The Effects Of High-mobility Group Box1on Status Epilepticus In Immature Rats

Epilepsy is one of the common neurological chronic disorders, with long-tern and repeated features. It has high morbidity in children. Frequently and seriously epileptic seizures may lead to further brain injury and persistent neurologic and psychogenic disorders, bringing troubles to the patients and family and society. Recent years, alought great evolution has been made in the etiology, pathology and treatment of epilepsy, the exact mechanism of epileptogenesis still remains unclear. Increasing evidence indicates that brain injury such as status epilepticus (SE), trauma, and stroke was accompanied by neuroinflammation, which may contribute to promoting neuronal excitability, neuronal death, and epileptogenesis. Accumulating data shows that many inflammatory factors participated in the development of epilepsy, and anti-inflammatory therapies could reduce seizures and neuropathological changes in some forms of epilepsy.High-mobility group box1(HMGB1) is a highly conserved, ubiquitous non-histone DNA-binding protein that participates in stabilization of nucleosome formation and regulation of gene transcription. HMGB1has recently been defined as a key cytokine. It is released into the extracellular milieu actively by immune cells or passively by necrotic or damaged cells and triggers inflammatory responses and tissue injury. HMGB1can bind to multiple receptors such as the receptor for advanced glycation end products (RAGE), toll-like receptor-4(TLR4) and TLR2. The pro-inflammatory effect of HMGB1is exerted by activating nuclear factor-κB (NF-κB) or other pathways which promotes the chemotaxis and the production of cytokines. Toll-like receptors are innate immune receptors and respond to microbial structures and endogenous danger factors. Among the TLRs, TLR4is one of receptors which are the focus of concern. TLR4was expressed widely in the nervous system, including in microglia, astrocytes and neurons, and participated in many neurological diseases.It has been reported that HMGB1contributes to the pathophysiology of sepsis, arthritis, respiratory disorders, pancreatitis, cerebral ischemia, traumatic brain injury, encephalitis and so on. The inhibitors of HMGB1such as anti-HMGB1antibody and Box A could alleviate inflammatory reaction and exert protective effects. On the contrary, injection recombinant HMGB1may promote inflammatory response and aggravate neuronal damage. For example, anti-HMGB1antibody could decrease the lethality of sepsis. HMGB1translocates from the nucleus into the cytoplasm and increases in cerebrospinal fluid and serum in cerebral ischemia and traumatic brain injury models. Anti-HMGB1antibody may inhibit the HMGB1translocation and inflammatory reaction and ameliorate brain infarction. However, recombinant HMGB1could prompt the induction of pro-inflammatory mediators and increase the neuronal death in vivo and in vitro.Recently, it has been reported that the HMGB1-TLR4axis is activated in adult mice models of acute and chronic seizure, and elevated serum levels of HMGB1are observed in febrile seizure patients. However, so far, the role of HMGB1in epilepsy of immature brain and the role of anti-HMGB1antibody in the neuronal damage and inflammation following SE have not been explored. In addition, experimental evidence indicated that the susceptivity and the neuropathological changes of epilepsy are age-dependently, moreover, maturational stage could also affect the expression pattern of HMGB1. Therefore, in order to investigate the effect of HMGB1after SE in immature brain, we used postnatal day21(P21) wistar rats to induce SE models by an intracerebroventricular kainic acid (KA). We firstly investigated the expression of HMGB1/TLR4signal pathway and neuronal damage in hippocampus during the early phase of KA-induced SE (from3h to7d). Further, we studied the effects of anti-HMGB1antibody on the hippocampal damage and inflammatory reaction after KA-induced SE by an intracerebroventricular injection of anti-HMGBl antibody. Chapter ⅠThe expression of HMGB1/TLR4signal pathway in immature hippocampus after status epilepticusObjectiveTo investigate the expression changes of HMGB1/TLR4in P21rat hippocampus after KA-induced status epilepticus.Materials and methods1. KA-induced SE modelP21male Wistar rats were used. Rats were anesthetized using chloral hydrate (400mg/kg, intraperitoneally) and injected stereotaxically with KA (2nmol in1ml0.01M PBS) into the lateral ventricle at the following coordinates from the bregma:0.7mm posterior,1.3mm lateral, and3.0mm deep. The control rats received equal volumes of PBS. Seizures were behaviorally recorded in five grades using Racine’s scale. The onset of SE was defined as the first grade4or greater seizure that progressed to similar repeated or prolonged behavioral seizures. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)2h after the onset of SE. Rats that did not reach grade4were removed from further experiments.The rats subjected to SE were further assigned randomly into six time point groups:3h,6h,12h,24h,3d and7d after the onset of SE. In addition to the above groups, normal-control group and PBS-control group (24h after injection) were also included.2. Nissl and Fluoro-Jade B stainings were used to observe the neuronal morphology and neuronal damage in the hippocampus of P21rats after SE.3. Immunohistochemistry was performed to examine the dynamic expression of HMGB1and TLR4in different subfields of hippocampus of P21rats after SE. The detection of HMGB1included the total HMGB1and cytoplasmic HMGB1.4. Western blot was used to detect the dynamic expression of HMGB1and TLR4proteins in the hippocampus of P21rats after SE.5. Double-labeling immunofluorescence staining was used to identify the cellular distribution (neuron, microglia and astrocytes) of HMGB1in the hippocampus of P21rats after SE. Results1.KA-induced SEWith in30min, all the rats showed polypnea, head nodding, facial automatisms, wet-dog shakes and forelimb clonus. Then, rats appeared rearing, generalized tonic-clonic seizures and transient loss of postural control.8%of rats died, and80%of the rats were died within the1hour after the intracerebroventricular injection of KA. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)2h after the onset of SE. No seizure was observed in normal rats and PBS-injected rats.2. Neuronal damage in the hippocampus of P21rats after KA-induced SENissl and Fluoro-Jade B staining showed that there was no neuronal damage in normal rats and PBS-injected rats. Neuronal damage was occurred early in the CA3and Hilus at6h after SE, it was showed that the Nissl-positive cell counts were decreased (P<0.01), and FJB positive cells appeared. The neuron cell counts analysis showed a time-dependent decrease of Nissl-positive neurons in the hippocampus after KA-induced SE. At24h after SE, there was a substantial neuronal damage in the CA3and Hilus. However, delayed neuronal damage of CA1pyramidal cell layer was appeared lightly at24h after SE (P<0.05), and the degree of damage was light in CA1than that in CA3and Hilus.3. Time-course expression of HMGB1in the hippocampus of P21rats after KA-induced SEWestern blot analysis showed that the total HMGB1protein level did not changed after SE, but the cytoplasmic HMGB1protein significantly upregulated after SE. The elevation of HMGB1was observed at3h after the onset of SE (P<0.01), peaked at24h (P<0.01), and decreased thereafter. At7days, a subtle elevation was still detected in the hippocampus (P<0.05). There was no significant difference between the normal rats and PBS-injected rats (P>0.05). Immunohistochemistry showed that in the normal and the PBS-injected rat hippocampus, HMGB1immunoreactivity was detected mostly in the nuclei. When rats were subjected to SE, HMGB1cytoplasmic translocation was observed in the hippocampus as early as3h after SE. From3h to12h after SE, the cytoplasmic translocation of HMGB1was obvioused mainly in the pyramidal neurons of CA1region, and after12h, the cytoplasmic HMGB1staining was occurred in the cell with glia morphology. In the pyramidal cell layer of CA3and hilus, HMGB1staining loss was observed from6h after SE, the staining loss region was in line with the region which undergoing degenerative changes at these time points.4. The colocalization of HMGB1with different cellular markers in the hippocampus of P21rats after KA-induced SEDouble-labeling immunofluorescence showed that in the normal rat hippocampus, HMGB1immunoreactivity was observed in the nuclei of both neurons and astrocytes, and there were no ED1-positive cells in normal hippocampus. At6h after SE, cytoplasmic HMGB1staining was present mainly in the neurons of pyramidal layer. At24h post SE, the percentages of HMGB1-positive astrocytes and the cytoplasmic HMGB1-positive astrocytes were substantially increased (P<0.05). Moreover, the immunoreactivity of ED1, a marker of activated microglia/macrophages, was appeared mainly in the CA3and hilus. And double-labeling assay detected that78.4±0.12%and81.2±0.08%of ED1-positive cells were double stained by HMGB1in the CA3and hilus, respectively.5. Time-course expression of TLR4protein in the hippocampus of P21rats after KA-induced SEWestern blotting analysis demonstrated that the expression of TLR4protein was very low in the normal and PBS control group. After SE, the expression of TLR4protein was progressively increased from3h (P<0.01) in the hippocampus, peaked at24h (P<0.01), and decreased thereafter.7days after SE, the elevation of TLR4was still detected in the hippocampus (P<0.01). Immunohistochemistry showed that in some of the normal rat hippocampus, faint immunoreactivity was observed in only a few scattered cells present in pyramidal layer. After SE, the immunostaining of TLR4protein in the hippocampus was significantly increased from3h after SE. before24h after SE, the TLR4-positive cells were mainly distributed in the pyramidal layer, and after24h, the TLR4immunoreactivity was also observed in the cells with glia morphology. In the subfields of hippocampus, the TLR4immunostaining was distributed mainly in the dentate hilus and CA3. Conclusion1. The expression of HMGB1/TLR4axis was transiently activated during the early phase of KA-induced SE in P21rats, and the elevation of HMGB1/TLR4was related with the neuronal damage.2. The translocation of HMGB1from nuclei to cytoplasm and the extracellular release was first occurred in neurons, and then occurred in the glias. Chapter ⅡThe effects of HMGB1antibody in juvenile rat hippocampus after kainic acid-induced status epilepticusObjective To investigate the effects of anti-HMGB1antibody on hippocampal damage andinflammatory reaction after KA-induced SE in postnatal day21rats.Materials and methods1. KA-induced SE modelThe model was induced according to the method in chapter I.2. Experimental designRats were selected randomly to receive anti-HMGB1antibody (1,2, and4μg, IgY subclass) or nonimmune control IgY (4μg) by an intracerebroventricular injection immediately after the termination of SE and12h after the onset of SE. The PBS-injected rats received control IgY (4μg) at the same time point. The dose schedule was selected on the basis of previous report and preliminary experiments. Rats were killed6h (n=6/group) after SE to quantify the cytokine production. To evaluate the microglial activation and neuronal damage, rats were killed3d after SE (n=6/group)3. Real-time quantitative PCRRats were decapitated at6h after SE, and the hippocampi were dissected and stored at-80℃. The hippocampal mRNA expression of IL-1β and TNF-a was investigated by real-time PCR.4. ImmunohistochemistryRats were decapitated at3d after SE, and the brain was dissected and made into paraffin sections. The immunoreactivities of Ibal、ED1and GFAP were investigated by immunohistochemistry.5. Nissl and Fluoro-Jade B stainingsRats were decapitated at3d after SE, and the brain was dissected and made into paraffin sections. The neuronal damage was detected by Nissl and Fluoro-Jade B staining.Results1. Effects of anti-HMGB1antibody on the gene expression of inflammatory factors in the hippocampus of P21rats at6h after SE. The levels of IL-1β and TNF-a mRNA were significantly increased in the KA group when compared with the PBS group (P<0.01). An intracerebroventricular injection of anti-HMGB1antibody (2and4μg) notably suppressed the mRNA expression of IL-1β and TNF-a (P<0.05). However, no significant decreases were observed in the group treated with1μg of antibody (P>0.05).2. Effects of anti-HMGB1antibody on glial activation in the hippocampus of P21rats at3d after status epilepticusImmunohistochemistry showed that the immunoreactivities of Ibal, ED1and GFAP were increased when compared with the PBS group (P<0.01). An intracerebroventricular injection of anti-HMGB1antibody (2and4μg) notably suppressed the immunoreactivities of Ibal, ED1and GFAP in the hippocampus (P<0.01), but no significant inhibition were observed in the group treated with1μg of antibody (P>0.05).3. Effects of anti-HMGB1antibody on the neuronal damage in the hippocampus of P21rats at3d after status epilepticusNissl and Fluoro-Jade B staining showed that there was no neuronal damage was observed in the PBS group. However, the neuronal damage was significant in the KA+IgY group, and the damage was obvious in CA3and DG. An intracerebroventricular injection of anti-HMGB1antibody (2and4μg) notably attenuated the neuronal damage (P<0.05), but no significant inhibition were observed in the group treated with1μg of antibody (P>0.05).Conclusions1. KA-induced SE markedly increased the mRNA expression of IL-1β and TNF-a, microglial activation, and neuronal damage in the hippocampus.2. An intracerebroventricular injection of anti-HMGB1antibody dose dependently inhibited the synthesis of cytokines, glial activation, and neuronal losses in the hippocampus after SE.