A).

image

Figure 1. Spatial navigation of rats during the acquisition training period in the Morris water maze. (A) Status epilepticus (SE). Diazepam (DZP). Ketamine (KET). Carbamazepine (CBZ). Phenytoin (PHN). Escape latencies (mean + S.E.M.) of saline control animals (saline Non-SE); pilocarpine-induced SE with thiopental as post-treatment (SE + Thio + Saline); pilocarpine-induced SE treated with thiopental + ketamine (SE + Thio + KET); thiopental + diazepam (SE + Thio + DZP); thiopental + carbamazepine (SE + Thio + CBZ) and thiopental + phenytoin (SE + Thio + PHN). (B) Control groups of control animals treated with saline and anti-epileptic drugs.

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On the first training day, the escape latencies of saline-treated animals were different from untreated status epilepticus and ketamine-treated animals (F[5,29] = 5.7, P = 0.006 and 0.022, respectively). On the second day, anova analysis pointed to significant differences of escape latencies of pilocarpine-treated rats in comparison with diazepam- and saline-treated healthy animals (F[5,29] = 9.762, P = 0.0001, in both the cases). In the third and fourth training days, rats treated with diazepam and carbamazepine reached platform as fast as control non-status epilepticus animals, and these latencies were significantly different from pilocarpine-treated animals (F[5,29] = 14.021 and 11.428, P < 0.01). Although escape latencies for both groups, ketamine- and phenytoin-treated status epilepticus animals were decreased along training sections, these treatments failed to reach statistical significance when compared to status epilepticus not treated animals.

Data from control non-status epilepticus animals treated with anti-epileptic drugs did not differ from saline control animals, regarding training trials. Thus, a significant effect of training trials was observed (F[3,12] = 60.317, P < 0.0001), whereas neither drugs nor training versus drugs interaction were found statistically significant (F[4,23] = 1.613, P = 0.205 and F[12,59] = 0.824, P = 0.625, respectively) (fig. 1B).

During probe trial with removed platform, escape latencies of healthy control rats were similar to status epilepticus rats treated with diazepam, carbamazepine and phenytoin (F[5,29] = 3.374, P = 0.0227), whereas no differences were observed for untreated status epilepticus and ketamine-treated animals (fig. 2A). With regard to spatial retention, the RM-anova revealed statistical significance among groups considering quadrant (F[3,20] = 23.038, P < 0.001), drug treatment (F[5,29] = 4.895, P = 0.0029) and quadrant versus treatment interaction (F[15,77] = 2.025, P = 0.027). Post hoc analyses showed that untreated status epilepticus animals did not show place preference to goal quadrant (P = 0.4649) (fig. 2B). In this case, only diazepam-treated animals and saline-treated non-status epilepticus animals spent more time in the goal quadrant (quadrant 1), differing from spatial occupation of the other groups (F[5,29] = 4.895, P < 0.05).

image

Figure 2. Mean escape latencies during the probe trial in the Morris water maze. (A) Status epilepticus (SE). Diazepam (DZP). Ketamine (KET). Carbamazepine (CBZ). Phenytoin (PHN). Escape latencies (mean + S.E.M.) of saline control animals (saline Non-SE); pilocarpine-induced SE with thiopental as post-treatment (SE +Thio + Saline); pilocarpine-induced SE treated with thiopental + ketamine (SE + Thio + KET); thiopental + diazepam (SE + Thio + DZP); thiopental + carbamazepine (SE + Thio + CBZ) and thiopental + phenytoin (SE + Thio + PHN). (B) Pool occupancy in quadrants (Quad) of animals with different anti-epileptic drugs. Data were analysed by RM-anova and Student–Newman–Keuls as post-test. *P < 0.05 and **P < 0.001, statistically different from saline non-SE animals.

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Histology.

Histopathologic analysis of neuronal tissues.  Qualitative analysis of brain sections in untreated status epilepticus animals revealed severe damage throughout the right (ipsilateral) dorsal hippocampus. Shrunken neurons, nuclear pycnosis and cytoplasmic vacuolar degeneration were found in all examined areas but mostly in the pyramidal cell layer of CA1 and CA3 (fig. 3). In contrast, only a moderate cellular damage was observed in the dentate gyrus granule layer. Moreover, tissue pieces of untreated status epilepticus rats exhibited extensive gliosis mostly in the hilar region of the dentate gyrus and disorganization of pyramidal cell layers of CA1 and CA3. Brains of status epilepticus rats treated with diazepam had pyramidal organized cell layers and few neurons with altered morphology. Status epilepticus rats treated with other drugs had various degrees of histological damage but generally preserved layer organization.

image

Figure 3. Cellular aspects of sections stained with routine haematoxylin and eosin technique showing hippocampal formation of rats with different treatments: (A) Saline non-SE; (B) SE + thiopental + saline; (C) SE + thiopental + ketamine; (D) SE + thiopental + diazepam; (E) SE + thiopental + carbamazepine; F. SE + thiopental + phenytoin. Scale bar equal to 200 µm. Arrows show areas of neural damage and gliosis.

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Quantification of neuronal damage.

Mean cell densities (number of cells/mm2) of each treatment in the hippocampal areas are shown in fig. 4. These data show that tissue pieces from pilocarpine untreated animals exhibited marked cell loss in all three examined areas; 89% in CA1, 90% in CA3 and 50% in dentate gyrus, as compared to mean cell densities of control non-status epilepticus animals. One-way anovas pointed to statistical significance among treatments regarding cell density in CA1 (F[5,37] = 17.51, P < 0.0001), CA3 (F[5,37] = 19.34, P < 0.0001) and dentate gyrus (F[5,37] = 14.24, P < 0.0001). Diazepam prevented the loss of pyramidal neurons in CA1, CA3 and dentate gyrus that exhibited 88%, 73.5%, and 75% of viable neurons (P < 0.0001, in CA1 and CA3 and P < 0.01 in dentate gyrus, as compared to status epilepticus untreated animals). Carbamazepine treatment led to partial neuroprotection of CA1 and dentate gyrus. In these regions, cell density estimates pointed to 65% and 69% of surviving pyramidal neurons (P < 0.05, in the two regions as compared to status epilepticus untreated animals). In CA3, only 44% of pyramidal neurons remained viable, not differing from cell densities status epilepticus untreated rats. Neuronal cell loss was slightly attenuated by ketamine and phenytoin treatments in comparison to control non-status epilepticus animals in CA1 (P < 0.001), CA3 (P < 0.05) but not in dentate gyrus. In the hippocampi of animals treated with these drugs, the percentages of surviving neurons were 50.5% and 59%; 45.5% and 42%, and 59% and 52%, for the three regions, respectively.

image

Figure 4. Neuronal densities in the three analysed regions of the hippocampus; CA1, CA3 and dentate gyrus of rats with different treatments. Status epilepticus (SE). Diazepam (DZP). Ketamine (KET). Carbamazepine (CBZ). Phenytoin (PHN). Data represent mean number of neurons per mm2 in brain sections of saline control animals (saline non-SE); pilocarpine-induced SE with thiopental as post-treatment (SE + Thio + Saline); pilocarpine-induced SE treated with thiopental + ketamine (SE + Thio + KET); thiopental + diazepam (SE + Thio + DZP); thiopental + carbamazepine (SE + Thio + CBZ) and thiopental + phenytoin (SE + Thio + PHN). Error bars demonstrate values for S.E.M. calculated by one-way anova and Student–Newman–Keuls as post-test. *P < 0.05; **P < 0.01 and ***P < 0.001. a, b, and c are statistically different from saline non-SE animals, SE + Thio + Saline and SE + Thio + DZP, respectively.

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CA1 of diazepam-treated rats had the higher number of neuronal cells (P < 0.0001, as compared to ketamine and phenytoin and P < 0.05, as compared to carbamazepine). In CA3, we detected statistical significance when comparing diazepam and phenytoin treatments (P < 0.5), whereas in dentate gyrus no statistical difference was detected among treatments (P < 0.05).

GFAP immunofluorescence detection.

In the hippocampi of untreated status epilepticus animals and those treated with phenytoin, strong GFAP immunolabelling was detected in the CA1, CA3 and dentate gyrus with the strongest labelling detected in the hilar region (fig. 5). Also, astrocytes of untreated status epilepticus animals and animals treated with phenytoin showed body hyperplasy and much of the immunostaining was detected in the cell processes rather than cellular body. Post status epilepticus animals treated with ketamine and carbamazepine exhibited moderate GFAP staining, whereas brain sections of diazepam-treated animals presented very weak GFAP staining, similarly to control non-status epilepticus animals.

image

Figure 5. GFAP-positive astrocytes in the hilar region of the hippocampus of rats 15 days after pilocarpine-induced status epilepticus. Animals post-treated with thiopental + saline (Pilo + Thio + Saline). Animals post-treated with thiopental + phenytoin (Pilo + Thio + PHN). Intense astrocyte staining was observed in brain sections of both groups of animals. Scale bar equal to 50 µm.

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

The present work investigated the effects of subchronic administration of diazepam, carbamazepine, phenytoin and ketamine after a 3-hr pilocarpine-induced status epilepticus, in which behavioural seizures were interrupted by the administration of thiopental. As we did not perform video-EEG monitoring, we can not discuss the effects of these associations on subclinical seizures. However, our data showed that the association of all drugs with thiopental inhibited to some extent the massive neuronal loss in the hippocampus of post-status epilepticus animals treated with the single administration of thiopental. However, only diazepam and carbamazepine ameliorated cognitive impairments observed in the water maze test run with status epilepticus animals. Moreover, reactive gliosis was reported in the hippocampus of rats submitted to status epilepticus and post-treated with all drugs except diazepam.

After a status epilepticus of sufficient duration, more than 2 hr, a massive neuronal loss may be seen in the hippocampus that undergoes synaptic reorganization leading to abnormal circuitries in a highly heterogeneous silent period that may last from 4–23 days [10,24]. During this seizure-free period, impairment in the spatial learning and memory of rats is often observed [25,26] being associated with the size of neuronal damage and severity and duration of the initial status epilepticus [27,28]. Therefore, in our work we chose to attenuate status epilepticus after 3 hr, allowing sufficient duration of status epilepticus to produce neuronal damage [24] and minimizing the differences among animals. However, as we did not use EEG monitoring, we can not rule out the effects of treatments over subclinical status epilepticus and we can not either assure that the anticonvulsant effects might be responsible for the observed effects.

In a previous work, single administration of diazepam 2 hrs after the onset of status epilepticus, induced by the electrical stimulation of the amygdala reduced cell loss and lowered the frequency of recurrent seizures [29]. Conversely, a recent work demonstrated that the combination of a single dose of diazepam after the onset of pilocarpine-induced status epilepticus, with daily topiramate, was only partially neuroprotective against cell loss (14 days after status epilepticus) and did not stop spontaneous seizure occurrence [30]. Our findings point to neuroprotective effects of diazepam that could be attributed to treatment, that is, daily injections rather than a single administration. Indeed, according to Fujikawa [24] cell death still occur up to 72 hrs after the end of status epilepticus, and in a recent work abnormal discharges were continuously observed as late as 72 hrs after pilocarpine-induced status epilepticus [31]. Another point is the reactive astrocytosis which is a common finding in brains of animals submitted to virtually all insults [32,33]. Our data showed that in untreated status epilepticus animals, reactive astrocytes were visualized in all regions of hippocampus, mostly in the hilar region of dentate gyrus. In this case, phenytoin, ketamine and carbamazepine did not prevent reactive gliosis, whereas only a slight increase in GFAP expression was noted in brains of rats treated with diazepam when compared to healthy animals. Our work also describes a partial neuroprotective effect for the daily administration of carbamazepine, similarly to that previously demonstrated by Capella and Lemos [34]. In addition, previous works have suggested the neuroprotective effects of carbamazepine in ischemic/hypoxic model of neuronal injury [35]. Regarding the NMDA antagonist ketamine, a neuroprotective effect was observed on neuronal density but not in GFAP staining. Moreover, animals treated with ketamine were able to find the hidden platform in the Morris test with short latencies only in the last day of training, indicating cognitive deficits. In this point, our findings differ from previous data by Hort et al. [26] that demonstrated that a single injection of a high dose of ketamine (100 mg/kg) 15 min. after the onset of pilocarpine-induced status epilepticus together with clonazepam (120 min. after status epilepticus) inhibits the deterioration of spatial memory in rats trained in the Morris water maze before induction of status epilepticus.

The development of disease-modifying drugs has long been a matter of debate. Pitkänen [1] raises a question about the real clinical benefit of using anti-epileptic drugs if most works point to the occurrence of recurrent seizures after status epilepticus despite drug treatment. In a more recent work, the same group concludes that even partial suppression of seizure activity improved the outcome of rats submitted to self-sustained status epilepticus [29]. Another interesting point is the effect of the drug on patients’ functional recovery. Hernandez [36] demonstrated the delaying effect of the administration of diazepam or phenobarbital on the recovery of sensorimotor performance after head trauma. In contrast, the administration of carbamazepine and vigabatrin induced no effect.

As epilepsy is an important limiting clinical condition, more research is necessary in order to investigate benefits of neuroprotective effects of anti-epileptic drugs. These studies should focus on the many aspects of epileptogenesis, including cognitive function, spontaneous seizures and side-effect profile.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

This work was supported by the Brazilian National Research Council (CNPq), Brazilian Council for Research (Capes) and São Paulo State Research Foundation (FAPESP). Authors thank Professor Norberto Cysne Coimbra for providing some antibodies and Professors Luiz Tadeu de Moraes Figueiredo and Victor Hugo Aquino for the use of fluorescence microscope.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures.
  4. Animals and surgery.
  5. Pilocarpine-induced status epilepticus and drug treatment.
  6. Morris water maze.
  7. Histological procedures.
  8. GFAP immunofluorescence detection.
  9. Quantification of neuronal damage.
  10. Statistical analysis.
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References