Behavioral, morphological and electrographic aspects of pilocarpine-injected Wistar male rats that did not develop status epilepticus

Aspectos comportamentais, morfológicos e eletrográficos de ratos Wistar machos injetados com pilocarpina que não desenvolveram estado de mal epiléptico

Abstract

In order to investigate the temporal lobe epilepsy in humans, the chronic experimental model of epilepsy in rats, which uses a single and systemic dose of pilocarpine, has been used worldwide. In this model, the occurrence of Status Epilepticus (SE) in the model’s acute phase, has been mandatory and a crucial step for the successful development of the model’s chronic phase, where spontaneous and recurrent seizures (SRS) occur. However, different authors have shown that SE may not be necessary as postulated until now, since rats that received Pilo, but did not evolve in to SE (Non-SE rats) presented mossy fiber sprouting and spontaneous and recurrent seizures in the model’s chronic phase. Although such works have shown similar findings, they also have lasted some gaps. Considering this, our work aimed to investigate possible behavioral, morphological and electrographic alterations in the Pilo’s model chronic phase in rats that did not evolve to SE. For that, video-monitoring analysis was performed for 20 months, EEG recordings and histological analysis were performed by the end of the 20^th month in non-SE and control rats. No SRS were observed during observation period in Non-SE and control groups. Additionally, no alterations were observed in the EEG recordings (sleep-wake cycling was regular in both groups). Moreover, the isotropic fractionator technique showed no differences in number of hippocampal neuronal and non-neuronal cells between Non-SE and control groups. No mossy fiber sprouting was observed in Non-SE animals in both analyzed regions (CA3 and dentate gyrus). Behavioral, electrographic and histological findings suggest that Non-SE male rats do not become epileptic in the Pilo’s model chronic phase. According to results we observed that SE development is still necessary to SRS occurrence in the Pilo-induced model of epilepsy in male rats.

Keywords:
pilocarpine; temporal lobe epilepsy; status epilepticus; hippocampus; seizures

Resumo

Com o intuito de investigar a epilepsia do lobo temporal em humanos, o modelo experimental crônico de epilepsia em ratos, que utiliza uma dose única e sistêmica de pilocarpina, tem sido utilizado mundialmente. Neste modelo, a ocorrência de Status Epilepticus (SE) na fase aguda do modelo tem sido obrigatória, além de um passo crucial para o desenvolvimento bem-sucedido da fase crônica do modelo, onde ocorrem crises epilépticas espontâneas e recorrentes (CRE). Apesar disto, diferentes autores têm demonstrado que o SE pode não ser necessário como postulado até o momento, uma vez que ratos que receberam Pilo, mas não evoluíram para o SE (ratos não-SE) apresentaram brotamento de fibras musgosas e crises epilépticas espontâneas e recorrentes na fase crônica do modelo. Embora tais trabalhos tenham mostrado achados semelhantes, também deixaram algumas lacunas. Considerando isso, este trabalho teve como objetivo investigar possíveis alterações comportamentais, morfológicas e eletrográficas na fase crônica do modelo da Pilo em ratos que não evoluíram para SE. Para isto, foram realizadas análises de vídeo-monitoramento por 20 meses, gravações de EEG e análises histológicas até o final do 20º mês em ratos não-SE e controle. Nenhuma CRE foi identificada durante o período de observação nos grupos Não-SE e controle. Adicionado a isto, os registros de EEG não apontaram alterações, além do que, o ciclo sono-vigília foi regular em ambos os grupos. A técnica de fracionamento isotrópico não mostrou diferenças no número de células neuronais e não neuronais no hipocampo entre os grupos Não-SE e controle. Nenhum brotamento de fibras musgosas foi observado em animais Não-SE em ambas as regiões analisadas (CA3 e giro dentado). Achados comportamentais, eletrográficos e histológicos sugerem que ratos machos Não-SE não se tornam epilépticos na fase crônica do modelo de Pilo. Os resultados observamos direcionam para a necessidade do SE para a ocorrência de CREs no modelo de epilepsia induzido por Pilo em ratos.

Palavras-chave:
pilocarpina; epilepsia do lobo temporal; status epilepticus; hipocampo; crises epilépticas

1. Introduction

Temporal lobe epilepsy (TLE) is known for the disruption of the brain’s normal activity owing to the neuronal hyperexcitability of the temporal lobe areas (Schwartzkroin, 1986). This neurological disorder is characterized by the presence of focal seizures including or not secondary generalization (Shorvon, 1990). TLE deserves special attention since it is the most prevalent epileptic syndrome among adults (Sander, 2003), reaching 30% of all cases of epilepsy and may also curse with a pharmaco-resistant profile (Reyes-Garcia et al., 2018).

Animal models of seizures and epilepsy have been largely used to understand the physiological and behavioral changes associated with the human TLE (Kharatishvili et al., 2006; Pitkanen and McIntosh, 2006; Arida et al., 2007). The pilocarpine (Pilo) model of epilepsy is probably the most studied model for TLE, since it reproduces the main features of the human condition in rats and mice (Cavalheiro, 1995). The Pilo-induced status epilepticus (SE) leads to cell loss in several hippocampal and extrahippocampal structures and to spontaneous and recurrent seizures (SRSs) appearance (Turski et al., 1983; Leite et al., 1990). Briefly, after Pilo injection, animals present facial automatisms, akinesia, forelimb clonus, rearing, limbic seizures and, finally, evolve to SE, which lasts for 24h, characterizing the model’s acute phase. In the following days animals present normalization of behavioral and electrographic alterations – model’s silent period. Starting from days to weeks after Pilo administration, animals exhibit SRSs which lasts for the rest of their lives – model’s chronic period (Turski et al., 1983; Cavalheiro, 1995; Arida et al., 1999).

Regarding seizures, it has long been recognized that the hippocampus is particularly vulnerable to damage by prolonged seizures (Villamizar-Torres et al., 2024). In general, seizures last a brief time but occasionally become more prolonged and are considered as a separate entity (the SE) that has a high morbidity and mortality in humans (Treiman and Walker, 2006; Chen and Wasterlain, 2006). Following SE patients may present associated cognitive and neurological deficits, which is also a risk factor for the development of epilepsy (Hesdorffer et al., 1998). MRI studies have demonstrated progressive atrophy of the hippocampus in patients following SE (Vespa et al., 2010). Postmortem studies in epileptic patients that died after a SE have shown significant acute neuronal loss in the hippocampi (DeGiorgio et al., 1992).

In contrast with these findings, authors have shown that SE may not be a mandatory step to a rat reach the chronic phase of the Pilo’s model of epilepsy. A study found that 50% of male rats induced by Pilo and did not show SE (Non-SE rats) lost cells from the entorhinal cortex, while 17% of them showed mossy fiber sprouting (Scharfman et al., 2001). Other group observed SRS in Non-SE male rats under telemetry, 6 - 8 months after Pilo administration. Authors also reported commitment of hippocampus, piriform cortex and thalamus in MRI analysis, 1 year after Pilo injection (Navarro Mora et al., 2009). Another study observed seizure-like behaviors in castrated and non-castrated female rats. Authors described behavioral manifestations that resembled stages 1-3 of Racine Scale (Dal Pai et al., 2022).

It is known among researchers that not all animals that receive systemic pilocarpine in a dose higher than 320 mg/kg will evolve to SE. However, until now the SE condition shown by animals injected with Pilo has been considered a crucial step for the successful development of this experimental model (Covolan and Mello, 2000; Scharfman et al., 2001).

Considering the controversial results presented in literature, the small number of studies concerning the SE phenomena in the Pilo model, along with its possible clinical relevance for studies in the epilepsy field, this work aimed to investigate the possible behavioral, morphological and electrographic alterations in Non-SE male rats that received Pilo injection.

2. Material and Methods

2.1. Animals

Male adult rats, 60 days old, weight range between 200-250g were randomly selected. Animal facilities room was maintained at 21 ± 2°C with a 12h light/dark schedule, and food and water ad libitum throughout the experiment. All measures were taken to minimize animals’ pain or discomfort. Experimental protocols were approved by the ethics committee of the UNIFESP (#54712002/15).

2.2. Pilocarpine administration

Forty- five male rats were induced to experimental model of TLE by receiving a single dose of systemic Pilo hydrochloride through injection (350 mg/kg i.p.; Sigma) (Turski et al., 1984). Scopolamine methylnitrate (1 mg/kg s.c.; Sigma) was administered 30 minutes before Pilo to limit peripheral cholinergic effects (Turski et al., 1984). Diazepam (1mg/kg, ip, Santisa) and Tiopental (30 mg/kg, ip., Cristália) were administered to rats that evolved or not in to SE 4 hours after Pilo treatment. The next step was the rats hydration with a 3 ml ip. and 1ml sc. saline solution 0.9%. SE and Non-SE animals were set alone in their cages and covered with newspaper. For the next days, SE animals were hydrated and fed by mouth manually, with feed bran mixed with water and fruits 4 - 5 times a day, until they were able to feed itself. Rats were considered SE when uninterrupted seizures were observed for 4 hours. Racine Scale (Racine, 1972) was used for seizures identification. Animals that evolved to SE were included in other study. To the control group, another 10 male rats received a single injection of saline, in the same amount of Pilo given to the experimental group.

2.3. Video monitoring room

Non-SE and control groups were taken to the video monitoring room, which is equipped with IR-light video cameras coupled to a recording system, VD16E480C model, Intelbras (Brazil). Both groups were continuously monitored over 24h per day for 20 months, aiming the observation and quantify of SRS (Figure 1).

Figure 1
Schema of experimental procedures. Control group was submitted to the same procedures, except for the pilocarpine injection.

2.4. EEG recordings

After video-monitoring observation, for 20 months, EEG recordings were applied continuously for 96 hours, in 3 Non-SE and 3 control rats. For that, animals were submitted to a stereotaxic surgery for electrodes implantation in the brain. Rats were first in deeply anesthetized with ketamine-xylazine (4:1 1.0 ml/kg) and placed in a stereotaxic apparatus (Bland et al., 2007). Body temperature was maintained during surgery by a heating pad and spontaneous breathing was observed. A referential electrode was implanted in the nasal bone. Two jeweler's screws used as electrodes, were placed bilaterally over the posterior parietal cortex in a coronal plane to neocortical EEG. One bipolar twisted electrode was placed into the right hippocampal formation (CA1). Stereotaxic coordinates for hippocampal electrode were -3.8 mm posterior to bregma, +2.5 mm lateral to midline, and 2.8 mm ventral to the skull surface (Paxinos and Watson, 2007). After those electrodes were soldered in a pin socket and fixed to the skull with dental acrylic cement. Operated animals were set into the EEG room after the 7^th day of post-surgery recovering. Such rats were observed by two researchers blinded to the experiment and designated to determine the electrographic seizure activity and its association with motor seizures, recorded for a period of 4 days, 24h/day. The sleep-wake cycling was also analyzed. The adequate electrodes position above dorsal pyramidal cell layer and underneath, were verified through histology, applied to all implanted rats.

2.5. Sprouting of mossy cells

To the observation of mossy cells sprouting 4 Non-SE and 4 control rats were taken. For that, animals were anesthetized and perfused transcardially with 25 ml of Millonigs’s buffer (MB), followed by 50ml of 0.1% solution of sodium sulfide (Na₂S solution) dissolved in MB (pH 7.4), 100ml of 3% solution of glutaraldehyde in 0.1M phosphate buffer (pH 7.4) and, 200ml of 0.1% solution of sodium sulfide diluted in MB. Coronal brain sections, with 20µm thickness, were obtained with a vibratome for the visualization of dentate gyrus (DG) and CA3 regions. Slices obtained were mounted on glass slides and dried before staining procedure using the Neo-Timm, for mossy fibers visualization (Babb et al., 1991). Mossy fiber sprouting was assessed semi-quantitatively based on a scoring method in which the distribution of Timm granules in the DG and CA3 regions are scored from 0 - 5 (0- no granules and 5- dense laminar band of granules) (Cavazos et al., 1991). Histological slices were verified by three observers blinded to the experimental condition. Each observer graded a total of 40 images (5 slices/animal) containing the DG and CA3.

2.6. Quantification of hippocampal cells

To the quantification of total number of cells in the hippocampus, 7 Non-SE and 6 controls rats were perfused. Two animals of each group died during the anesthesia and perfusion procedures, resulting in 5 Non-SE and 4 controls. The counting of neuronal and non-neuronal cells was made using the isotropic fractionator technique (Herculano-Houzel and Lent, 2005). Briefly, rats were deeply anesthetized (Tionembutal, 50 mg-kg, i.p.) and perfused transcardially with a solution of 0.01M phosphate-buffered saline (PBS), followed by 4% formaldehyde in 0.1M phosphate-buffered (PB), pH 7.4. After perfusion, rats’ brain was removed immediately from the skull and post fixed in 4% paraformaldehyde in PB for 24h. Then, the hippocampal formation was dissected and mechanically dissociated in a saline solution with 0.1% Triton X-100 and turned into an isotropic suspension of isolated nuclei, kept homogeneous by agitation. The total number of cells was estimated by determining the number of nuclei in small aliquots stained with the fluorescent DNA marker 4’-6-diamidino-2-phenylindole dihydrochloride (DAPI) utilizing a Zeiss Axiovert 100 microscope with a 40x objective, using a hemocytometer for quantification (Neubauer chamber). To determine neuronal and non-neuronal cell number, samples were then incubated with the primary antibody against the neuron-specific nuclear protein (NeuN; 1:1000; Chemicon, MAB377B) at 4°C overnight and, subsequently, with secondary antibody conjugated to AlexaFluor® 555 (Molecular Probes) diluted in PBS (1:200) and 10% normal goat serum for 2h. The neuronal fraction in each sample was estimated by counting NeuN-labeled nuclei in at least 500 DAPI-stained nuclei and the number of non-neuronal nuclei was obtained by subtraction.

2.7. Statistical analysis

A non-parametric test was applied to compare the groups' neuronal and non-neuronal cell numbers. All results were presented as means and standard deviation (M ± SD) and significance was established at p<0.05 level. Analyses were performed using an IBM SPSS version 20.0 (IBM, Chicago, Armonk, NY, USA).

3. Results

3.1. Pilo injection

Ten male rats out of 45 treated with Pilo evolved into tonic-clonic seizures and died. Twenty-one showed behavioral SE for 4 hours and were included in another study. Fourteen animals did not present SE after treatment with Pilo. SE animals showed repeatedly limbic behaviors until behavioral SE was observed.

3.2. Behavioral analysis

Video-monitoring analysis of 14 Non-SE and 10 control rats were performed. As expected, the control group did not present seizures during the analyzed period. Moreover, animals from Non-SE group also did not present seizures.

3.3. EEG recording

EEG analysis, performed in 3 Non-SE and 3 controls, did not show electrographic activity suggestive of seizure. Sleep-wake cycling of both groups were analyzed but no differences were observed. The wakefulness phase was characterized by desynchronization, high frequency with low amplitude waves in the cerebral cortex. Theta hippocampal activity were also similar in both groups. During the slow-wave sleep phase, cortical synchronization of high amplitude with low frequency waves and hippocampal delta waves were observed in Non-SE and control animals. Moreover, no alterations in the paradoxical sleep were observed. Cortical desynchronization and a modulated theta rhythm in the hippocampus, presenting a discrete oscillation of voltage was observed (Figure 2).

Figure 2
Electroencephalographic recordings of sleep–wake cycle from no-SE and control rats. (A, B) Wakefulness phase, characterized by desynchronization with high frequency and low amplitude waves in the cerebral cortex, and theta activity in the hippocampus (similar in both groups). (C, D) Slow-wave sleep phase with cortical synchronization of high amplitude and low frequency waves, and hippocampal delta waves (also similar in both groups). (E, F) Paradoxal sleep, characterized by cortical desynchronization and a modulated theta rhythm in the hippocampus, presenting a discrete oscillation of voltage (similar in both groups). In none of the electroencephalographic recordings were observed epileptiform discharges.

3.4. Quantification of hippocampal cells

The isotropic fractionator technique performed for cell counting in histological slices of 5 Non-SE and 4 controls did not show significant differences in number of hippocampal neuronal cells (p= 0,686) and non-neuronal cells (p= 0,402) between groups (Figure 3).

Figure 3
Number of non-neuronal and neuronal cells in the hippocampal formation. No significant differences in the number of cells were observed.

3.5. Neo-Timm staining

Neo-Timm staining was performed in 4 Non-SE and 4 control animals. The scoring method of Cavazos et al. (1991) to assess the distribution of Timm granules graded level zero to animals from both groups, meaning that no mossy fiber sprouting was observed (Figure 4 and 5).

Figure 4
Neo Timm’s staining showing no mossy fiber sprouting in dentate gyrus for both groups.

Figure 5
Neo Timm’s staining showing no mossy fiber sprouting in CA3 region for both groups.

4. Discussion

This study investigated the long-term (20 months) observation of behavioral seizure occurrence, electrographic seizures and hippocampal morphological alterations in male wistar rats that did not develop behavioral SE after Pilo-induced model of epilepsy. Behavioral manifestations of SE rats were like that described for male rats in the Pilo’s model acute phase (Cavalheiro, 1995; Arida et al., 1999). As stated in literature, in the Pilo’s model chronic phase animal are expected to present SRS in both, male (Arida et al., 1999) and female (Vannucci Campos et al., 2017; Dal Pai et al., 2022), with no long-term remission of seizures (Lopim et al., 2016). In this work, inconsistent behavioral seizures were not shown by Non-SE animals after Pilo’s treatment, different from other authors (Scharfman et al., 2001; Dal Pai et al., 2022).

Non-SE male rats were found to show SRS on telemetry from 8 to 10 months after Pilo-induced seizures (Navarro Mora et al., 2009), which were not observed in our rats’ EEG recordings. However, this group did not observe neuronal degeneration on Nissl technique in their experiment, which corroborates our findings. Despite that, commitment of the hippocampus, piriform cortex and thalamus in the MRI analysis was observed 1 year after Pilo injection (Navarro Mora et al., 2009).

Seizure-like behaviors that resemble Racine scale 1-3, were described in Non-SE female rats through video-monitoring, which were not observed in this work. Authors suggest that such manifestations were observed because slow speed analysis of recordings was applied, since less severe seizures cannot be recognized and may be confused as a usual animal movement (Dal Pai et al., 2022).

The sprouting of the mossy fibers is one of the main histopathological features observed in pilocarpine-induced model of epilepsy (Mello et al., 1992; Mello et al., 1993). Mossy fiber sprouting is a synaptic plasticity event (i.e. granular cell axons in the DG project inputs to their own dendrites) that promotes an excitatory aberrant reorganization of the mossy fibers (Tauck and Nadler, 1985). Additionally, this event is also considered one of the leading bases for hyperexcitability and seizures (Babb et al., 1991; Isokawa et al., 1991; Tauck and Nadler, 1985; Chen et al., 2013). A study with male Non-SE rats found that 50% had a small degree of cell loss in the entorhinal cortex from 1 to 7 months after Pilo injection (seizures were not observed). Additionally, 17% of such animals showed mossy fiber sprouting, suggesting that functioning abnormalities of these cells may arise without SE and may be related to sprouting (Scharfman et al., 2001). Another study considered Non-SE mice those that showed seizure 1-2 scores of Racine’ scale and up to two generalized seizures, scores 3-5, within 2 hours after Pilo treatment. SRS and cell loss were not observed in such animals. However, a very small degree of mossy fiber sprouting was verified (Shibley and Smith, 2002).

Histological analysis of the hippocampal complex of epileptic animals has revealed physiological and anatomical alterations (Mello et al., 1992; Mello et al., 1993; Kersanté et al., 2013) like those found in patients with TLE (Babb et al., 1991; Isokawa et al., 1991). Controversially to these reports, a postmortem study revealed that patients who have suffered from SE episodes did not show evidence of hippocampus’ injury, suggesting that SE itself may not be sufficient to cause neuronal damage (Thom et al., 2005).

The occurrence of an SE event has been thought of as one of the main importance for the development of chronic epilepsy. Authors agree that during the Pilo-induced model’s silent period (i.e. epileptogenic period) that occurs between the brain injury and the first SRS, is rich in cellular and molecular alterations that culminate with the establishment of the epileptic condition, right after the first SRS (Williams et al., 2007; Navarro Mora et al., 2009). Nevertheless, as observed in studies with rats and mice, animals that did not evolve into SE showed behavioral seizures after Pilo treatment, leading to findings observed in model’s chronic phase. Such animals may not show the same behavioral, electrographic and histological aspects presented in epileptic rats, however, the observed alterations pointed in the literature suggests that: 1) possibly seizures per se (and not only SE) may led animals do present some aspects of the Pilo’s chronic phase, even when these animals do not become epileptic; 2) such animals and their “resistance” to evolve into SE should serve as a model for the study and comprehension of the epileptogenic period.

5. Conclusions

The absence of behavioral, electrographic and histological findings observed in this work suggest that Non-SE animals do not become epileptic in the Pilo’s model chronic phase. Additionally, SE development is still necessary to SRS occurrence in the Pilo-induced model of epilepsy.

Data Availability Statement

Data collected in this research are available for all interested

• Ethics and consent
  Experimental protocols used is this research were approved by the ethics committee of UNIFESP (#54712002/15).

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Edited by

• Editor:
  Marcelo A.M. Esquisatto

Publication Dates

• Publication in this collection
  17 July 2026
• Date of issue
  2026

History

• Received
  29 Oct 2025
• Accepted
  21 Apr 2026