A Molecular Mechanism That Astrocytic P-connexin43Promotes Neuronic Autophagy In The Hippocampus Following Traumatic Brain Injury In Rats And Neuronic Autophagy Contributes To Astrocytic P-connexin43Degradation

Traumatic brain injury (TBI) is the leading cause of mortality in theyoung aged population and is one of the major reasons for hospital admissionsin modern life. Mechanical disruption of neurons triggers a cascade of eventsleading to tissue edema, neuronal cell death, and impaired motor and cognitivefunctions after TBI. Neuronal autophagy is increased after TBI, which can leadto brain damage and neurologic outcome deficits. However, few experimentalstudies have addressed the mechanism of regulating autophagy in traumaticdamage and neurologic outcome. At present, the domestic and foreignresearch mainly concentrated on the signal transduction pathways in neuron.The mechanisms that astrocytes regulates neuronal autophagy have not beenfully elucidated. Moreover, the effects of neuronal autophagy on astrocyteshave not been reported.The recent studies have shown that P-Cx43expression in the hippoc-ampal astrocytes was significantly induced after TBI. Gap junctions areconductive channels formed by membrane proteins termed connexins (Cx),which permit the intercellular exchange of metabolites, ions, and smallmolecules. Activation of P2X7receptors in astrocytes promotes cerebraledema and neurological injury after TBI. TBI down-regulates glial glutamatetransporter (GLT-1) proteins in rat brain, which contributed to the increasedextracellular glutamate that may mediate the excitotoxic neuronal damage.However, a molecular mechanism that astrocytic p-Cx43promotes neuronicautophagy and reduces postsynaptic potentials via activation of P2X7receptorand down-regulation of glial glutamate transporter expression in thehippocampus following traumatic brain injury in rats has not been elucidated. At the same time, the mechanism that neuronic autophagy contributes toastrocytic p-Cx43degradation has not been determined. If these mechanismshave been established, it demonstrates that the nervous system has a selfregulating, which has a neuroprotective function. This paper is divided intothree parts to elaborate the idea.The first part Astrocytic p-connexin43regulates expression levels ofneuronic autophagy in the hippocampus following traumatic brain injuryin ratsObjective: According to Marmarou’s falling model in1994reproducedby male SD rats, this part of study detects whether astrocytic p-connexin43regulates expression levels of neuronic autophagy in the hippocampusfollowing traumatic brain injury in rats.Method: Rat diffuse TBI models were reproduced by using the improvedMarmarou’s falling method. A total of192male Sprague-Dawley (SD) rats(300-350g) were used in this study. By randomized block method, rats wererandomly divided into four groups: Sham-operated (n=48), TBI (n=48), Salinegroup (n=48) and CBX group (n=48). Each group was further divided into3h,6h,24h and48h. To observe the following indexes: H﹠E stains observedthe hippocampal region pathological morphological changes. The cellularlocalization and expression of P-Cx43and LC3protein in the hippocampuswere observed by immunohistochemistry. The expression of P-Cx43and LC3protein was evaluated by Western blotting. The colocalization of P-Cx43andLC3protein was observed by immunofluorescence microscope. Immunohisto-chemical stained sections were analyzd with IOD values detected by Imageproplus6.0digital medical image analysis system. The films of western blotwere scanned and quantified using the Image Lab4.1and Image J analysissoftware. Statistical analysis was performed via the SPSS16.0statisticssoftware. All data were expressed as mean±standard error. Statistical analysiswas performed using analysis of variance (ANOVA) and followed by Dunnett t-test. P value of less than0.05was considered statistically significant.Results:1The sections of TBI rats were observed by light microscope1.1The TBI rat brains were generally observed: We can observed subarac-hnoid hemorrhage and a small amount of intraventricular hemorrhage in TBImodel. Petechial hemorrhage located in the dorsal brainstem parenchyma.Cerebral vascular significant congestion, brain tissue swelling and no localcerebral contusion were observed.1.2Pathological changes in the hippocampus: Light microscopy level of H﹠Eindicated that the histomorphology of Sham-operated rat brain and cerebralblood vessel was normal with abundance of integrity neuron cells. In TBI24hand Saline group, there were severe neurons swelling, cytoplasm acidophilicstain weaken, nucleolus shrinkage, a larger gap existed around cells, anddegeneration and necrosis in the hippocampus. When treated with CBX, thesechanges were weakened at the corresponding time points after TBI (P <0.05).2P-Cx43and LC3results in immunochemistry in the hippocampusThe positive cells of p-Cx43were stained brown, and mainly located inthe cytoplasm of astrocytes. In Sham group, we can seldom see a positive cell,and the positive cell was light stained. The immunoreactivity of P-Cx43increased significantly in TBI24h and Saline group, compared with Shamgroup (P<0.05). The immunoreactivity of P-Cx43declined significantly inCBX group (P<0.05). The positive cells of LC3were stained brown, andmainly located in the cytoplasm of neurons. In Sham group, we canoccasionally see a positive cell, and the positive cell was light stained. Theimmunoreactivity of LC3increased significantly in TBI24h and Saline group,compared with Sham group (P<0.05). The immunoreactivity of LC3declinedsignificantly in CBX group (P<0.05).3p-Cx43and LC3results in western blot in the hippocampusWestern blot results indicated that sham group has only a little expressionof p-Cx43and there were no changes in each point. The P-Cx43protein inTBI group and Saline group began to increase obviously at3h with peaking at 6h (P<0.05), then decreased, but still higher than the basic level till to24h(P<0.05). In CBX group, the change of P-Cx43expression was similar withTBI group and Saline group at corresponding time points. While treated withCBX, expression of P-Cx43was weakened compared with TBI groups andSaline group at corresponding time points (P<0.05).There were no changes inthe expression of P-Cx43total protein at each point following TBI (P>0.05).In Sham group, the expression of LC3Ⅱ has no changes at each point. TheLC3Ⅱ protein in TBI group and Saline group began to increase obviously at6h with peaking at24h (P<0.05), then decreased, but still higher than the basiclevel till to48h (P<0.05), compared with Sham group. In CBX group, thechange of LC3Ⅱ expression was similar with TBI group and Saline group atcorresponding time points. While treated with CBX, expression of LC3Ⅱ wasweakened compared with TBI groups and Saline group at corresponding timepoints (P<0.05).4LC3and NeuN (neuronal marker) double la belling result in immunofluo-rescence in the hippocampus following TBI; P-Cx43and GFAP (astocyticmarker) double labelling result in immune of luorescence in the hippocampusfollowing TBIObserved with confocal microscopy, we can clear see the greenfluorescence of LC3labeled by DyLight488accumulated in the cytoplasmand the red fluorescence of NeuN labeled by DyLight594located in neuronalnucleus. In the hippocampus, we can see there were almost entire overlappingof green and red fluorescence, presented as orange color at24h after TBI,suggesting that autophagy is located in neurons. Furthermore, we can alsoobserve the green fluorescence of P-Cx43labeled by DyLight488accumul-ated in the cytoplasm and the red fluorescence of GFAP labeled by DyLight594located in astrocytes with confocal microscopy. In the hippocampus, therewere entire overlapping of green and red fluorescence, presented as orangecolor after TBI, indicating that P-Cx43located in astrocytes.Summary: Marmarou’s modified diffuse TBI model fits this study. Theexpression of P-Cx43and LC3protein significantly increased in the hippocampus following TBI. P-Cx43located in astrocytes and LC3located inneurons in the hippocampus. Pretreatment with CBX (gap junction blocker)can both significantly attenuate the protein expression and immunoreactivityof P-Cx43and LC3, which improved neuron edema and nucleolarconcentration, suggested that astrocytic P-Cx43regulated neuronic autophagyin the hippocampus.The second part Astrocytic p-connexin43promotes neuronic autophagyvia activation of P2X7receptors and down-regulation of GLT-1expres-sion in the hippocampus following traumatic brain injury in ratsObjective: To identify whether astrocytic p-connexin43promotes neur-onic autophagy and reduces field excitatory postsynaptic potentials via active-ation of P2X7receptors and down-regulation of glial glutamate transporterexpression in the hippocampus following traumatic brain injury in rats.Method: The modified Marmarou’s falling method was used toreproduce TBI model. A total of156male SD rats were used in this study. Byrandomized block method, animals were randomly divided into six groups:Sham group (n=36), TBI group (n=66), TBI24h+Saline (n=24), TBI24h+CBX (n=6), TBI24h+OxATP (n=12), TBI24h+Ceftriaxone (n=12). Eachgroup was further divided into3h,6h,24h and48h. The cellular localizationand expression of P2X7and GLT-1protein in the hippocampus were observedby immunohistochemistry. The location of p-Cx43, P2X7and GLT-1proteinwas observed by immunofluorescence. The expression of P-Cx43, P2X7andGLT-1protein was evaluated by Western blotting analysis. Another30ratsrecorded fEPSPs in the hippocampal brain slice, which were used to evaluatedthe change of LTP. Field excitatory postsynaptic potentials (fEPSPs) wereanalyzed with Clampfit10.0.30rats were randomly divided into five groups:Sham group (n=6), TBI24h group (n=6), TBI24h+Saline (n=6), TBI24h+CBX (n=6) and TBI24h+3-MA (n=6). Furthermore, another30ratsundergone morris water maze test to detect their changes of the ability of learning and memory.30rats were randomly divided into five groups: Shamgroup (n=6), TBI14d group (n=6), TBI14d+Saline (n=6), TBI14d+CBX(n=6) and TBI14d+3-MA (n=6). The latency was analyzed with Smart.Quantitative analysis and statistics are the same as mentioned previously.Result:1Morris water maze resultMorris water maze test was use to detect the changes of learning andmemorizing ability. The obviously prolonged delitescence in searching safetyisland existed in TBI14d and Saline groups compared with Sham group(P<0.05). Search moving tracks showed that search strategy altered moreideally with time in CBX group and3-MA than that in TBI group and Salinegroup (P<0.05).2Long-term potentiation (LTP) resultIt is important to evaluate the long-term memory by LTP. The fEPSPamplitude and slope values significantly (P<0.05) decreased in hippocampalslices from TBI24h and Saline group compared with Sham group. CBX and3-MA significantly increased fEPSP amplitude and slope values (P<0.05vs.TBI and Saline group).3P2X7and GLT-1results in immunochemistry in the hippocampusThe positive cells of P2X7were stained brown, and mainly located in thecell membrane of astrocytes in the hippocampus. In Sham group, we can seemany positive cell. The immunoreactivity of P2X7has no significant increasein TBI24h and Saline group, compared with Sham group (P>0.05). Theimmunoreactivity of P2X7has no obvious decline in OxATP group (P>0.05).The positive cells of GLT-1were stained brown, and mainly located in the cellmembrane of astrocytes. In Sham group, we can see some positive cell. Theimmunoreactivity of GLT-1decreased significantly in TBI24h and Salinegroup, compared with Sham group (P<0.05). The immunoreactivity of GLT-1increased significantly in Cef group (P<0.05).4In the hippocampus CA3region, p-Cx43、P2X7、GLT-1and GFAP immu-nofluorescent results By confocal microscopy, we can detect the purple fluorescence of p-Cx43labeled by DyLight647accumulated in the cytoplasm, the green fluorescenceof P2X7labeled by DyLight488located in the membrane and the redfluorescence of GFAP labeled by DyLight594located in astrocytes in thehippocampus. In the hippocampus, we can see the overlapping of purple,green and red fluorescence, presented as yellow color at24h after TBI,suggesting that p-Cx43and P2X7are located in astrocytes. Furthermore, wecan also observe the purple fluorescence of GLT-1labeled by DyLight647accumulated in the membrane, the green fluorescence of P2X7labeled byDyLight488located in membrane and the red fluorescence of GFAP labeledby DyLight594located in astrocytes in the hippocampus. In the hippocampus,the merge of purple, green and red fluorescence presented as yellow color at24h after TBI, indicating that GLT-1and P2X7are located in astrocytes.5Western blot resultsTBI increased LC3Ⅱ expression, compared with Sham-operated rat at24h (P<0.05). Treatment with CBX, OxATP and Ceftriaxone reduced thepost-traumatic expression of LC3Ⅱ (P<0.05vs. TBI and Saline group).GLT-1protein level significantly decreased at6h, peaked at24h in TBI24hand Saline group in the hippocampus compared with the Sham-operatedcontrol (P<0.05). A significant increase in post-traumatic GLT-1expressionwas observed in TBI24h+CBX and TBI24h+OxATP group, as comparedwith TBI and Saline group (P<0.05). The expression of P2X7was notincreased following TBI at3h,6h,24h and48h, as compared to Sham-operated rats (P>0.05). CBX administration had no significant effect on P2X7expression (P>0.05vs. TBI at24h and Saline group).Summary: Astrocytic p-connexin43promotes neuronic autophagy andreduces fEPSPs via activation of P2X7receptors and down-regulation of glialglutamate transporter expression in the hippocampus following traumaticbrain injury in rats, which impaired learning and memorizing ability. The third part Molecular mechanism that neuronic autophagy contr-ibutes to the degradation for astrocytic p-Connexin43in the hippoca-mpus following traumatic brain injury in ratsObjective: Through a study that neuronic autophagy contributes to thedegradation for astrocytic P-Connexin43in the hippocampus followingtraumatic brain injury in rats, we can clarify that neurons have effect on theastrocytes except that astrocytes regulate neurons in the hippocampus.Method: The120male SD rats were randomly divided into four groups:Sham (n=30), TBI (n=36), TBI treated with Salin（en=24）and TBI treated with3-MA group (n=30). Every subgroup was composed of6rats and the rats werekilled at the point of3h,6h,24h, and48h after TBI. The expression andlocation of GFAP, LC3and P-Cx43protein were observed by confocalmicroscopy. Western-blot detected the expression of LC3and P-Cx43protein.Transmission electron microscopy was used to detect neuronal autophagydegradation for gap junction. Quantitative analysis and statistics are the samementioned previously.Result:1LC3and p-Cx43protein expression was analyzed by western blot analysisin the hippocampusThe ratio of LC3-Ⅱ/β-actin was identified at low levels in thehippocampus in the Sham group. The immunoreactivity for LC3-Ⅱ/β-actin inthe hippocampus was significantly induced at6h after injury, peaked at24h,then slightly decreased afterward (P＜0.05vs. sham). The comparison of datain TBI group and Saline group had no statistically significant (P＞0.05).3-MA pretreatment significantly inhibited the upregulation of LC3-Ⅱ/β-actincompared with the TBI groups at6h,24h and48h (P＜0.05). The expressionof p-Cx43protein in the hippocampus was significantly up-regulated at3hafter TBI, persisted at a high level until24h after injury (P＜0.05vs. Shamgroup). There were no changes of p-Cx43expression between TBI and Salinegroup. Pretreatment with3-MA significantly increased the relative protein abundance of p-Cx43in the hippocampus (P <0.05vs. TBI6h,24h andSaline group).2GFAP, LC3and p-Cx43immunofluorescent results in the hippocampusfollowing TBIBy confocal microscopy, we can detect the green fluorescence of GFAPlabeled by DyLight488located in astrocytes, the purple fluorescence of LC3labeled by DyLight673accumulated in the cytoplasm, and the redfluorescence of p-Cx43labeled by DyLight594located in the cytoplasm inthe hippocampus. Blue represents4′,6-diamidino-2-phenylindole (DAPI)staining of nuclei. The immunofluorescent intensity of LC3and P-Cx43in thehippocampus was significantly up-regulated in TBI, compared with Shamgroup. The immunofluorescent intensity of LC3in the hippocampus recucedby pretreatment with3-MA, compared with TBI. The TBI-induced increase inimmunofluorescent intensity of P-Cx43in the hippocampal astrocytes wassignificantly upregulated by pretreatment with the autophagy inhibitor3-MA.The purple immunofluorescence of LC3located in the hippocampalneuron. We observed some orange overlap between GFAP and p-Cx43in thehippocampus astrocytes, suggesting that P-Cx43strongly colocalized with thehippocampal astrocytes.3Transmission electron microscopy resultGap junction ultrastructure structures were found between astrocyte andneuron in the rat hippocampus after24h of TBI by transmission electronmicroscopy. Autophagosome structures were observed in the neuroniccytoplasm, which probably disrupts gap junction.Summary: The expression of astrocytic P-Cx43increased when pretre-atment with3-MA inhibited neuronal autophagy in the hippocampus followingTBI. We observed that autophagosome appeared near the gap junctionbetween neuron and astrocytes in the hippocampus by transmission electronmicroscopy. Conclusion: Astrocytic p-connexin43promotes neuronic autophagy viaactivation of P2X7receptors and down-regulation of GLT-1expression in thehippocampus following traumatic brain injury in rats. Inhibition of thepathway can improve long-term potentials and memorizing ability. Further-more, we found neuronal autophagy degradation for astrocytic P-Cx43,suggesting the nervous system with self regulation.