© 2009 The Authors Doi: 10.1111/j.1742-7843.2009.00403.

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Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 470–477

Neuroprotective Effects of Diazepam, Carbamazepine, Phenytoin and

Blackwell Publishing Ltd

Ketamine after Pilocarpine-induced Status Epilepticus

Alexandra Olimpio Siqueira Cunha, Márcia Renata Mortari, José Luiz Liberato and Wagner Ferreira dos Santos
Neurobiology and Venoms Laboratory, Department of Biology, University of São Paulo, Ribeirão Preto, SP, Brazil
(Received 4 June 2008; Accepted 27 November 2008)

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 of the drug after the insult diminishes the frequency and
by unpredicted, recurrent and spontaneous seizures that severity of recurrent seizures but does not prevent the
often have an impact on patients’ quality of life [1–3]. In most occurrence of epilepsy [1,4,9].
cases, treatments consist of keeping patients seizure-free Chronic models of temporal lobe epilepsy consist of
with chronic use of anti-epileptic drugs. However, none of applying a chemical or electrical stimulus, which triggers a
the currently used anti-epileptic drugs were developed to long-lasting ictal activity [10]. The administration of the
alter the natural course of disease, that is, the epileptogenic cholinergic agonist, pilocarpine, systemically or centrally in
process through which the brain undergoes as seizures rats, elicits a status epilepticus that may last for several
occur [4]. Indeed, the examination of the neuronal tissue of hours and in some cases lead to the death of the animal [11].
experimental animals as well as epileptic patients has Therefore, in order to diminish the incidence of death among
pointed to irreversible morphological, physiological, and experimental animals, most researchers stop behavioural
biochemical changes [5–7]. In this respect, the most commonly status epilepticus with the administration of anaesthetics
observed alterations are: extensive neuronal loss, hippocampal [12,13]. After status epilepticus, animals go through a highly
mossy fiber sprouting, tissue hyperexcitability, and changes variable silent or latent period in which epileptogenesis is
in receptor subunit composition [8]. Whether these alterations considered to occur, and at the end, spontaneous and recurrent
occur during the epileptogenic process or due to repetitive seizures start in a period commonly named as chronic phase
seizures is still a matter of discussion [6,9]. [11]. Post-status epilepticus recurrent seizures differ from the
A number of studies using animal models in experimental initial status epilepticus in behaviour and pharmacology, as
research have gathered information on the neuroprotective they result from the hyperexcitation of different neural sites
properties of established and novel anti-epileptic drugs, with [4,14].
special reference to the self-sustained status epilepticus Most studies apply single doses of neuroprotective drugs
and the amygdala-kindling models, which mimic the most before and after status epilepticus or focus on the treatment
common type of epilepsy; the temporal lobe epilepsy [1,4]. of recurrent seizures. In contrast, our work aims at the
In most of these studies, the administration of a single dose examination of the efficacy of sub-chronic treatment
immediately after the pilocarpine-induced status epilepticus
using four commonly used drugs with distinct modes of
Author for correspondence: Wagner Ferreira dos Santos, Department
action. As parameters, we chose to analyse the status
of Biology, FFCLRP, University of São Paulo, Av. Bandeirantes,
14040-090, Ribeirão Preto, São Paulo, Brazil (fax +55 16 3602 4886, epilepticus-induced neuronal damage as well as, rodents’ spatial
e-mail [email protected]). learning and memory deficits in the Morris water maze.
 EFFECTS OF DRUG ON STATUS EPILEPTICUS 471

Experimental procedures. Morris water maze.

This work was approved by the Ethics Committee for In these experiments, rats were submitted to a training
Experimental Animals at the University Campus (CEUA- period of 4 days in the Morris water maze in order to check
RP) that follows the Guidelines of the Brazilian College of cognitive impairments in spatial learning and memory [22].
Animal Experimentation; Guiding Principles for Research The water maze apparatus consisted of a white polyethylene
involving Animals and Human Beings; American Physiological circular pool (diameter 140 cm, depth 50 cm), filled with
Society and Ethical Guidelines for investigations of Experi- water and milk up to 25 cm to the boards and was divided
mental Pain in Conscious Animals. Also, every effort was made in four quadrants. An invisible platform was placed in the
to avoid unnecessary stress and pain to the experimental animals. centre of a fixed quadrant (goal quadrant) and was not
removed until the completion of the test.
Animals and surgery. Each training trial consisted of putting the animals on
Adult male Wistar rats (220–250 g) from the animal housing randomly chosen quadrants of the pool, except the platform
of the University Campus of Ribeirão Preto were used in quadrant. Animals were allowed to explore the pool for
the assays. The animals were kept in wire-mesh cages in a 90 sec. after which they were gently placed over the platform
room with a 12-hr dark/light cycle (lights on at 7:00 a.m.) in order to rest for 30 sec. Animals were submitted to six
with standard laboratory rat food and water ad libitum. attempts and the latencies to find the platform in the
Also, conditions of luminosity and temperature (22°) were training periods were recorded. After the last training
kept constant in the housing and experimental rooms. attempt, the platform was removed from the pool for the
Two days after arrival, animals were anaesthetized with probe trial session when the occupancy in each quadrant of
sodium thiopental 40 mg/kg (Cristalia, Brazil) for stereotaxic the pool was quantified.
implantation of stainless steel guide cannulae in the dorsal
hippocampus following coordinates: AP: −3.8 mm, ML: Histological procedures.
−2.6 mm, DV: −2.8 mm, from bregma [15]. Cannulae were At the end of the experimental period, animals were deeply
attached to the skull with acrylic resin, anchored with anaesthetized with an overdose of thiopental and perfused
stainless steel screws and temporarily sealed with a stainless in the left ventricle with saline 0.9% solution (40 ml at 4°)
steel wire to protect it from obstruction. Animals were followed by paraformaldehyde 4% (200 ml, phosphate-
allowed to rest for 5–7 days to recover from surgery. buffered saline 0.5 M, pH 7.4, cooled) for 15 min. in a constant
pressure of 50 mmHg. Next, brains were removed, disconnected
Pilocarpine-induced status epilepticus and drug treatment. from cerebellum and olfactory bulb and fixed in a fresh
After recovering from surgery, some rats were injected with fixative paraformaldehyde solution (4%) for 12 hrs. After
pilocarpine (2.4 mg/µl; l µl/1 min., Sigma-Aldrich, USA) in that, routine paraffin embedding was performed and all
the right dorsal hippocampus and observed for the onset of brains were identically processed in order to minimize the
status epilepticus, which consisted of one non-interrupted effects of differential shrinkage. Next, tissues were cut in
generalized seizure or seizures without recovery [16,17]. 10 µm-sections using an 820″ Spencer microtome (American
Seizures were behaviourally assessed using Racine scale for Optical Corporation, USA). Some sections were stained
limbic seizures [18]. Three hours after the onset of status with haematoxylin and eosin, whereas other sections were
epilepticus, thiopental was administered (30 mg/kg, via i.p., submitted to immunofluorescence protocol for GFAP.
Cristalia, Brazil) in order to attenuate behavioural seizures.
One hour after thiopental administration, groups of animals GFAP immunofluorescence detection.
(n = 6, each) were then submitted to different treatments. Before immunolabelling, slides were deparaffinized (at 58°),
Treatments consisted of daily injections of ketamine (50 mg/kg, hydrated and washed in phosphate-buffered saline (0.05 M,
i.p., Ketalar®, Parke Davis Warner Lambert, Brazil), diazepam pH 7.6). Next, slides were immersed in the first serum
(2 mg/kg, i.p., Sanofi-Synthelabo, Brazil), carbamazepine blocking solution (phosphate-buffered saline, BSA 2%,
(120 mg/kg, i.p., Tegretol®, Novartis Biosciences, Brazil) Triton X 0.5%) overnight at 4°. In the morning after, slides
and phenytoin (Hidantal® 60 mg/kg, i.p., Hoechst Marion were immersed in the second serum blocking solution
Roussel, Brazil), whereas status epilepticus-untreated animals (phosphate-buffered saline, BSA 1%, Triton X 0.1%) at
were given saline injections (i.p.). Drugs and saline were 37°, for 30 min. After blockade, sections were incubated
injected in the same volume (0.2 ml) at 4, 28, 52, and 76 hrs with mouse primary antibody anti-GFAP (anti-human,
after status epilepticus onset. All doses were chosen from Dakocytomation, Denmark) diluted in the first serum
previous data on anticonvulsant screening [19 –21]. blocking solution (1 : 500 µl) at 37% for 2 hrs and then washed
Treatment lasted for 4 days after which, 2 days were three times in phosphate-buffered saline (10 min. each).
allowed before cognitive testing started in order to avoid Next, slides were incubated with fluorescein isothiocyanate
drug-induced impairments. Control groups of animals (FITC)-conjugated goat secondary antibody (anti-mouse IgG,
(n = 5, each group), not injected with pilocarpine, where Dakocytomation, Denmark) diluted in second serum blocking
injected with all other drugs in order to assess the effects of solution (1 : 200 µl) for 1 hr, after which the slides were
treatments over spatial memory. washed three times in phosphate-buffered saline (10 min. each)

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Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 470–477
472 ALEXANDRA OLIMPIO SIQUEIRA CUNHA ET AL.

and further mounted with coverslips using a solution con- Student–Newman–Keuls post-test. All data are demonstrated
taining phosphate-buffered saline and glycerol (1 : 1, v/v) by means ± S.E.M. These analyses were performed using
and examined in a fluorescence microscope (Leica DM 4500  software, version 13.0 (USA, 2004).
B microscope, Leica Microsystems, Germany). Immunoflu-
orescence was evaluated qualitatively. All used reagents were Results
purchased from Sigma-Aldrich.
Pilocarpine-induced status epilepticus and drug treatment.
Quantification of neuronal damage. Within approximately 30 min. after hippocampal injection,
Cell density estimates were performed in the sections (5 70% of rats developed behavioural seizures. The first signs
sections per each animal) selected at the same stereotaxic consisted of orofacial automatisms, hypersalivation, body
coordinates for all animals (AP: −2.8), according to the tremors that evolved to rearing, falling and generalized clonus.
Atlas of Paxinos and Watson [15] in the hippocampal CA1, In most animals, a single injection of thiopental (35 mg/kg,
CA3 and dentate gyrus. The estimated number of neurons, i.p.) attenuated behavioural seizures, whereas in a small
diameter of the nucleus of each neuron and the size of percentage of animals (5%) one additional injection of
histological areas were obtained using the software Q-Win thiopental (10 mg/kg, i.p.) was required. One hour after the
(Leica Microsystems). The images were acquired with a interruption of behavioural status epilepticus, the treatment
Leica DM 4500 B microscope (Leica Microsystems) in was initiated, at which time animals were unresponsive to
400-fold enlargements in 3 adjacent regions for each slice environmental stimulation and partially anaesthetized. We
and were digitized by the Leica DFC 300FX camera (Leica did not observe any strong side-effects due to daily drug
Microsystems). treatment, except for ketamine that induced strong depressant
Data from these experiments were normalized using the effects over respiration and nervous system.
Abercrombie correction method [23] according to the fol-
lowing formula: Morris water maze.
Data from all training trials are summarized in fig. 1 (A and
n[T / (T + D )]
N per mm 2 = B). Saline-injected control animals rapidly learned to find
A(mm 2 )
the hidden platform, as demonstrated by the progressive
In this formula, N stands for the real number of cells in each decrease in escape latencies, whereas it took longer for
mm2, n the observed number of neurons, T section thickness, untreated status epilepticus animals reach the platform.
D mean diameter of neuronal nucleus and A the area of cell RM- pointed to significant effects of training trials
counts in mm2. (F[3,27] = 24.094, P < 0.0001) and drugs (F[5,29] = 20.407,
P < 0.0001), whereas training versus drugs interaction did
Statistical analysis. not reach statistical significance (F[15,77] = 1.759, P = 0.057)
Data from cell counts for each hippocampal area were (fig. 1A).
submitted to one-way  followed by Student–Newman– On the first training day, the escape latencies of saline-treated
Keuls post-test. Data from Morris water maze were analysed animals were different from untreated status epilepticus and
by  with repeated measures (RM-), followed by ketamine-treated animals (F[5,29] = 5.7, P = 0.006 and 0.022,

Fig. 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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Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 470–477
 EFFECTS OF DRUG ON STATUS EPILEPTICUS 473

respectively). On the second day,  analysis pointed to animals did not show place preference to goal quadrant
significant differences of escape latencies of pilocarpine- (P = 0.4649) (fig. 2B). In this case, only diazepam-treated
treated rats in comparison with diazepam- and saline- animals and saline-treated non-status epilepticus animals
treated healthy animals (F[5,29] = 9.762, P = 0.0001, in both spent more time in the goal quadrant (quadrant 1), differing
the cases). In the third and fourth training days, rats treated from spatial occupation of the other groups (F[5,29] =
with diazepam and carbamazepine reached platform as fast 4.895, P < 0.05).
as control non-status epilepticus animals, and these latencies
were significantly different from pilocarpine-treated animals Histology.
(F[5,29] = 14.021 and 11.428, P < 0.01). Although escape Histopathologic analysis of neuronal tissues. Qualitative analysis
latencies for both groups, ketamine- and phenytoin-treated of brain sections in untreated status epilepticus animals
status epilepticus animals were decreased along training sec- revealed severe damage throughout the right (ipsilateral)
tions, these treatments failed to reach statistical significance dorsal hippocampus. Shrunken neurons, nuclear pycnosis
when compared to status epilepticus not treated animals. and cytoplasmic vacuolar degeneration were found in all
Data from control non-status epilepticus animals treated examined areas but mostly in the pyramidal cell layer of
with anti-epileptic drugs did not differ from saline control CA1 and CA3 (fig. 3). In contrast, only a moderate cellular
animals, regarding training trials. Thus, a significant effect damage was observed in the dentate gyrus granule layer.
of training trials was observed (F[3,12] = 60.317, P < Moreover, tissue pieces of untreated status epilepticus rats
0.0001), whereas neither drugs nor training versus drugs exhibited extensive gliosis mostly in the hilar region of the
interaction were found statistically significant (F[4,23] = dentate gyrus and disorganization of pyramidal cell layers
1.613, P = 0.205 and F[12,59] = 0.824, P = 0.625, respec- of CA1 and CA3. Brains of status epilepticus rats treated
tively) (fig. 1B). with diazepam had pyramidal organized cell layers and few
During probe trial with removed platform, escape laten- neurons with altered morphology. Status epilepticus rats
cies of healthy control rats were similar to status epilepticus treated with other drugs had various degrees of histological
rats treated with diazepam, carbamazepine and phenytoin damage but generally preserved layer organization.
(F[5,29] = 3.374, P = 0.0227), whereas no differences were
observed for untreated status epilepticus and ketamine- Quantification of neuronal damage.
treated animals (fig. 2A). With regard to spatial retention, Mean cell densities (number of cells/mm2) of each treatment
the RM- revealed statistical significance among in the hippocampal areas are shown in fig. 4. These data
groups considering quadrant (F[3,20] = 23.038, P < 0.001), show that tissue pieces from pilocarpine untreated animals
drug treatment (F[5,29] = 4.895, P = 0.0029) and quadrant exhibited marked cell loss in all three examined areas; 89%
versus treatment interaction (F[15,77] = 2.025, P = 0.027). in CA1, 90% in CA3 and 50% in dentate gyrus, as compared
Post hoc analyses showed that untreated status epilepticus to mean cell densities of control non-status epilepticus

Fig. 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-
 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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474 ALEXANDRA OLIMPIO SIQUEIRA CUNHA ET AL.

Fig. 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.

animals. One-way s pointed to statistical significance CA3, we detected statistical significance when comparing
among treatments regarding cell density in CA1 (F[5,37] = diazepam and phenytoin treatments (P < 0.5), whereas in
17.51, P < 0.0001), CA3 (F[5,37] = 19.34, P < 0.0001) and dentate gyrus no statistical difference was detected among
dentate gyrus (F[5,37] = 14.24, P < 0.0001). Diazepam treatments (P < 0.05).
prevented the loss of pyramidal neurons in CA1, CA3 and
dentate gyrus that exhibited 88%, 73.5%, and 75% of viable GFAP immunofluorescence detection.
neurons (P < 0.0001, in CA1 and CA3 and P < 0.01 in dentate In the hippocampi of untreated status epilepticus animals
gyrus, as compared to status epilepticus untreated animals). and those treated with phenytoin, strong GFAP immunola-
Carbamazepine treatment led to partial neuroprotection of belling was detected in the CA1, CA3 and dentate gyrus
CA1 and dentate gyrus. In these regions, cell density estimates with the strongest labelling detected in the hilar region
pointed to 65% and 69% of surviving pyramidal neurons (fig. 5). Also, astrocytes of untreated status epilepticus animals
(P < 0.05, in the two regions as compared to status epilepticus and animals treated with phenytoin showed body hyperplasy
untreated animals). In CA3, only 44% of pyramidal neurons and much of the immunostaining was detected in the cell
remained viable, not differing from cell densities status processes rather than cellular body. Post status epilepticus
epilepticus untreated rats. Neuronal cell loss was slightly animals treated with ketamine and carbamazepine exhibited
attenuated by ketamine and phenytoin treatments in com- moderate GFAP staining, whereas brain sections of diazepam-
parison to control non-status epilepticus animals in CA1 treated animals presented very weak GFAP staining, similarly
(P < 0.001), CA3 (P < 0.05) but not in dentate gyrus. In the to control non-status epilepticus animals.
hippocampi of animals treated with these drugs, the percentages
of surviving neurons were 50.5% and 59%; 45.5% and 42%,
Discussion
and 59% and 52%, for the three regions, respectively.
CA1 of diazepam-treated rats had the higher number of The present work investigated the effects of subchronic
neuronal cells (P < 0.0001, as compared to ketamine and administration of diazepam, carbamazepine, phenytoin and
phenytoin and P < 0.05, as compared to carbamazepine). In ketamine after a 3-hr pilocarpine-induced status epilepticus,

© 2009 The Authors

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 EFFECTS OF DRUG ON STATUS EPILEPTICUS 475

Fig. 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  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.

in which behavioural seizures were interrupted by the epilepticus animals. Moreover, reactive gliosis was reported
administration of thiopental. As we did not perform in the hippocampus of rats submitted to status epilepticus
video-EEG monitoring, we can not discuss the effects of and post-treated with all drugs except diazepam.
these associations on subclinical seizures. However, our data After a status epilepticus of sufficient duration, more than
showed that the association of all drugs with thiopental 2 hr, a massive neuronal loss may be seen in the hippocampus
inhibited to some extent the massive neuronal loss in the that undergoes synaptic reorganization leading to abnormal
hippocampus of post-status epilepticus animals treated with circuitries in a highly heterogeneous silent period that may
the single administration of thiopental. However, only last from 4–23 days [10,24]. During this seizure-free period,
diazepam and carbamazepine ameliorated cognitive impair- impairment in the spatial learning and memory of rats is
ments observed in the water maze test run with status often observed [25,26] being associated with the size of

Fig. 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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476 ALEXANDRA OLIMPIO SIQUEIRA CUNHA ET AL.

neuronal damage and severity and duration of the initial work, the same group concludes that even partial suppression
status epilepticus [27,28]. Therefore, in our work we chose to of seizure activity improved the outcome of rats submitted
attenuate status epilepticus after 3 hr, allowing sufficient to self-sustained status epilepticus [29]. Another interesting
duration of status epilepticus to produce neuronal damage point is the effect of the drug on patients’ functional recovery.
[24] and minimizing the differences among animals. How- Hernandez [36] demonstrated the delaying effect of the
ever, as we did not use EEG monitoring, we can not rule out administration of diazepam or phenobarbital on the recovery
the effects of treatments over subclinical status epilepticus of sensorimotor performance after head trauma. In contrast,
and we can not either assure that the anticonvulsant effects the administration of carbamazepine and vigabatrin induced
might be responsible for the observed effects. no effect.
In a previous work, single administration of diazepam As epilepsy is an important limiting clinical condition,
2 hrs after the onset of status epilepticus, induced by the more research is necessary in order to investigate benefits of
electrical stimulation of the amygdala reduced cell loss and neuroprotective effects of anti-epileptic drugs. These studies
lowered the frequency of recurrent seizures [29]. Conversely, should focus on the many aspects of epileptogenesis,
a recent work demonstrated that the combination of a single including cognitive function, spontaneous seizures and
dose of diazepam after the onset of pilocarpine-induced side-effect profile.
status epilepticus, with daily topiramate, was only partially
neuroprotective against cell loss (14 days after status Acknowledgements
epilepticus) and did not stop spontaneous seizure occurrence This work was supported by the Brazilian National
[30]. Our findings point to neuroprotective effects of Research Council (CNPq), Brazilian Council for Research
diazepam that could be attributed to treatment, that is, daily (Capes) and São Paulo State Research Foundation (FAPESP).
injections rather than a single administration. Indeed, Authors thank Professor Norberto Cysne Coimbra for
according to Fujikawa [24] cell death still occur up to 72 hrs providing some antibodies and Professors Luiz Tadeu de
after the end of status epilepticus, and in a recent work Moraes Figueiredo and Victor Hugo Aquino for the use of
abnormal discharges were continuously observed as late as fluorescence microscope.
72 hrs after pilocarpine-induced status epilepticus [31].
Another point is the reactive astrocytosis which is a common
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