The Study On Biological Role And Mechanisms Of MIP-1α/CCR5Axis In The Epileptogenesis In Immature Rats

BackgroudEpilepsy is one of the most common neurological disorders, affecting the life-quality of patients and making the people's welfare dangerous. In recent years.although great endeavor has been made on the etiology.pathology and treatment of temporal epilepsy.the precise mechanism of the disease, namely epileptogenesis,still remains unclear. Accumulating data shows that external insults such as brain trauma,hypoxia and drugs can trigger a cascade of molecular and cellular events, which eventually, close to epileptogenesis, lead to the development of spontaneous seizures and epilepsy. The activation of inflammatory cascades during the acute and subacute changes in epileptogenesis following initial insults raises the significance that inflammatory mediators may contribute to the development of epileptic process.Chemokines, a family of small proteins involved in the activation and migration of immune cells, has been widely explored in the inflammatory response of central nervous system (CNS) disorders. As a vital member of (3-chemokine subfamily, macrophage inflammatory protein-1alpha (MIP-1α) is produced by a variety of cells, including lymphocytes, monocytes/macrophages, mast cells, epithelial cells, and fibroblasts, and stimulates the secretion of several cytokines and attracts immune cells. Limited information manifested that MIP-la was dramatically over-expressed in brain lesion areas of neurological diseases, but its role and cellular sources stay unidentified. Chemokines exert their biological effects through binding specific high-affinity G-protein-coupled receptors. The C-C chemokine receptor5(CCR5) is an identifiably main receptors of MIP-1α in the CNS and distributed to glial and neuronal cells. However, the controversial expression pattern of CCR5in the rodent models of excitotoxic brain injury has limited our understanding of MIP-1α/CCR5contribution to pathogenesis of epileptic seizure. Further, Growing evidence indicates that lots of signal molecules, such as rnitogen-actived protein kinases (MAPKs), phosphatidylinositol3-kinase and nuclear factor κB (NF-κB), are constructively expressed in the CNS, and external stimuli including seizure could activate them and subsequently modulate the gene transcription. Current studies in vitro demonstrate that activated MAPKs and NF-κB could mediate the expression of chemokines and their receptors in cultured glial and neuronal cells. However, up to now, there was limited information as to the molecular mechanism of expression of MIP-la/CCR5axis. In all, such queries prompt us carry out further research to explore the expression,significance and regulation of MIP-la/CCR5axis in the pathogenesis during epileptogenesis.In addition, experimental evidence in animals indicated that seizures lead to neuropathological changes in the hippocampus in an age-as well as region-specific manner and maturational stage could affect the expression of chemokines or their receptors in some pathological conditions. Earlier studies specifically for MIP-1α, moreover, mostly focused on the neonatal and adult rat brain. Such facts, along with the high clinical prevalence of epilepsy in childhood, prompted us to recruited immature rats as research subjects. We chose a reproducible seizure model by intracerebroventricular (i.c.v.) injection of kainic acid (KA), and evaluated the development of chronic spontaneous seizures by the continuous record of video-electroencephalogram (EEG). Further, we investigated the characterization of the expression of MIP-1α and CCR5in the hippocampus in the early stages following KA-induced status epilepticus (SE). Additionally, we also explored the biological role of MIP-la/CCR5axis in epileptogenesis and identified its potential regulatory mechanisms based on the appropriate intervention measures using the inhibitors of MAPKs (PD98059or SB203580) or NF-κB (PDTC).Part Ⅰ The study on the expression of MIP-la/CCR5axis in the epileptogenesis in immature ratsObjective:To identify the expression features of MIP-1α and CCR5in immature rat hippocampus and explored the potential link between this axis and neuropathological alterations following SE initiated by i.c.v. KA injection.Methods:1. Seizures were induced through i.c.v. KA by a stereotaxic apparatus, postnatal day21(P21) wistar rats were randomly divided into normal group, control group and groups at4h,8h,16h,24h,3d and5d after KA treatment. Referring the findings in preliminary study, phosphate-buffered saline (PBS)-injected controls were further divided into8h and24h post-injection groups for the analysis of total RNA and24h post-injection group for protein analysis. Additionally, one study including15KA-treated and7PBS-treated rats was performed to evaluate the seizure behaviour and EEG of animals2months later.2. Hematoxylin-eosin (HE), Nissl and Fluoro-Jade B (FJB) staining were used to examine the evolution of neuronal damage in different subfields of rat's hippocampus, including the dentate hilus, CA3and CA1subfield.3. Immunohistochemistry was performed to examine the dynamic expression of OX-42, MIP-1α and CCR5in different hippocampal subfields of animals.4. Real-time PCR was used to detect the mRNA expression of MIP-1α and its CCR5receptor in the hippocampus in each group.5. MIP-1a and CCR5protein in the hippocampus in each group were quantified by ELISA and Western blot respectively. 6. Double-labelling immunofluorescence was performed to determine the cellular distributions of MIP-1a and CCR5in the hippocampus.Results:1. The construction of KA rat models of epilepsy. Following deep breathing, head nodding and forelimb clonus within15-30min after KA injection, rats gradually showed rearing and generalized tonic-clonic seizures within30-40min, which lasting for2-4h. To evaluate the chronic spontaneous seizures behavior, additional study showed that during the7days of continuous recording, eight of12(67%) KA-treated rats exhibited chronic spontaneous seizures, whereas no seizure was observed in the PBS-treated rats.2. Neuronal damage and microglial reaction in the hippocampus after KA. HE, Nissl and FJB staining results:As compared with the normal rats, there was only scattered injured neurones in the hippocampal CA3pyramidal cell layer and the dentate hilus at8h after KA treatment. Thereafter, neuronal damage was especially pronounced in the CA3at24h. However, delayed neuronal damage of the hippocampal CA1pyramidal cell layer appeared was highly significant at3days post-KA treatment. OX-42immunohistochemical results:In normal P21rats, OX-42positive cells was diffusely low in the normal hippocampus. After KA treatment, amoeboid-shaped microglia were apparent within the hippocampal pyramidal cell layer and the dentate hilus undergoing degenerative changes at24h. Immunostaining of microglia was further enhanced at3days in those degenerating regions.3. MIP-1α expression in the hippocampus, real time PCR results:After KA treatment, hippocampal MIP-1α mRNA showed a significant increase at4h (P<0.01), peaked at8-16h (P<0.01), and was observed highly for as long as3days (P<0.05). ELISA results:As compared with normal rats, the level of MIP-1α was significantly elevated from8to16h (P<1.01), and peaked at24h (P<0.01) and declined to near baseline levels at5days (P<0.05) after KA treatment. Immunohistochemical results: There was undetectable immunostaining for MIP-1α in normal hippocampus. After KA, MIP-1α immunoreactivity was markedly elevated particularly in the CA3at16h, and reached a peak in the CA3and dentate hilus at24h. At3days, MIP-1α immunoreactivity was markedly reduced in the CA3and hilus when compared with24h post-KA group, and was decreased gently in the CA1. Double-labelling immunofluorescence results:Induced MIP-1α immunoreactivity was expressed in OX-42-rather than glial fibrillary acidic protein (GFAP)-labeled immunoreactive cells.4. CCR5expression in the hippocampus. real time PCR results:A marked increase of CCR5mRNA in the hippocampus was initiated at8h (P<0.05), and reaching a peak at24h after KA treatment (P<0.01). Western blot results:As compared with normal rats,a significant increase in CCR5was observed at16h (P<0.01), peaked at24h after KA (P<0.01), and decreased thereafter. Immunohistochemical results: CCR5immunoreactive cells in normal rats were distributed in the whole hippocampus, with a round or oblong shape. Some immunoreactivity was also observed in the endothelium of blood vessel. At24h after KA, there was prominent immunoreactivity of CCR5in the dentate hilus and the CA3. However, in the CA1, CCR5immunoreactivity appeared abundant at3days. Double-labelling immunofluorescence results:In normal rats, CCR5immunoreactive cells were colocalized with faint OX-42immunoreactivity as well as GFAP immunoreactivity. After KA, CCR5immunoreactive cells were mainly colocalized with OX-42.Conclusions:1. SE in immature rats induced by i.c.v. KA injection could result in chronic spontaneous seizures.2. The expression of MIP-1α/CCR5axis was transiently increased after KA-induced SE in the immature rat hippocampus after KA-induced SE. Moreover, the presence of the MIP-1α and CCR5almost paralleled the accumulation of activated microglia in injured hippocampal subfields. Part Ⅱ The potential biological role of MIP-la/CCR5axis in the epileptogenesis in immature ratsObjective:To explore the effects of MIP-la/CCR5axis on the microglial reaction, blood-brain barrier alteration and neuronal damage in the early stages of epileptogenic process through the appropriate intervention measures.Methods:1. To examine the biological function of MIP-la, P21wistar rats were randomly divided into PBS+IgG group, KA+IgG group and KA+anti-MIP-la group. In each group, rats were sacrificed at24h and3d after KA or PBS treatment, and tissues were collected. The drug of anti-MIP-1or normal immunoglobulin (IgG) was administered immediately and12h after KA or PBS injection.2. To examine the biological function of CCR5, P21wistar rats were randomly divided into PBS group, KA+saline group and KA+DAPTA (D-ala-peptide T-amide, CCR5antagonist) group. All rats were sacrificed at3d after KA or PBS treatment, and tissues were collected. The drug of DAPTA was administered immediately,24h, and48h after KA injection. Saline was also used as a control of drug treatment.3. To examine the effect of biological axis on spontaneous recurrent seizures, P21wistar rats were randomly divided into KA+saline group and KA+DAPTA group. The drug of DAPTA was administered immediately,1d,2d,3d,4d and5d after KA injection. Saline was also used as a control of drug treatment. All rats were evaluated the development of chronic spontaneous seizures by the continuous record of video-electroencephalogram2m later, and subsequently scarified to collect tissues.4. Immunohistochemistry was performed to examine the expression of OX-42, endothelial barrier antigen (EBA) and ZO-1in the hippocampus in each group.5. Western blot was used to detecte the level of ZO-1and occludin protein in each group.6. FJB staining was used to examine the neuronal damage in different subfields of rat's hippocampus.7. Double-labelling immunofluorescence was performed to determine the alteration of proliferation rate of microglia through observing the colocalization of proliferating cell nuclear antigen (PCNA) and ED-1(marker of activated microglia) in the hippocampus.Results:1. The effect of anti-MIP-1α on microglia. Immunohistochemical results:as compared with the results of KA+IgG group, the number of microglia was reduced significantly (P<0.05). However, we failed to detect obvious alteration in the morphorlogy and proliferation rate of microgila between KA+IgG group and KA+anti-MIP-1α group.2. The effect of anti-MIP-1α on the integrity of blood-brain barrier. Evans blue (EB) qualitative and quantitative analysis:As compared with KA+IgG group, the staining intensity and concentration for EB (P<0.05) were significantly decreased in the hippocampus. EBA immunohistochemical results:In PBS+IgG group, EBA staining was distributed to the blood vessels, showing continuous structure. However, in KA+IgG group, immunostaining had almost disappeared and no structure resembling blood vessels was observeded. In KA+anti-MIP-1α group, EBA staining was obvious and structure was detected successively. Western blot results:As compared with KA+IgG group, the expression of both ZO-1and occludin in KA+anti-MIP-1α group was evidently increased (P<0.05).3. The effect of anti-MIP-1α on neuronal cells. FJB staining results:As compared with KA+IgG group, the loss of neuronal cells was distinctly decreased in the hippocampal subfields of KA+anti-MIP-1α rats (P<0.05).4. The impact of DAPTA on the microglial response, integrity of blood-brain barrier and neuronal survival in hippocampus. Expectedly, as compared with KA+saline group, microglial immunoreactivity, EB concentration and neuronal damage were decreased obviously in the hippocampus of KA+DAPTA rats (P<0.05).5. The impact of DAPTA on spontaneous recurrent seizures. As compared with KA+saline group, average frequency of seizure per day (2.4±0.9Vs1.5±0.5) and interictal spike frequency per day (5.4±0.7Vs2.8±0.7) were decreased obviously in KA+DAPTA group.Conclusions:1. After SE induced by i.c.v. KA injection, the biological axis of MIP-la/CCR5leads to the accumulation and migration of microglia in the hippocampus, but fails to influence on microglial activity and proliferation.2. After SE induced by i.c.v. KA injection, the biological axis of MIP-1α/CCR5can inhibit the expression of ZO-1and occludin, and prompt blood-brain barrier disruption.3. After SE induced by i.c.v. KA injection, the biological axis of MIP-la/CCR5can cause neuronal damage, which may be mediated by activated microglia.4. In the early stages of epileptogenesis following SE initiated by i.c.v. KA injection, the biological axis can prompt the development of chronic epilepsy, based on its potential contribution to the accumulation of microglia, blood-brain barrier disruption and neuronal damage, suggesting a potential therapeutic role for MIP-1α/CCR5inhibitors in chronic epilepsy. Part Ⅲ The study on the mechanisms of the induction of MIP-la/CCR5axis in the epileptogenesis in immature ratsObjective:To explore the potential regulatory mechanisms of the induction of MIP-1α/CCR5axis through the interruption of MAPKs or NF-κB signal pathwayMethods:1. To detect the time course changes of the activation of MAPKs and NF-kB in hippocampus, P21wistar rats were randomly divided into normal group, control group and groups at4h,8h,16h,24h,3d after KA treatment. Referring the findings in preliminary study, phosphate-buffered saline (PBS)-injected controls were designed to 24h post-injection group.2. To detect the effect of MAPKs on the hippocampal MIP-la/CCR5expression, P21wistar rats were randomly divided into PBS+DMSO group, KA+DMSO group, KA+PD98059group, and KA+SB203580group. The interventional drug of dimethyl sulfoxide (DMSO), PD98059or SB203580was i.c.v. adminstered30min prior to KA/PBS injection. To detect the effect of NF-κB on MIP-1α/CCR5in hippocampus, P21wistar rats were randomly divided into PBS+saline group, KA+saline group, and KA+PDTC group. saline or PDTC was i.p. administered immediately and12h after KA/PBS injection. All rats were sacrificed at24h after KA/PBS treatment, and tissues were collected.3. Immunohistochemistry was performed to examine the expression of P-ERK1/2, P-p38MAPK,P-CREB and OX-42in each group.4. Western blot was used to detecte the level of phosphorylation and nonphosphorylation of MAPKs, NF-κB and IκBα, as well as CCR5protein in the hippocampus in each group5. ELISA was used to quantify the MIP-1α protein in the hippocampus in each group.Results:1. Western blot results:As compared with normal rats, a significant increase in P-ERK1/2was observed at4h (P<0.05), peaked at8h (P<0.05) after KA and decreased thereafter. P-p38MAPK was markedly elevated at8h (P<0.05) and further increased from24to72h after KA (P<0.05). Whereas, we fail to detect the significant differences in the level of P-JNK among groups in the present study. Immunohistochemical results:The expression of P-ERK1/2and P-p38MAPK immunoreactivity tallied with the findings of Western blot, and their immunostaining was to some extent coincided with the distribution of up-regulated MIP-1α/CCR5. As to NF-κB. A increase of P-IκBα in the hippocampus was initiated at4h (P<0.05), and reached a maximum at16-24h (P<0.05) after KA and decreased thereafter. P-NF-κB/p65was distinctly increased at8h (P<0.05) and still remained elevated at 24h(P<0.05)after KA.The expression of P-NF-κB/p65immunoreactivity was consistent with the findings of Westem blot.2.After the administration of MAPKs or NF-κB inhibitor,Western blot results:As compared with KA+DMSO group,the pre-treatment of PD98059or SB203580decreased MIP-1α/CCR5levels and microglial accumulation induced by KA.The MIP-1α/CCR5levels in the hippocampus was reduced obviously in KA+PDTC group than KA+saline group(P<0.05). In addition,the pre-treatment of PD98059or SB203580also decreased the levels of P-NF-κB/p65and P-CREB induced by KA when compared with KA+DMSO group(P<O.05).Conclusions:1.In the SE model initiated by KA injection,the pretreatment of PD98059, SB203580or PDTC by i.c.v.injection could inhibit the signalling pathway of ERK1/2, p38MAPK or NF-κB respectively.2.In the SE model initiated by KA injection,MAPKs(ERK1/2and p38MAPK) and NF-κB have at least a partial impact on the induction of MIP-1α/CCR5axis in the early stages of epileptogenesis.3.NF-κB and CREB may contribute to the epileptogenesis as a pathway down-stream of MAPKs(ERK1/2and p38MAPK).