Pilocarpine-induced Epilepsy In Rats Research Articles

Ketogenic Diets Alter the Gut Microbiome, Resulting in Decreased Susceptibility to and Cognitive Impairment in Rats with Pilocarpine-Induced Status Epilepticus.

A ketogenic diet (KD) is a high-fat, low-carbohydrate, and low-protein diet that exerts antiepileptic effects by attenuating spontaneous recurrent seizures, ameliorating learning and memory impairments, and modulating the gut microbiota composition. However, the role of the gut microbiome in the antiepileptic effects of a KD on temporal lobe epilepsy (TLE) induced by lithium-pilocarpine in adult rats is still unknown. Our study provides evidence demonstrating that a KD effectively mitigates seizure behavior and reduces acute-phase epileptic brain activity and that KD treatment alleviates hippocampal neuronal damage and improves cognitive impairment induced by TLE. We also observed that the beneficial effects of a KD are compromised when the gut microbiota is disrupted through antibiotic administration. Analysis of gut microbiota components via 16S rRNA gene sequencing in fecal samples collected from TLE rats fed either a KD or a normal diet. The Chao1 and ACE indices showed decreased species variety in KD-fed rats compared to TLE rats fed a normal diet. A KD increased the levels of Actinobacteriota, Verrucomicrobiota and Proteobacteria and decreased the level of Bacteroidetes. Interestingly, the abundances of Actinobacteriota and Verrucomicrobiota were positively correlated with learning and memory ability, and the abundance of Proteobacteria was positively correlated with seizure susceptibility. In conclusion, our study revealed the significant antiepileptic and neuroprotective effects of a KD on pilocarpine-induced epilepsy in rats, primarily mediated through the modulation of the gut microbiota. However, whether the gut microbiota mediates the antiseizure effects of a KD still needs to be better elucidated.

Gut microbiota and serum metabolomic alterations in modulating the impact of fecal microbiota transplantation on ciprofloxacin-induced seizure susceptibility.

The gut microbiota and the microbiota-gut-brain axis have gained considerable attention in recent years, emerging as key players in the mechanisms that mediate the occurrence and progression of many central nervous system-related diseases, including epilepsy. In clinical practice, one of the side effects of quinolone antibiotics is a lower seizure threshold or aggravation. However, the underlying mechanism remains unclear. We aimed to unravel the intrinsic mechanisms through 16S rRNA sequencing and serum untargeted metabolomic analysis to shed light on the effects of gut microbiota in ciprofloxacin-induced seizure susceptibility and lithium pilocarpine-induced epilepsy rat models. We observed that ciprofloxacin treatment increased seizure susceptibility and caused gut dysbiosis. We also found similar changes in the gut microbiota of rats with lithium pilocarpine-induced epilepsy. Notably, the levels of Akkermansia and Bacteroides significantly increased in both the ciprofloxacin-induced seizure susceptibility and lithium pilocarpine-induced epilepsy rat models. However, Marvinbryantia, Oscillibacter, and Ruminococcaceae_NK4A214_group showed a coincidental reduction. Additionally, the serum untargeted metabolomic analysis revealed decreased levels of indole-3-propionic acid, a product of tryptophan-indole metabolism, after ciprofloxacin treatment, similar to those in the plasma of lithium pilocarpine-induced epilepsy in rats. Importantly, alterations in the gut microbiota, seizure susceptibility, and indole-3-propionic acid levels can be restored by fecal microbiota transplantation. In summary, our findings provide evidence that ciprofloxacin-induced seizure susceptibility is partially mediated by the gut microbiota and tryptophan-indole metabolism. These associations may play a role in epileptogenesis, and impacting the development progression and treatment outcomes of epilepsy.

Neuroprotective effects of quinpirole on lithium chloride pilocarpine-induced epilepsy in rats and its underlying mechanisms

IntroductionEpilepsy is a common neurological disorder that presents with challenging mechanisms and treatment strategies. This study investigated the neuroprotective effects of quinpirole on lithium chloride pilocarpine-induced epileptic rats and explored its potential mechanisms.MethodsLithium chloride pilocarpine was used to induce an epileptic model in rats, and the effects of quinpirole on seizure symptoms and cognitive function were evaluated. The Racine scoring method, electroencephalography, and Morris water maze test were used to assess seizure severity and learning and memory functions in rats in the epileptic group. Additionally, immunohistochemistry and Western blot techniques were used to analyze the protein expression levels and morphological changes in glutamate receptor 2 (GluR2; GRIA2), BAX, and BCL2 in the hippocampi of rats in the epileptic group.ResultsFirst, it was confirmed that the symptoms in rats in the epileptic group were consistent with features of epilepsy. Furthermore, these rats demonstrated decreased learning and memory function in the Morris water maze test. Additionally, gene and protein levels of GluR2 in the hippocampi of rats in the epileptic group were significantly reduced.Quinpirole treatment significantly delayed seizure onset and decreased the mortality rate after the induction of a seizure. Furthermore, electroencephalography showed a significant decrease in the frequency of the spike waves. In the Morris water maze test, rats from the quinpirole treatment group demonstrated a shorter latency period to reach the platform and an increased number of crossings through the target quadrant. Network pharmacology analysis revealed a close association between quinpirole and GluR2 as well as its involvement in the cAMP signaling pathway, cocaine addiction, and dopaminergic synapses.Furthermore, immunohistochemistry and Western blot analysis showed that quinpirole treatment resulted in a denser arrangement and a more regular morphology of the granule cells in the hippocampi of rats in the epileptic group. Additionally, quinpirole treatment decreased the protein expression of BAX and increased the protein expression of BCL2.ConclusionThe current study demonstrated that quinpirole exerted neuroprotective effects in the epileptic rat model induced by lithium chloride pilocarpine. Additionally, it was found that the treatment not only alleviated the rats' seizure symptoms, but also improved their learning and memory abilities. This improvement was linked to the modulation of protein expression levels of GLUR2, BAX, and BCL2. These findings provided clues that would be important for further investigation of the therapeutic potential of quinpirole and its underlying mechanisms for epilepsy treatment.

MiR-101a-3p Attenuated Pilocarpine-Induced Epilepsy by Downregulating c-FOS.

This study aimed to explore the effects and function of microRNA-101a-3p (miR-101a-3p) in epilepsy. Rat model of pilocarpine-induced epilepsy was established and the seizure frequency was recorded. Expression of miR-101a-3p and c-Fos in hippocampus tissues of Rat models were detected by qRT-PCR and western blot. Besides, we established a hippocampal neuronal culture model of acquired epilepsy using Mg2+ free medium to evaluate the effects of miR-101a-3p and c-Fos in vitro. Cells were transfected with miR-101a-3p mimic, si-c-FOS, miR-101a-3p + c-FOS and its corresponding controls. MTT assay was used to detect cell viability upon transfection. Flow cytometry was performed to determine the apoptosis rate. Western blot was performed to measure the protein expression of apoptosis-related proteins (Bcl-2, Bax, and cleaved caspase 3), autophagy-related proteins (LC3 and Beclin1) and c-FOS. The targeting relationship between miR-101a-3p and c-FOS was predicted and verified by TargetScan software and dual-luciferase reporter assay. The role of miR-101a-3p was validated using epilepsy rat models in vivo. Another Rat models of pilocarpine-induced epilepsy with miR-NC or miR-101a-3p injection were established to evaluate the effect of miR-101a-3p overexpression on epilepsy in vivo. MiR-101a-3p was downregulated while c-FOS was increased in hippocampus tissues of Rat model of pilocarpine-induced epilepsy. Overexpression of miR-101a-3p or c-FOS depletion promoted cell viability, inhibited cell apoptosis and autophagy. C-FOS was a target of miR-101a-3p and miR-101a-3p negatively regulated c-FOS expression to function in epilepsy. Overexpression of miR-101a-3p attenuated pilocarpine-induced epilepsy in Rats in vivo. This study indicated that miR-101a-3p could attenuate pilocarpine-induced epilepsy by repressing c-Fos expression.

Anticonvulsant effect of piperine ameliorates memory impairment, inflammation and oxidative stress in a rat model of pilocarpine-induced epilepsy.

The primary active component of black pepper is piperine, which is purified and used to treat epilepsy, achieving higher efficiency when purified. The present study was conducted to evaluate whether the anticonvulsant effect of piperine ameliorates pilocarpine-induced epilepsy, and to investigate the mechanism underlying these effects. Epilepsy was induced in Sprague Dawley rats using pilocarpine. Pilocarpine-induced epilepsy in the rats was treated with 40 mg/kg piperine for 45 consecutive days. Status epilepticus and a Morris water maze test were used to analyze the anticonvulsant effects of piperine in the epileptic rats. Inflammation and oxidative stress were then measured using commercially-available kits following piperine treatment. Lastly, the activity of caspase-3 and the protein expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were evaluated using commercially-available kits and western blot analysis, respectively. The results demonstrated that treatment with piperine was able to reduce the status epilepticus and prevented memory impairment following pilocarpine-induced epilepsy in rats. The anticonvulsant effects of piperine decreased inflammation and oxidative stress following pilocarpine-induced epilepsy in rats. The upregulated activity of caspase-3 and expression levels of Bax/Bcl-2 were suppressed following treatment with piperine in the rats with pilocarpine-induced epilepsy. These results suggest that the anticonvulsant effects of piperine ameliorate memory impairment, inflammation and oxidative stress in a rat model of pilocarpine-induced epilepsy.

Electrical stimulation of left anterior thalamic nucleus with high-frequency and low-intensity currents reduces the rate of pilocarpine-induced epilepsy in rats

PurposeBilateral electrical stimulation of anterior nuclei of thalamus (ANT) has shown promising effects on epileptic seizures. However, bilateral implantation increases the risk of surgical complications and side effects. This study was undertaken to access the effectiveness of a stimulation paradigm involving high frequency and low intensity currents to stimulate the left ANT in rats. MethodsMale Sprague-Dawley rats were implanted with electroencephalogram (EEG) electrodes, and an additional concentric bipolar stimulation electrode into either the left or right ANT. The stimulus was a train of pulses (90μs duration each) delivered with a frequency of 200Hz and a current intensity of 50μA. Thalamic stimuli were started 1h before the first intraperitoneal pilocarpine injection (i.p., 300mg/kg), and were applied for 5h. ResultsEEG documented seizure activity and status epilepticus (SE) developed in 87.5% of rats treated with no ANT stimulation after a single dose of pilocarpine. Left ANT stimulation significantly increased the tolerance threshold for pilocarpine-induced EEG seizure activity; 20% of rats developed their EEG documented seizure activity after receiving the first dose, whereas 50%, 10% and 20% of rats did not develop seizure activity until they had received the 2nd, 3rd and 4th pilocarpine injection at 1-h intervals. Moreover, left thalamic stimulation reduced the occurrences of both EEG documented seizure activity and SE induced by single-dose pilocarpine to 25%. However, our result demonstrated that little effect on the occurrence rate of seizures and SE was found when rats received right ANT stimulation. ConclusionsThese results suggest that continuously 5-h left ANT stimulation with high frequency and low intensity currents, beginning from 1h before the pilocarpine administration, may successfully reduce the occurrence rate of EEG documented seizure activity and SE development in rats.

Down-regulation of cerebellar 5-HT2C receptors in pilocarpine-induced epilepsy in rats: Therapeutic role of Bacopa monnieri extract

Epilepsy is a syndrome of episodic brain dysfunction characterized by recurrent unpredictable, spontaneous seizures. Cerebellar dysfunction is a recognized complication of temporal lobe epilepsy and it is associated with seizure generation, motor deficits and memory impairment. Serotonin is known to exert a modulatory action on cerebellar function through 5HT2C receptors. 5-HT2C receptors are novel targets for developing anti-convulsant drugs. In the present study, we investigated the changes in the 5-HT2C receptors binding and gene expression in the cerebellum of control, epileptic and Bacopa monnieri treated epileptic rats. There was a significant down regulation of the 5-HT content (p<0.001), 5-HT2C gene expression (p<0.001) and 5-HT2C receptor binding (p<0.001) with an increased affinity (p<0.001). Carbamazepine and B. monnieri treatments to epileptic rats reversed the down regulated 5-HT content (p<0.01), 5-HT2C receptor binding (p<0.001) and gene expression (p<0.01) to near control level. Also, the Rotarod test confirms the motor dysfunction and recovery by B. monnieri treatment. These data suggest the neuroprotective role of B. monnieri through the upregulation of 5-HT2C receptor in epileptic rats. This has clinical significance in the management of epilepsy.

Estrogen effects on pilocarpine-induced temporal lobe epilepsy in rats

Objetive To evaluate the effects of conjugated equine estrogens (CEE) on the pilocarpine-induced epilepsy in rats. Study design 40 female rats were divided into: GPC (positive control) presented “ status epilepticus” (SE) induced by pilocarpine; GOC (ovariectomized control) only castrated; GNC (negative control) received only saline solution; GPE received pilocarpine, presented SE, castrated and received 50 μg/kg CEE treatment; GPV received pilocarpine, castrated and received propylene glycol (vehicle). The animals were monitored by a video system. At the end of observation, the brains removed for later histologic analysis using Neo-Timm and Nissl methods. Results The GPE presented a reduction in number of seizures compared to GPV. The Neo-Timm analysis showed that GPV had greater sprouting of mossy fibers, with a denser band in the area of the dentate gyrus hilum compared to GPE. On Nissl staining, GPE showed evident neuronal loss in the CA3 area. GPV presented loss in CA1 and dentate gyrus. Conclusion Estrogen may have a protecting effect on the central nervous system.

Volumetric Magnetic Resonance Imaging of Functionally Relevant Structural Alterations in Chronic Epilepsy after Pilocarpine‐induced Status Epilepticus in Rats

After pilocarpine-induced epilepsy in rats, volumetric magnetic resonance imaging (MRI) reveals significant morphologic changes in functionally relevant structures of the brain. To relate structural changes to functional alteration, we studied the correlation of regional brain atrophy (e.g., of the hippocampus) with lesion-induced learning deficits in the Morris water maze. MRI experiments were performed on an MR scanner at 4.7 Tesla. For volumetric analysis, various cerebral structures were segmented in horizontal and coronal T(2)-weighted MR images. Before the MRI investigations, animals were trained for 10 days in a Morris water maze. Volumetric MRI revealed a significant loss in hippocampal size in both the dorsal and ventral parts, correlated with an increase in ventricular size. Furthermore, significant losses were found in the relative size of thalamus, putamen, cortex, and the combined areas of perirhinal, entorhinal, and piriform cortices adjacent to the hippocampus. A significant correlation of learning performance in the Morris water maze with the relative hippocampal area and not with other areas tested was observed in pilocarpine-treated animals. The data provide a quantitative analysis of functionally relevant structural alterations in rats with chronic epilepsy. Water maze performance of pilocarpine-treated animals correlates with the degree of hippocampal but not with the degree of cortical damage, demonstrating the potential of this method for the investigation of cognitive impairments in relation to cerebral changes. In addition, the data point to an important role of even the residual hippocampus in memory formation.

Activity-Induced Expression of Common Reference Genes in Individual CNS Neurons

T he quantitative study of gene expression in neurons under physiological and pathophysiological conditions has gained considerable importance. Consequently, a variety of different techniques to quantify gene expression have evolved. Generally, such approaches can be subdivided into strategies that provide absolute versus relative quantitation of a target transcript. Quantitative procedures such as quantitative Northern blots or conventional quantitative reverse transcription polymerase chain reaction (RT-PCR) methods have several disadvantages. For instance, Northern blots require substantial amounts of RNA, rendering investigation of mRNA levels from single cells impossible. Comparability of results is extremely dependent upon identical amounts of starting mRNA used in different samples, a condition that cannot usually be achieved with small cell numbers or minute amounts of tissue. To circumvent these obstacles, various techniques have been developed to quantify gene expression relative to a reference gene (Fink et al, 1998; Waha et al, 1998). This obviates the need to perform an accurate determination of initial mRNA concentrations. On the other hand, a suitable reference gene is essential as an internal control for relative mRNA quantitation. This internal control is crucial for a quantitative comparison of gene expression between different tissue types, at various developmental stages, and between control and disease tissue. Ideally, internal control transcripts should show constant, highlevel expression independent of experimental conditions. The genes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and b-actin have been commonly used as reference genes. In recent years, cyclophilin A and hypoxanthine phosphoribosyl-transferase (HPRT) have also been adopted for this purpose. However, several classical reference genes may exhibit considerable changes in expression in cell types or under disease conditions (Suzuki et al, 2000). Here, we have analyzed several of these genes as reference transcripts for activity-dependent chronic changes in central nervous system (CNS) gene expression. Pilocarpine-induced epilepsy in rats was employed as experimental paradigm. We find that GAPDH is not suitable for this purpose, whereas the neuron-specific gene synaptophysin appears remarkably stable following intense neuronal activity. To study activity-dependent alterations in reference gene expression, seizure activity was induced in vivo by applying a single dose of the convulsant pilocarpine (male Wistar rats 150–200 g, 300–380 mg/kg, ip) after prior administration of methyl-scopolamine (1 mg/kg, sc, 30 minutes before pilocarpine, ip). Seizures were terminated after 2 hours by application of diazepam (0.1 mg/kg, sc). Age-matched rats, injected with saline served only as controls. Thirty days after pilocarpine/saline injection, the brains of pilocarpinetreated and control animals (n 5 5 each) were removed. One hippocampus was immediately frozen in liquid nitrogen for in situ hybridization, whereas the contralateral hippocampus was used for preparation of 400-mm transverse vibratome slices. From these slices, single CA1 neurons were harvested with a glass pipette in less than 10 ml of extracellular solution (Beck et al, 1999). mRNA was isolated using the Dynabead mRNA Direct Micro Kit (Dynal, Hamburg, Germany) according to the manufacturer’s protocol. For real-time RT-PCR fluorescent hybridization, oligonucleotides and primers were chosen for the five putative reference genes: GAPDH, b-actin, cyclophilin A, HPRT, and synaptophysin (Eurogentec, Seraing, Received March 9, 2001. This work is supported by the Deutsche Forschungsgemeinschaft (SFB 400), the BONFOR program of the University of Bonn Medical Center, and the German-Israel Program of the Bundesministerium fur Bildung und Fors and Ministry of Science. Address reprint requests to: Dr. Albert J. Becker, Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud Str. 25, 53105 Bonn, Germany. E-mail: albert_becker@uni-bonn.de 0023-6837/01/8106-913$03.00/0 LABORATORY INVESTIGATION Vol. 81, No. 6, p. 913, 2001 Copyright © 2001 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A.