Region-Specific Long-Term Transcriptional Changes in the Plasminogen Activation System and Neuroinflammation in the Rat Brain After Status Epilepticus: Association with Depressive-like Behavior

Lithium pilocarpine-induced status epilepticus in postnatal day 20 rats results in greater neuronal injury in ventral versus dorsal hippocampus

The hippocampus

Background: Studies explored the therapeutic role of agents inhibiting RAS in epilepsy. Fewer studies addressed the electrophysiological changes associated with angiotensin converting enzyme inhibitors (ACEIs) in terms of sustained seizures (status epilepticus). Sodium valproate (SVPA), a broad-spectrum anticonvulsant, has been associated with adverse cardiac events upon long-term use, in contrast to the beneficial role of ACEIs in cardiovascular disorders. This work explored the potential effects of ramipril, an ACEI, compared to SVPA, on the behavior, and electrophysiology of the brain and heart in a rat model of status epilepticus. The dose dependent pattern of the presumed ramipril activities was investigated. Methods: Adult male rats were assigned into seven groups, controls, IP pyridostigmine (36 mg/kg)-induced status epilepticus (PISE), oral SVPA (5 mg/kg), and three groups receiving oral ramipril at respective doses of 5 (R5), 10 (R10), and 20 mg/kg (R20). Rat behavior was assessed using Racine’s motor convulsion scoring for 10 minutes. Blood pressure was recorded, and electroencephalography (EEG) and electrocardiography (ECG) were performed on the sedated rats 24 hours after recovery. Results: Despite the partial behavioral improvement of motor convulsions with R5 and R10 exhibited epileptogenic activity, as indicated by the increased relative power of fast and slow gamma waves and total EEG power. R10 triggered arrhythmia and cardiac ischemia as indicated by absence of P wave, along with ST elevation and tall T wave, slowed heart rate and prolonged QRS, QTc, and RR intervals. Conclusion: PISE was resistant to sodium valproate and ramipril. Ramipril at low and moderate doses induced epileptogenic activity and, especially at moderate dose, precipitated cardiac ischemia and arrhythmia. Summary The debatable role of ramipril in epilepsy was studied in a rat model of pyridostigmine-induced status epilepticus, compared to sodium valproate. Increasing ramipril doses did not resolve status epilepticus in rats. Instead, low and moderate doses exhibited epileptogenic activity, opposite to high dose ramipril and sodium valproate. Blood pressure was dose-dependently reduced with ramipril. Electrocardiography showed evidence of cardiac arrythmia and ischemia, especially with the moderate ramipril dose. The behavioral and EEG indices correlated with systolic blood pressure and ECG changes.

Alterations in mRNA expression of glutamate receptor subunits and excitatory amino acid transporters following pilocarpine-induced seizures in rats

After seizures, the hyperactivation of extracellular signal-regulated kinases (ERK1/2) causes mitochondrial dysfunction. Through the guidance of dynamin-related protein 1 (DRP1), ERK1/2 plays a role in the pathogenesis of several illnesses. Herein, we speculate that ERK1/2 affects mitochondrial division and participates in the pathogenesis of epilepsy by regulating the activity of DRP1. LiCl-Pilocarpine was injected intraperitoneally to establish a rat model of status epilepticus (SE) for this study. Before SE induction, PD98059 and Mdivi-1 were injected intraperitoneally. The number of seizures and the latency period before the onset of the first seizure were then monitored. The analysis of Western blot was also used to measure the phosphorylated and total ERK1/2 and DRP1 protein expression levels in the rat hippocampus. In addition, immunohistochemistry revealed the distribution of ERK1/2 and DRP1 in neurons of hippocampal CA1 and CA3. Both PD98059 and Mdivi-1 reduced the susceptibility of rats to epileptic seizures, according to behavioral findings. By inhibiting ERK1/2 phosphorylation, the Western blot revealed that PD98059 indirectly reduced the phosphorylation of DRP1 at Ser616 (p-DRP1-Ser616). Eventually, the ERK1/2 and DRP1 were distributed in the cytoplasm of neurons by immunohistochemistry. Inhibition of ERK1/2 signaling pathways downregulates p-DRP1-Ser616 expression, which could inhibit DRP1-mediated excessive mitochondrial fission and then regulate the pathogenesis of epilepsy.

Children are highly vulnerable to the neurotoxic effects of organophosphates (OPs), which can cause neuronal developmental defects, including intellectual disability, autism, epilepsy, and related comorbidities. Unfortunately, no specific pediatric OP neurotoxicity model currently exists. In this study, we developed and characterized a pediatric rat model of status epilepticus (SE) induced by the OP diisopropylfluorophosphate (DFP) and examined its impact on long-term neurological outcomes. Postnatal day 21 rats were exposed to a DFP regimen with standard antidotes. Progressive behavioral deteriorations were assessed over a three-month period. Development of epileptic seizures, ictal discharges, high-frequency oscillations (HFOs), and interictal spikes were monitored by video-electroencephalography recordings. Histology-stereology analysis was performed to assess neurodegeneration, neuroinflammation, and morphologic abnormalities. DFP-exposed, post-SE animals exhibited significantly elevated levels of anxiety and depression than age-matched controls at 1, 2, and 3 months post-exposure. DFP-exposed animals displayed aggressive behavior and a marked decline in object recognition memory, as well as prominent impairment in spatial learning and memory. DFP-exposed animals had striking electrographic abnormalities with the occurrence of displayed epileptic seizures, ictal discharges, HFOs, and interictal spikes, suggesting chronic epilepsy. Neuropathological analysis showed substantially fewer principal neurons and inhibitory interneurons with a marked increase in reactive microglia and neuroinflammation in the hippocampus and other brain regions. DFP-exposed animals also exhibited mossy fiber sprouting indicating impaired network formations. Long-term epileptic seizures and neuropsychiatric functional deficits induced by DFP were consistent with neuropathological defects. Collectively, this pediatric model displays many hallmarks of chronic sequelae reminiscent of children exposed to OPs, suggesting that it will be a valuable tool for investigating pathologic mechanisms and potential treatment strategies to attenuate long-term OP neurotoxicity. SIGNIFICANCE STATEMENT: Millions of children are exposed to organophosphates (OPs) used in agriculture or chemical incidents. This study investigated the long-term impact of neonatal exposure to the OP chemical diisopropylfluorophosphate (DFP) on neurobehavioral and neurodevelopmental outcomes in adulthood. DFP exposure caused long-lasting behavioral abnormalities, epileptic seizures, and bilateral brain defects with an array of neurological sequelae seen in children's OP neurotoxicity. Thus, this model provides a novel tool to explore therapeutic interventions that mitigate long-term neurotoxic effects of children exposed to OP-induced seizures and status epilepticus.

A rat model of organophosphate-induced status epilepticus and the beneficial effects of EP2 receptor inhibition

Epileptogenesis may be responsible for both of recurrent seizures and comorbid depression in epilepsy. Disease-modifying treatments targeting the latent period before spontaneous recurrent seizures may contribute to the remission of seizures and comorbid depression. We hypothesized that pre-treatment with 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a soluble epoxide hydrolase (sEH) inhibitor, which has anti-inflammatory and neuroprotective effects might rescue status epilepticus (SE)-induced dendritic spine loss and alleviate depressive behaviours. Rats were either pre-treated with TPPU (0.1 mg/kg/d) intragastrically or with vehicle (40% polyethylene glycol 400) from 7 days before to 7 days after SE that was induced with lithium chloride and pilocarpine intraperitoneally. Rats in the Control group were given saline instead. The forced swim test (FST) was performed on the 8th day after SE to evaluate the depression-like behaviours in rats. The results showed that seizures severity during SE was significantly decreased, and the immobility time during FST was significantly increased through TPPU pre-treatment. Moreover, pre-treatment with TPPU attenuated inflammations including microglial gliosis and the level of proinflammatory cytokine IL-1β in the hippocampus; in addition, neuronal and dendritic spine loss in the subfields of hippocampus was selectively rescued, and the expression of NR1 subunit of N-methyl-D-aspartate (NMDA) receptor, ERK1/2, CREB, and their phosphorylated forms involved in the dendritic spine development were all significantly increased. We concluded that pre-treatment with TPPU attenuated seizures severity during SE and depressive behaviours during the period of epileptogenesis probably by rescuing dendritic spine loss in the hippocampus.

Differential regulation of adenylate cyclase activity in rat ventral and dorsal hippocampus by rat galanin

BackgroundThe current objective is to evaluate the effect of frankincense oil on the convulsions and the associated neurochemical alterations produced in pilocarpine-induced status epilepticus rat model.MethodsRats were divided randomly into: control, status epilepticus rat model and rat model of status epilepticus pretreated with frankincense oil daily for 5 days before pilocarpine treatment. On the fifth day, after pilocarpine injection, rats were observed to evaluate the severity of seizures for 2 h. The oxidative stress parameters malondialdehyde, reduced glutathione and nitric oxide, the proinflammatory cytokines interleukin-6 and interleukin-1β and acetylcholinesterase were determined in the cortex, hippocampus and striatum. Dopamine, norepinephrine and serotonin were measured in the cortex and striatum.ResultsThe status epilepticus model exhibited repetitive seizures in the form of generalized tonic- clonic convulsions after 30 min. of pilocarpine injection. This was associated with a significant increase in the levels of malondialdehyde and nitric oxide and a significant decrease in reduced glutathione in the three regions. A significant increase was also observed in interleukin-1β, interleukin-6 and acetylcholinesterase. In the cortex and striatum, a significant decrease was recorded in monoamine levels. Pretreatment of rat model of status epilepticus with frankincense oil decreased the severity of seizures that appeared in the form of tremors and facial automatisms and prevented the increase in malondialdehyde, nitric oxide, interleukin-1β, interleukin-6 and acetylcholinesterase and the decrease in reduced glutathione induced by pilocarpine in the studied brain regions. Frankincense oil failed to restore the decreased level of cortical serotonin and dopamine. In the striatum, frankincense oil improved the levels of serotonin and norepinephrine but failed to restore the decreased dopamine levels.ConclusionIt is clear from the present results that frankincense oil reduced the severity of seizures induced by pilocarpine. This could be mediated by its potent antioxidant and anti-inflammatory effects.

This study aims to establish pilocarpine-induced rat model of status epilepticus (SE), observe the activity of calpain I in the rat hippocampus and the subsequent neuronal death, and explore the relationship between calpain I activity and neuronal death in the hippocampus. Fifty-eight adult male Wistar rats were assigned randomly into either control group (n = 8) or epilepsy group (n = 50). SE was induced in the epilepsy group using pilocarpine. Before the injection, the rats were given atropine sulfate to reduce the side effect of pilocarpine. All rats in the seizure group were grouped into either SE or non-SE, depending on whether they developed convulsive seizures. The rats in SE group were treated with chloral hydrate to stop seizures after 60 min. Control animals were treated with the same dose of 0.9 % saline. All rats were monitored for seizures. At 24 h after SE, the rats' left brain tissues were stained by HE and TUNEL. Neuronal necrosis and apoptosis in the hippocampal CA3 area were observed. Calpain I activity in the right hippocampus was also observed using western blotting. Eighty percent of the rats in the seizure group developed SE, of which 35 % died. No rat died in both the control and non-SE groups. At 24 h after SE, the number of HE-stained neurons decreased (SE group: 55.19 ± 8.23; control group: 102.13 ± 3.73; non-SE group: 101.2 ± 2.86) and the number of TUNEL-positive neurons increased (SE group: 4.91 ± 1.35; non-SE and control group: 0). No obvious changes were observed in the neurons of the control and non-SE group animals. The 76 kDa cleavage of calpain I (the average optical density ratio is 0.096 ± 0.015) emerged in the SE group. Neuronal death has a direct relationship with calpain I activity. There is high success rate and lower death rate for pilocarpine to induce SE. At 24 h after SE, activity of calpain I, neuronal necrosis and apoptosis increased in the hippocampus. Neuronal death has a direct relationship with calpain I activity, which suggests that calpain I plays an important role in neuronal damage during SE.

Most antiepileptic drug therapies are symptomatic and adversely suppress normal brain function by nonspecific inhibition of neuronal activity. In recent times, growing evidence has suggested that neuroinflammation triggered by epileptic seizures might be involved in the pathogenesis of epilepsy. Although the potential effectiveness of anti-inflammatory treatment for curing epilepsy has been extensively discussed, the limited quantitative data regarding spatiotemporal characteristics of neuroinflammation after epileptic seizures makes it difficult to be realized. We quantitatively analyzed the spatiotemporal changes in neuroinflammation in the early phase after status epilepticus in rats, using translocator protein (TSPO) positron emission tomography (PET) imaging, which has been widely used for the quantitative evaluation of neuroinflammation in several animal models of CNS disease. The second-generation TSPO PET probe, [18F]DPA-714, was used for brain-wide quantitative analysis of neuroinflammation in the brains of rats, when the status epilepticus was induced by subcutaneous injection of kainic acid (KA, 15 mg/kg) into those rats. A series of [18F]DPA-714 PET scans were performed at 1, 3, 7, and 15 days after status epilepticus, and the corresponding histological changes, including activation of microglia and astrocytes, were confirmed by immunohistochemistry. Apparent accumulation of [18F]DPA-714 was observed in several KA-induced epileptogenic regions, such as the amygdala, piriform cortex, ventral hippocampus, mediodorsal thalamus, and cortical regions 3 days after status epilepticus, and was reversibly displaced by unlabeled PK11195 (1 mg/kg). Consecutive [18F]DPA-714 PET scans revealed that accumulation of [18F]DPA-714 was focused in the KA-induced epileptogenic regions from 3 days after status epilepticus and was further maintained in the amygdala and piriform cortex until 7 days after status epilepticus. Immunohistochemical analysis revealed that activated microglia but not reactive astrocytes were correlated with [18F]DPA-714 accumulation in the KA-induced epileptogenic regions for at least 1 week after status epilepticus. These results indicate that the early spatiotemporal characteristics of neuroinflammation quantitatively evaluated by [18F]DPA-714 PET imaging provide valuable evidence for developing new anti-inflammatory therapies for epilepsy. The predominant activation of microglia around epileptogenic regions in the early phase after status epilepticus could be a crucial therapeutic target for curing epilepsy.

In the latent cue preference (LCP) task, water-deprived rats alternately drink a salt solution in one distinctive compartment of a conditioned cue preference (CCP) apparatus and water in the other compartment over 8 days (training trials). They are then given a choice between the two compartments with no solutions present (preference test). Previous findings showed that this training procedure results in two parallel forms of learning: conditioning to water-paired cues (a water-CCP) and latent learning of an association between salt and salt-paired compartment cues (a salt-LCP). Experiment 1 examined these two types of learning in isolation. Results showed that expression of the salt-LCP required salt deprivation during testing, but expression of the water-CCP did not require a deprivation state during testing. Other results showed that salt-LCP learning itself involves two distinct components: (1) the latent association among neutral cues in the salt-paired compartment, and (2) motivational information about salt deprivation during testing. Previous findings also demonstrated roles for the dorsal hippocampus (DH), ventral hippocampus (VH), and entorhinal cortex (EC) in salt-LCP learning. Experiment 2 examined the involvement of these structures during acquisition or expression of salt-LCP learning. Rats with cannulas aimed at DH, VH, or EC were given infusions of muscimol, either before exposure to the salt-paired, but not the water-paired, compartment during training or before the preference test. Inactivation of the DH or EC impaired both acquisition and expression of the association between salt and salt-paired compartment cues, while inactivation of the VH disrupted the influence of motivational information about salt deprivation required to express the salt-LCP. These results suggest unique roles for the EC-DH circuit and VH in salt-LCP learning, as well as a functional dissociation between the DH and VH.

Galanin differentially regulates acetylcholine release in ventral and dorsal hippocampus: a microdialysis study in awake rat

In vivo glutamate decline associated with kainic acid-induced status epilepticus