Antiseizure And Neuroprotective Efficacy Of Chronic Intermittent Hypobaric Hypoxia In The Rat Pilocarpine-induced Epilepsy Model

Epilepsy is one of the most common neurological disorders, afflictingmore than9million individuals in China according to domesticepidemiological investigations. Among patients with epilepsy, about25%-40%suffers from intractable epilepsy resistant to currently available antiepilepticdrugs (AEDs). Many such cases are of temporal lobe epilepsy (TLE),characterized by spontaneous recurrent seizures (SRS) and progressivehippocampal sclerosis. Clinical investigations have shown that over half ofepilepsy patients suffer from cognitive impairments due to neurologicaldamage associated with frequent seizures and (or) the side-effects oflong-term AED use. This cognitive dysfunction imposes an even heavierburden on epileptic individuals, their families, and society.The rat pilocarpine (PILO)-induced seizure model exhibits many of theclinical, behavioral, and neuropathological hallmarks of TLE and statusepilepticus (SE), and so is advantageous for preclinical testing of noveltreatment strategies.The pathogenesis of TLE is complex and may involve a combination ofrecurrent mossy fiber sprouting, excitotoxicity induced by neuronal calciuminflux, abnormal glutamate and γ-aminobutyric acid (GABA) receptorstructure and function, and oxidative stress with ensuing apoptosis. Oxidativestress occurs when production of reactive oxygen species (ROS) exceeds thecapacity of endogenous cellular antioxidant defense systems, resulting inmitochondrial dysfunction and destruction of macromolecules such asmembrane lipids. Status epilepticus is often associated with severe brainhypoxia. Reperfusion of hypoxic tissue is associated with ROS accumulationin mitochondria, which can damage mitochondrial membranes, decreasing the mitochondrial transmembrane potential, inducing mitochondrial cytochrome crelease into the neuronal cytoplasm, and activating the caspase cascadeleading to apoptosis. Reducing ROS production during SE to protectmitochondria and inhibit neuronal apoptosis is a possible replacement oradjuvant therapy for intractable epilepsy.A variety of pretreatment or preconditioning methods can induce hypoxicand oxidative stress tolerance, including mild transient ischemia, hyperbaricoxygen, hyperthermia, hypothermia, sleep deprivation. However, clinicalapplication of many of these methods is limited by potential side effects.Severe hypoxia often leads to pathological changes, but recent studies havethat shown moderate transient hypoxia is a physiologically importantmechanism for maintaining or enhancing endogenous cytoprotection.Hypoxic preconditioning can inhibit tumor necrosis factor alphaexpression while raising erythropoietin, vascular endothelial growth factor,and anti-apoptotic Bcl-2expression in hippocampal CA1and CA3subfields.In vivo, hypoxic preconditioning can prevent hippocampal damage fromischemia/hypoxia and prevent associated cognitive dysfunction. Accumulatingevidence indicates that chronic intermittent hypobaric hypoxia (CIHH)enhances cardiac tolerance against subsequent ischemia/hypoxia, reducesheart damaged due to ischemia/reperfusion, inhibits ultrastructural damageand calcium overload in cardiac myocytes, maintains myocyte calciumhomeostasis, and prevents arrhythmia. In addition, CIHH has been shown toprotect nerve, liver, kidney, and other organs against subsequent severehypoxic insults. Furthermore, CIHH lowers blood pressure, reduces oxidativestress and apoptosis, and enhances immune function. In a rat model ofischemic cerebrovascular disease, CIHH enhanced ischemic resistance byupregulating brain expression of glutamate transporter-1(GLT-1). However,whether CIHH preconditioning may protect against SE or not, there was noreported. In this study, we examined the effects of CIHH preconditioning onbehavioristics of rat lithium-pilocarpine model of TLE, hippocampal neuronsurvival (morphology), and molecular biology in order to further explore the underling mechanisms.PartⅠ Neuroprotective effect of CIHH preconditioning on pilocarpine-induced seizures in ratsObjective: To establish a pilocarpine (PILO)-induced SE rat model andevaluate the efficacy of CIHH preconditioning for preventing SE-associateddeath of hippocampal neurons and preserving hippocampal function.Methods:(Experiment1) Healthy adult male Sprague-Dawley rats wererandomly divided into six groups of10: a normal control (CON group) and5PILO groups (SE groups) sacrificed3h,12h,24h,72h, and7days post-SE(SE3h, SE12h, SE24h, SE72h and SE7d groups, respectively). Nissl stainingand flow cytometry were used to assess morphological damage and apoptosisof hippocampal neurons post-SE.(Experiment2) According to previousreports,72h post-SE is the peak time of SE-associated brain damage, so thistime point was selected to examine the neuroprotective effects of CIHHpreconditioning. Animals were randomly divided into four groups: CON (n=7),SE (n=13), CIHH+SE (n=13), CIHH (n=7). Rat mortality from0-72h post-SEwas determined and electroencephalograms (EEGs) were recorded to measurethe frequency, severity, and duration of epileptiform discharges. Hippocampaltissues were then isolated to assess morphological damage and neuronalsurvival by Nissl staining and neuronal apoptosis rate by flow cytometery.Results:(Experiment1)1Status epilepticus was induced by PILO in41of50rats (success rate,82%). Seven SE group rats died during the procedure. The remaining34PILO-treated rats were analyzed and compared to the10(surviving) CONgroup rats.2Nissl staining: In the CON group, there was almost no visible neuronalloss in CA1and CA3subfields. Similarly, SE3h and SE12h groups showed noobvious neuronal loss and neuronal morphology resembled that of the CONgroup. Neuronal loss was apparent in the CA1and CA3regions of the SE24hgroup. In the SE72h group, neuron loss appeared even greater, especially in CA1. Neuronal loss in the CA1of the SE7d group was clearly lower than inthe SE72h group. Cell counting revealed that the numbers of survivingneurons in the SE3h and SE12h groups were not significantly different fromthe CON group (P>0.05), while significant reductions were observed in theSE24h, SE72h, and SE7d groups compared to the CON group (P<0.05).Neuronal survival was significantly higher in all other SE groups compared tothe SE72h group (P<0.05).3Flow cytometry: Compared to the CON group, neuronal apoptosis rateswere significantly higher in the SE24h, SE72h, and SE7d groups (P<0.05), butnot in the SE3h and SE12h groups (P>0.05). Apoptosis rates in SE24h andSE7d groups were not significantly different (P>0.05) but were bothsignificantly lower compared than in the SE72h group (P<0.05). Thus, PILOinduced neuronal death in the hippocampus, peaking at72h post-SE.(Experiment2)1Mortality was23.1%in the SE group, while no deaths were observed inthe CIHH+SE group. Compared to the SE group, the latency to reach SE waslonger (P<0.05) and the frequency of EEG-defined epileptic discharges lower(P<0.05) in the CIHH+SE group. There was no mortality or seizures in theCON and CIHH groups.2Nissl staining: Neurons in CA1and CA3subfields of the CON groupwere clearly stained and there was almost no neuronal loss, while neuronal losswas clearly visible in the SE group. Neuronal cell death was partially reversedin the CIHH+SE group and fewer neurons showed morphological signs ofdamage in both CA1and CA3subfields compared to the SE group. There wasvery slight neuronal loss in the CIHH group. Cell counting revealed asignificant decrease in neuronal survival in the SE group compared to the CONgroup (P<0.05), while the number of surviving neurons was significantlyhigher in the CIHH+SE group compared to the SE group (P<0.05). Althoughthe number of surviving hippocampal neurons was lower in the CIHH groupthan the CON group, there was no significant statistical difference (P>0.05).3Flow cytometry: The numbers of apoptotic neurons were significantly higher in the SE and SE+CIHH groups compared to the CON group (P<0.05).However, apoptotic rate was significantly lower in the CIHH+SE groupcompared to the SE group (P<0.05), indicating partial rescue from apoptosisby CIHH preconditioning. The rate of neuronal apoptosis was slightly higher inthe CIHH group compared to the CON group, but the difference was notstatistically significant (P>0.05).Conclusion: The PILO-induced SE rat model allows for dynamicobservation of progressive hippocampal neuronal injury and apoptosis. At72hafter SE, damage was more severe in the CA1. Preconditioning with CIHHprolonged SE latency and reduced seizure frequency, PILO-induced mortality,morphological signs of hippocampal neuronal damage, and neuronalapoptosis.PartⅡ Effects of different hypoxic preconditioning protocols on thehippocampal damage of status epileptic ratsObjective: Chronic intermittent normobaric hypoxia (CINH）has beenproved playing an important role in protecting the damaged brain of rats. Inthis study, we compared the effects of different hypoxic preconditioningprotocols (CIHH and CINH) on spontaneous recurrent seizures (SRS),intracellular free calcium concentration ([Ca2+]i), and apoptosis rate followingPILO-induced status epilepticus (SE).Methods: Adult male Sprague-Dawley rats were randomly divided intofour groups: normal control group (CON group, n=18), PILO group (SE group,n=34), CIHH+SE group (CIHH-Pre, n=24) and CINH+SE group (CINH-Pre,n=24). The rats in each group were divided into two parts: a part of that wascompared by video monitoring of behavioral seizure activity (frequency, rank,and duration); another part of that were used to examine changes in themorphology of hippocampal pyramidal neurons by menas of Nissl stainingand Fluoro-Jade B (FJB) staining, and further to detect the quantification of[Ca2+]i and cell apoptosis by means of flow cytometry at SE72hour.Results:1Mortality and SRS of rats induced by PILO: In the SE group, mortality rate was47.1%, while in the CIHH-Pre group and CINH-Pre group, mortalityrate was16.7%and20.8%, respectively.SRS by the V-EEG: There was a significant decrease in the number,mean severity, and duration of seizures in both CIHH-treated andCINH-treated SE rats compared to those treated by PILO (P<0.05). However,the CIHH-Pre protocol induced a greater reduction in both seizure frequencyand severity compared to the CINH-Pre group (P<0.05). There was no SRSseizures and animal died in the CON group.2Comparation of the [Ca2+]i: A substantial increase was measured in theSE group compared to the CON group (P<0.05), while both CIHH-Pre andCINH-Pre significantly reduced the post-SE [Ca2+]i elevation compared to theSE group (P<0.05). Hippocampal [Ca2+]i was lower in the CIHH-Pre groupthan that of the CINH-Pre group (P<0.05).3Nissl staining: There was a marked loss of neurons in CA1and CA3subfields of SE group compared to the CON group. Compared to the SE group,there was a significantly reduced neuronal loss in the CIHH-Pre andCINH-Pre groups.The number of surviving neurons in both subfields of the hippocampus inSE group obviously decrease compared to the CON group (P<0.05).Compared to the SE group, the number of neurons was significantly increasein the CIHH-Pre and CINH-Pre groups (P<0.05), while the average CA1pyramidal neuron density was lower in the CINH-Pre group than in theCIHH-Pre group (P<0.05).4FJB staining: No FJB-positive cell was observed in the CON group.Many FJB-positive cells with kelly fluorescent were observed in the CA1andCA3subfields in the SE group. CIHH-Pre and CINH-Pre groups showed asignificant reduction in the number of FJB-positive cells in the CA1and CA3subfiels (P<0.05). The average numbers of FJB-positive cells in the CA1subfield was lower in the CIHH-Pre group than in the CINH-Pre group(P<0.05).5Apoptosis rate of the hippocampus: Both the number of cells in early apoptosis (Q4) and total apoptotic cells (Q2+Q4) were significantly higher inthe SE group than in the CON group (P<0.05). Both the CIHH-Pre group andthe CINH-Pre group exhibited markedly reduced apoptosis rate compared tothe SE group (P<0.05). Moreover, apoptosis rate was lower in the CIHH-Pregroup than in the CINH-Pre group (P<0.05).Conclusion: SE rats induced by PILO prone to SRS leads to neuronaldamage of hippocampus by increasing the [Ca2+]i. CIHH-Pre and CINH-Precan effectively inhibit the SRS behavior and reduce the post-SE [Ca2+]i so asto rescue the hippocampal neurons. What’s more, hypobaric preconditioningmay be more effective.Part Ⅲ Effect of CIHH on the antioxidant defense system of thehippocampus in epileptic ratsObjective: To examine whether CIHH preconditioning protects againstoxidative stress and mitochondrial damage induced by SE and to explore theunderlying mechanisms.Methods: Animals were randomly divided into three groups: CON(n=20), SE (n=20), and CIHH+SE (n=20). Following these treatments,homogenates were prepared from hippocampus to measure MDAaccumulation, an index of oxidative stress, as well as the activities of theantioxidants superoxide dismutase (SOD) isoforms and glutathione (GSH)using commercial assay kits72hour post-SE. Second, levels of ROS and themitochondrial transmembrane potential (△Ψm) in the hippocampus weremeasured by flow cytometry. Third, Cytochrome c, activated Caspase3, Bcl-2,and Bax proteins in cytoplasm were measured by Western Blot. Theultrastructure of mitochondria under the three experimental conditions wasexamined by transmission electron microscopy.Results:1Compared to the CON group, the level of MDA was significantlyhigher (P<0.05), total SOD and SOD2activities lower (P<0.05), and GSHconcentration lower (P<0.05) in the SE group. These “pro-oxidant” changes inthe SE group were partially reversed in the CIHH+SE group (P<0.05). There were no significant group differences in SOD1activity (P>0.05).2Compared to the SE group, ROS was significantly lower (P<0.05) and△Ψm significantly greater (P<0.05) in the hippocampus of the CIHH+SEgroup.3Compared to the CON group, the protein expression levels ofCytochrome c, Caspase3, and Bax, as well as the Bax/Bcl-2ratio weresignificantly higher in the SE group (P<0.05), while expression of Bcl-2wassignificantly lower (P<0.05). These pro-apoptotic changes were partiallyreversed by CIHH preconditioning prior to SE (CIHH+SE group)(P<0.05).4Compared to the CON group, mitochondria from the SE groupexhibited obvious morphological abnormalities, such as vacuolization, ridgefracture, and membrane damage, effects partially reversed by CIHHpreconditioning.Conclusion: Mitochondria were damaged by SE, possibly due to freeradical generation and oxidative stress. Preconditioning by CIHH inhibitedoxidative stress and maintained mitochondrial structure and function, possiblyaccounting for the neuroprotection and inhibition of apoptosis observed in theCIHH+SE group compared to the SE group.Part Ⅳ Effect of CIHH on cognitive impairment in SE ratsObjective: To examine if CIHH can reduce cognitive impairments inSE-treated rats with spontaneous recurrent seizures (SRSs) and to determinethe underlying mechanisms.Methods: Animals were randomly divided into three groups: CON(n=10), SE (n=20), and CIHH+SE (n=20). According to results of ourpreliminary study, SRSs occur1442days after SE. Thus, the Morris watermaze (MWM) test of hippocampus-dependent spatial reference memory wasperformed at day42post-SE. After the MWM test, synaptic ultrastructure inthe hippocampal CA1was examined by electron microscopy and theexpression levels of synaptic proteins PSD-95and Kalirin-7were detected byWestern Blot.Results: As some rats in the SE group died, the numbers of rats examined by the MWM on day42post-SE were as follows: CON group(n=10), PILO-induced SE group (n=9), CIHH+SE group (n=14).1In the Morris water maze, all animals exhibited a progressive decline inescape latency starting on the third day of training (P<0.05). Rats in theCIHH+SE group exhibited a significantly shorter escape latency than the SEgroup (P<0.05). On the probe trial, the number of platform crossing wasmarkedly lower (P<0.05) in the SE group and these rats spent significantlyless time in the target quadrant compared to the CON and CIHH+SE group(P<0.05). Thus, SE group rats demonstrated both impaired (hippocampus-dependent) spatial learning during training and poor spatial reference memory.These deficits were partially reversed in the CIHH+SE group as indicated bysignificantly shorter escape latencies and a longer mean time in the targetquadrant compared to the SE group (P<0.05). There were no significant groupdifferences in escape latency and swimming speed in the visible platformversion of the MWM test (P>0.05), indicating that motor and sensory effectscannot account for the effects of CIHH preconditioning on cognition.2The width of synaptic cleft: The PILO-treated rats exhibitedsignificantly wider synaptic clefts compared to the CON group (P<0.05)(SE:25.20±0.78nm, CIHH+SE:23.13±0.89nm, CON:20.27±0.45nm). AlthoughCIHH reduced the synaptic cleft width, there was no statistical differencebetween SE and CIHH+SE groups (P>0.05).The thickness of the postsynaptic density (PSD): PILO-treated ratsexhibited a significant decrease in PSD compared to the CON group (P<0.05)(SE:22.67±0.76nm,CIHH+SE:27.87±0.77nm,CON:30.27±0.69nm). However,PSD thickness was significantly greater in the CIHH+SE group compared tothe SE group (P<0.05).3Expression levels of synaptic proteins: Compared to the CON group(mean density,0.72±0.02), expression levels of PSD-95were significantlylower in the SE and CIHH+SE groups (P<0.05), but expression wassignificantly higher in the CIHH+SE group compared to the SE group(0.40±0.02vs.0.21±0.02, P<0.05). Similarly, compared to the CON group (0.68±0.02), protein expression levels of kalirin-7were significantly lower inthe SE and CIHH+SE groups (P<0.05), but higher in the CIHH+SE groupthan SE group (0.34±0.02vs.0.26±0.02, P<0.05).Conclusion: PILO-induced SE rats demonstrated significant cognitiveimpairments and changes in hippocampal synaptic ultrastructure.Preconditioning by CIHH partially ameliorated hippocampus-dependentlearning and memory deficits, changes in synaptic ultrastructure, and impairedexpression levels of PSD-95and kalirin-7in the hippocampus. Rescue ofsynaptic dysfunction by CIHH preconditioning may be one of the underlyingmechanisms for improved cognition function.