Kainic Acid-induced Epileptic Rats Research Articles

Tandem mass tag-based proteomics reveals the antiepileptic mechanism of steroidal saponins from Anemarrhena asphodeloides in Kainic acid induced epileptic rat model.

Epilepsy (EP) is one of the most common neurological diseases in the world. Anemarrhena asphodeloides Bunge. (AA), as a typical heat-cleaning medicine, has been proven to possess the antiepileptic effect in clinical and experimental studies. Anemarrhena asphodeloides steroidal saponins (AAS) are main components. However, the therapeutic effects and underlying mechanisms of AAS against EP are not been fully elucidated. In this study, 63 steroidal saponins were discovered in AAS by UPLC-Q-TOF/MS analysis. Pharmacological and behavioral analysis demonstrated that AAS could significantly lower the Racine classification and reduce the frequency of generalized spike rhythm the rate of tetanic seizures in kainic acid-induced epileptic rats. Hematoxylin and eosin and Nissl staining-indicated AAS could significantly improve hippocampal injury and neuron loss in epileptic rats. TMT proteomic analysis discovered 26 different expressed proteins (DEPs), which were identified as the rescue proteins. After bioinformatic analysis, Heat Shock Protein 90 Alpha Family Class B Member 1 (Hsp90ab1) and Tyrosine 3-Monooxygenase (Ywhab) were screened as key DEPs and verified by western blotting. AAS could significantly inhibited the up-regulation of Hsp90ab1 and Ywhab in EP rats; these two proteins might be the key targets of AAS in treating EP.

Silencing miR-155–5p alleviates hippocampal damage in kainic acid-induced epileptic rats via the Dusp14/MAPK pathway

Epilepsy with recurrent seizures is characterized by neuronal damage and glial proliferation induced by brain inflammation. Recurrent seizures can lead to changes in the microRNA (miRNA) spectrum, significantly influencing the inflammatory response of microglia. MiR-155–5p, as a pro-inflammatory miRNA, is increased in the epileptic brain. However, its specific role in acute seizures remains unknown. The study aimed to develop a new strategy for treating epilepsy by investigating how silencing of miR-155–5p initiated its anticonvulsive mechanism. The level of miR-155–5p was up-regulated in the hippocampus of epileptic immature rats induced by kainic acid (KA). The use of antago-miR-155–5p exerted significant beneficial effects on the seizure scores, brain discharges and cognition in immature rats following KA-induced epilepsy. Antago-miR-155–5p also inhibited neuron damage and microglial activation. Moreover, the silencing of miR-155–5p significantly inhibited the Dual-specificity phosphatase 14 (Dusp14)/ mitogen-activated protein kinase (MAPK) axis in vivo. MiR-155–5p interacted with dusp14 to regulate MAPK signaling way expression, verified by a dual-luciferase reporter assay. The results suggested that the silencing of miR-155–5p might reduce hippocampal damage in epileptic immature rats induced by KA via Dusp14/MAPK signaling way. This implied that miR-155–5p could serve as a therapeutic tool to prevent the development of epilepsy.

Effects of scorpion venom heat-resistant peptide on the hippocampal neurons of kainic acid-induced epileptic rats.

Scorpion venom is a Chinese medicine for epilepsy treatment, but the underlying mechanism is not clear. Scorpion venom heat-resistant peptide (SVHRP), a peptide isolated from the venom of Buthus martensii Karsch, has an anti-epileptic effect by reducing seizure behavior according to a modified Racine scale. The present study aimed to investigate the molecular mechanism of SVHRP on temporal lobe epilepsy. The hippocampus and hippocampal neurons from kainic acid-induced epileptic rats were treated with SVHRP at different doses and duration. Quantitative RT-PCR and immunoblotting were used to detect the expression level of brain-derived neurotrophic factor (BDNF), neuropeptide Y (NPY), cAMP-response element binding protein (CREB), stromal interaction molecule (STIM), and calcium release-activated calcium channel protein 1 (ORAI1). In the hippocampal tissues and primary hippocampal neuron cultures, SVHRP treatment resulted in increased mRNA and protein levels of BDNF and NPY under the epileptic condition. The upregulation of BDNF and NPY expression was positively correlated with the dose level and treatment duration of SVHRP in hippocampal tissues from kainic acid-induced epileptic rats. On the other hand, no significant changes in the levels of CREB, STIM, or ORAI1 were observed. SVHRP may exhibit an anti-epileptic effect by upregulating the expression of BDNF and NPY in the epileptic hippocampus.

Downregulation of MicroRNA-34c-5p facilitated neuroinflammation in drug-resistant epilepsy

Drug-resistant epilepsy patients has aberrant inflammatory mediator levels. However, the mechanism of which is remains unillustrated. Here the molecular mechanism underlying the neuroinflammatory process in patients with drug-resistant epilepsy were investigated. Bioinformatics analysis revealed that miR-34c-5p was significantly downregulated in patients with drug-resistant epilepsy, compared to control population. Then, luciferase reporter assays indicated that HMGB1, inflammation-related mediators, was the target gene of miR-34c-5p. The kainic acid-induced epileptic rats were established and divided into drug-sensitive epilepsy and drug-resistant epilepsy according to their seizure behavior and EEG after antiepileptic drug administration. Downregulation of miR-34c-5p, elevated expression of HMGB1 and IL-1β had been found in rats with drug-resistant epilepsy, compared to drug-sensitive epilepsy rats. Aggravated hippocampal neuron loss was demonstrated in rats with drug-resistant epilepsy. The results from epileptic rats were subsequently validated from children with drug-resistant epilepsy. Analysis manifested that miR-34c-5p was obviously decreased, while HMGB1 was increased on serum of children with drug-resistant epilepsy. Our study highlights that decreased miR-34c-5p in drug-resistant epilepsy exacerbates neuroinflammation, which aggravates hippocampal neuron loss in epileptogenesis. Thus, miR-34c-5p could be considered as a potential noninvasive biomarker and shed novel light on the development of an effective therapeutic strategy for children with drug-resistant epilepsy in the future.

Neuroprotective and anti-inflammatory effects of isoliquiritigenin in kainic acid-induced epileptic rats via the TLR4/MYD88 signaling pathway.

Epileptogenesis is a complex pathological process that occurs after an initial brain injury and involves a series of molecular events. Isoliquiritigenin (ISL), a flavonoid in licorice, is reported to have anti-inflammatory and antioxidant effects in various experimental models, but its specific roles and molecular mechanisms in the epileptogenic process following kainic acid (KA) treatment remain unclear. The purpose of this study was to explore the effects of ISL pretreatment in KA-induced epileptic rats and the underlying mechanisms. Our findings show that ISL pretreatment significantly attenuated the KA-induced expression of ionized calcium-binding adapter molecule 1 (IBα1)-labeled microglia (F(3, 20) = 97.29, p < 0.01, ηp2 = 0.94) and glial fibrillary acidic protein (GFAP)-positive astrocytes (F(3, 20) = 72.48, p < 0.01, ηp2 = 0.92), and the release of inflammatory mediators, such as TNF-α (F(3, 20) = 133.14, p < 0.01, ηp2 = 0.95), IL-1β, and C-C motif chemokine ligand 3 (CCL3). ISL pretreatment given before KA also significantly prevented apoptotic neuronal injury by upregulating the activities of superoxide dismutase and glutathione peroxidase. It also significantly suppressed the protein levels of Toll-like receptor 4 (TLR4) (F(3, 20) = 63.23, p < 0.01, ηp2 = 0.91) and its downstream molecules, myeloid differentiation primary response 88 (MYD88), phosphorylated (p-)IκBα, and p-NF-κB. Blocking TLR4/MYD88 signaling also attenuated KA-induced neuroinflammation and neuronal damage in the hippocampus. Overall, our study demonstrates that ISL pretreatment plays neuroprotective and anti-inflammatory roles in KA-induced epileptogenesis, which may be mediated by the TLR4/MYD88 signaling pathway.

Anterior nucleus of thalamus stimulation inhibited abnormal mossy fiber sprouting in kainic acid-induced epileptic rats

BackgroundDeep brain stimulation (DBS) of the anterior nucleus of the thalamus (ANT) has demonstrated antiepileptic efficacy, especially for mesial temporal lobe epilepsy (MTLE). Mossy fiber sprouting (MFS) is involved in the pathogenesis of MTLE, and Sema-3A and GAP-43 are pivotal regulators of MFS. This study investigated the effects of ANT-DBS on MFS and expression levels of Sema-3A and GAP-43 as a possible mechanism for seizure suppression. MethodsAdult male Sprague-Dawley rats were randomly divided into four groups: (1) control (saline injection), (2) KA (kainic acid injection), (3) KA + Sham-DBS (electrode implantation without stimulation), and (4) KA + DBS (electrode implantation with stimulation). Video electroencephalography (EEG) was used to ensure model establishment and monitor seizure frequency, latency, and severity (Racine stage). Chronic ANT stimulation was conducted for 35 days in the KA + DBS group, and MFS compared to the other groups by quantitative Timm staining. Sema-3A and GAP-43 expression levels in the hippocampal formation were evaluated in all groups with western blot. ResultsThe latency period was significantly prolonged and spontaneous seizure frequency reduced in the KA + DBS group compared to KA and KA + Sham-DBS groups. Staining scores for MFS in CA3 and dentate gyrus (DG) were significantly lower in the KA + DBS group. The KA + DBS group also exhibited decreased GAP-43 expression and increased Sema-3A expression compared to KA and KA + Sham-DBS groups. ConclusionThese results suggest that ANT-DBS extends the latent period following epileptogenic stimulation by impeding MFS through modulation of GAP-43 and Sema-3A expression.

Anterior thalamic nuclei deep brain stimulation reduces disruption of the blood–brain barrier, albumin extravasation, inflammation and apoptosis in kainic acid-induced epileptic rats

ObjectiveThe therapeutic efficacy of anterior thalamic nuclei deep brain stimulation (ATN-DBS) against seizures has been largely accepted; however, the effects of ATN-DBS on disruption of the blood–brain barrier (BBB), albumin extravasation, inflammation and apoptosis still remain unclear.MethodsRats were distributed into four treatment groups: physiological saline (PS, N = 12), kainic acid (KA, N = 12), KA-sham-DBS (N = 12) and KA-DBS (N = 12). Seizures were monitored using video-electroencephalogram (EEG). One day after surgery, all rats were sacrificed. Then, samples were prepared for quantitative real-time PCR (qPCR), western blot, immunofluorescence (IF) staining, and transmission electron microscopy to evaluate the disruption of the BBB, albumin extravasation, inflammation, and apoptosis.ResultBecause of the KA injection, the disruption of the BBB, albumin extravasation, inflammation and apoptosis were more severe in the KA and the KA-sham-DBS groups compared to the PS group (all Ps < 0.05 or < 0.01). The ideal outcomes were observed in the KA-DBS group. ATN-DBS produced a 46.3% reduction in seizure frequency and alleviated the disruption of the BBB, albumin extravasation, inflammatory reaction and apoptosis in comparison to the KA-sham-DBS group (all Ps < 0.05 or < 0.01).Conclusion(1) Seizures can be reduced using ATN-DBS in the epileptogenic stage. (2) ATN-DBS can reduce the disruption of the BBB and albumin extravasation. (3) ATN-DBS has an anti-inflammatory effect in epileptic models.

Stimulation of Anterior Thalamic Nucleus Protects Hippocampus Neural Injury in Kainic Acid-Induced Epileptic Rats

Anterior nucleus of thalamus (ANT) stimulation has been proved to be effective in the treatment of refractory epilepsy, but the underlying mechanisms remain to be elucidated. We examined the role of ANT stimulation on hippocampal neuron death after seizures induced by kainic acid (KA). Our data showed that ANT stimulation could significantly rescue neurons from death induced by seizures, by reducing the release of cytochrome c (cyto c) and also via apoptosis-inducing factor (AIF) induced by seizures through inhibiting the activated caspase-9 and caspase-3. Our data suggest that ANT stimulation may protect against neuronal loss and reduce neuronal injury in the ipsilateral CA3 region of the hippocampus in the KA-induced epileptic rats, and the underlying mechanism may be mediated by inhibiting mitochondrial caspase-dependent (cyto c release and the subsequent cleavage of caspase-) and caspase-independent (nuclear translocation of AIF) apoptosis pathways.

Effects of hippocampal stimulus on α₅ subunit of extrasynaptic GABA(A) receptor in kainic acid-induced epileptic rats

To observe the effects of electrical hippocampal stimulation of α₅ subunit of extrasynaptic GABA(A) receptor in kainic acid-induced epileptic rats, explore the optimal therapeutic parameters and elucidate the possible mechanism of electrical stimulation for hippocampal epilepsy. A total of 40 healthy male Sprague Dawley (SD) rats were randomly divided into 5 groups of epilepsy, sham operation, low-frequency stimulation (LFS), high-frequency stimulation (HFS) and normal control (n = 8 each). The expression of α₅ subunit of extrasynaptic GABA(A) receptor as well as the association with the curative effects of LFS were evaluated by behavioristics, real-time fluorogenic quantitative polymerase chain reaction (PCR) and Western blot. The kainic acid kindling rats had class V attack with a modeling success rate of 67%. After treatment, as compared with sham operation and epilepsy groups, the number of seizures decreased significantly in LFS and HFS treatment groups (P < 0.01). But LFS and HFS treatment groups were compared, the number of seizures has no statistical significance (P > 0.05). When sham operation and epilepsy groups were compared, the number of seizures was not statistically significant (P > 0.05). The paired inter-group comparisons showed that LFS and HFS treatment groups and sham operation and epilepsy groups had significant differences in α₅ subunit mRNA and protein (P < 0.01). No statistical significance existed between LFS and HFS treatment groups (P > 0.05) or sham operation and HFS treatment groups (P > 0.05). As compared with normal control group, LFS treatment and HFS groups had significant difference (P < 0.01). Electrical hippocampal stimulation can effectively inhibit seizures. Both LFS and HFS are equally effective, but the former offers greater benefits. And an inhibition of extrasynaptic α₅ subunit plays an important role in seizure during hippocampal stimulation.

Stimulation of Anterior Thalamic Nuclei Protects Against Seizures and Neuronal Apoptosis in Hippocampal CA3 Region of Kainic Acid-induced Epileptic Rats.

Background:The antiepileptic effect of the anterior thalamic nuclei (ANT) stimulation has been demonstrated; however, its underlying mechanism remains unclear. The aim of this study was to investigate the effect of chronic ANT stimulation on hippocampal neuron loss and apoptosis.Methods:Sixty-four rats were divided into four groups: The control group, the kainic acid (KA) group, the sham-deep brain stimulation (DBS) group, and the DBS group. KA was used to induce epilepsy. Seizure count and latency to the first spontaneous seizures were calculated. Nissl staining was used to analyze hippocampal neuronal loss. Polymerase chain reaction and Western blotting were conducted to assess the expression of caspase-3 (Casp3), B-cell lymphoma-2 (Bcl2), and Bcl2-associated X protein (Bax) in the hippocampal CA3 region. One-way analysis of variance was used to determine the differences between the four groups.Results:The latency to the first spontaneous seizures in the DBS group was significantly longer than that in the KA group (27.50 ± 8.05 vs. 16.38 ± 7.25 days, P = 0.0005). The total seizure number in the DBS group was also significantly reduced (DBS vs. KA group: 11.75 ± 6.80 vs. 23.25 ± 7.72, P = 0.0002). Chronic ANT-DBS reduced neuronal loss in the hippocampal CA3 region (DBS vs. KA group: 23.58 ± 6.34 vs. 13.13 ± 4.00, P = 0.0012). After chronic DBS, the relative mRNA expression level of Casp3 was decreased (DBS vs. KA group: 1.18 ± 0.37 vs. 2.09 ± 0.46, P = 0.0003), and the relative mRNA expression level of Bcl2 was increased (DBS vs. KA group: 0.92 ± 0.21 vs. 0.48 ± 0.16, P = 0.0004). The protein expression levels of CASP3 (DBS vs. KA group: 1.25 ± 0.26 vs. 2.49 ± 0.38, P < 0.0001) and BAX (DBS vs. KA group: 1.57 ± 0.49 vs. 2.80 ± 0.63, P = 0.0012) both declined in the DBS group whereas the protein expression level of BCL2 (DBS vs. KA group: 0.78 ± 0.32 vs. 0.36 ± 0.17, P = 0.0086) increased in the DBS group.Conclusions:This study demonstrated that chronic ANT stimulation could exert a neuroprotective effect on hippocampal neurons. This neuroprotective effect is likely to be mediated by the inhibition of apoptosis in the epileptic hippocampus.

Study on protective effect of ketogenic diet against hippocampal neurons of kainic acid-induced epileptic rats

Objective To investigate the protective effects of ketogenic diet on hippocampal neurons of kainic acid (KA)-induced epileptic rats. Methods A total of 40 healthy male specific pathogen free (SPF) Sprague-Dawley (SD) rats were randomly divided into 4 groups, with each group containing 10 rats. Epileptic rat models were formed by injection of KA through lateral ventricle with brain stereotactic instrument. According to Racine classification, epileptic seizures in rats above grade Ⅳ were defined successful. Then the rats were given different dietary treatment: Group C with normal saline and normal diet, Group K with normal saline and ketogenic diet, Group E with KA and normal diet, Group EK with KA and ketogenic diet. All rats were observed for 21 d, and were recorded each body weight. The epileptic seizure frequency and duration were observed at 12:00-15:00 daily. At the 21st day, all rats were put to death, and the brain hippocampus tissues were separated. Neuron injury of rat hippocampal CA3 region in Group E and EK was observed by HE staining. Normal neuron number of rat hippocampal CA3 region in Group E and EK was counted by Nissl staining. Results Group C and K had no epileptic seizures, and the neuron number in hippocampal CA3 region was normal. Rats in both Group E and EK had grade Ⅳ or Ⅴ seizures. The number of seizures in Group EK [(17.90 ± 4.12) times] after 21-day ketogenic diet was decreased significantly compared to Group E [(30.50 ± 4.40) times] after 21-day normal diet ( t = 6.606, P = 0.000). The seizure duration in Group EK [(212.70 ± 17.75) s] after 21-day ketogenic diet was shortened compared to Group E [(335.00 ± 14.21) s] after 21-day normal diet ( t = 17.011, P = 0.000). The normal neuron number in hippocampal CA3 region in Group EK (117.67 ± 7.51) was more than those in Group E (71.33 ± 6.11), with statistically significant difference ( t = 9.614, P = 0.000). Conclusions Ketogenic diet has protective effect on hippocampal neurons of kainic acid-induced epileptic rats. DOI: 10.3969/j.issn.1672-6731.2015.11.011

A new model for studying focal and generalized chronic seizures in anesthetized rats

Seizure activity in anesthetized animals can be recorded in a number of different animal models. One such model uses urethane as an anesthetic and kainic acid as a convulsant. In addition, it is known that animals can be made epileptic using kainic acid by causing a period of status epilepticus and waiting for epileptogenesis to occur. We report an animal model to study seizure activity in kainic acid‐induced epileptic rats under urethane anesthesia. Sprague‐Dawley rats were injected with sufficient kainic acid to cause a period of status epilepticus. At ≥8 months post status epilepticus, at a time when behavioral seizures were evident, the rats were anesthetized with urethane. EEG recordings from CA1 and CA3 were performed together with recordings of EKG, BP, and vagus nerve. All rats exhibited abnormal spiking in the EEG and 42% (5/12) experienced spontaneous focal seizures while under anesthesia. Spontaneous generalized seizure activity occurred in a subset of these animals. In 56% (5/9 tested), focal and generalized seizures could be triggered with brief trains of electrical stimuli. Vagus nerve activity was found to be increased during focal and generalized seizures. These results demonstrate a new animal model to study seizures, especially focal seizures of limbic cortical origin, in anesthetized epileptic rats with a variety of applications, including the study of autonomic activity during seizures.