| Most of the central nervous system diseases, including neural degenerative diseases, may be associated with peripheral inflammatory process, but there is no comprehensive report about early morphological features, occurrence, development and outcome during central hippocampal damage induced by acute inflammation. Studies have shown that central nervous inflammation induced by lipopolysaccharide (LPS) damaged the cortex and hippocampus, and rats showed obvious decline of learning and memory in behavior. It is not known yet which partitions and which cell types in the hippocampus are involved in the acute inflammation injury, also, almost nobody knows what pathological physiology reaction follows with the injury, and what is the related internal mechanism. It needs to be elucidated.Phagocytosis is special interaction between cells, if apoptotic cells are not removed timely, they can cause secondary necrocytosis, nuclear membrane and nuclear cracking, cell broken up, thus releasing toxic contents, leading to severe inflammation and autoimmune disease. Therefore, it is essential to clear apoptotic cells rapidly by phagocytosis for preventing inflammation, also it is the key for our body to repair ourselves. It has been reported drosophila membrane connexin Junctophilin(JP) analogue Undertaker(UTA) is involved in phagocytosis of apoptotic cells by affecting calcium homeostasis during development, suggesting that JP may be involved phagocytosis of dead cells in nerve tissue. Given JP subtype 3 and 4 are mainly distributed in the hippocampus, my study focus on evaluating whether JP is involved in phagocytosis in acute inflammatory hippocampus induced by LPS.Mammalian central nervous system has some characteristics of endogenous regeneration, in some areas like brain and spinal cord, it can be regenerated after some injury. Studies show that after pathological changes happened in the central nervous system, neural stem cells migrated to the injury site and replaced deficient cells specifically, connected with other neurons, thus recovered the damaged brain tissue anatomically and functionally, which brought the hope for the treatment of central nervous diseases. However, it is rarely reported whether the body has nerve regeneration after the acute central nervous system injuries caused by the outer peripheral inflammatory process, my study will further observe the repair and regeneration after acute hippocampal damage induced by LPS, and is expected to provide experimental evidence for clinically relevant predictive assessment.The First PartCharacterization of hippocampus damage and restoration in inflammation model caused by intraperitoneal injection of lipopolysaccharide in ratsObjects:To explore the characterization of acute injury and restoration in different partitions of rat hippocampus after intraperitoneal injection of LPS.Methods:After modeling by intraperitoneal injection of saline or LPS (2 mg/kg) to male Sprage-Dawley rats respectively, we collected brain tissue at the first, fourth, seventh, tenth, thirteenth day respectively. Biopsy samples were taken after brain perfusion fixation, and protein sample were directly taken and stored at -80 ?. Using HE staining, we observed cell morphology in different hippocampal partitions. Using TUNEL we analyzed apoptosis. After immunofluorescence staining we used confocal scanning to observe the number of pyramidal neurons and axonal integrity in different hippocampal region. Also, we used immunohistochemistry to achieve quantitative analysis. At the same time, we used Western Blotting to detect the expression of apoptosis-related protein like p-c-Jun, Bax/Bcl-2, TNF-? and so on. Using Western Blotting to detect expression of Nestin, the neural precursor cell marker, ?-catenin and p-STAT3. Using immuno fluorescence staining to detect the distribution of Nestin-positive cells in different areas of the hippocampus.Results:HE staining showed that for saline group, hippocampal pyramidal cells arranged in order, with synaptic integrity, large nuclear, prominent nucleolus, cytoplasm full and uniform, cell borders clear. At the fourth day after injecting LPS to rat, the pyramidal cells showed nuclear contraction, and a large number of tissue vacuoles appeared. What's more, cell numbers reduced, at the same time, pyramidal cells arranged in irregular with significant axonal injury. However, at the seventh day after injecting LPS, there appeared the reversal of damage mentioned above, and at the thirteenth day, they recovered fundamentally. These changes were obvious most in hippocampal CA1 and CA4 region, therefore, our study mainly evaluated this two regions. TUNEL and Western Blot test results showed at the fourth day after injecting LPS, apoptosis in hippocampal CA1 and CA4 region increased, what's more, apoptosis related protein Bax/Bcl-2 ratio, TNF-oc up-regulated, suggesting apoptotic cells in the hippocampus region. Confocal laser scanning results showed, at the fourth day after injecting LPS, for hippocampal CA1 pyramidal neurons, numbers reduced, arranged in disorder. Above phenomena were more obvious in CA4 region, what's more, axons were damaged and cell layer was missing. At the seventh day, the injury began to recover. At the tenth day or thirteenth day, for model group, there was no significant difference about the number of neurons or neuron arrangement compared with the control group in the CA1 and CA4 region. After immunohistochemistry staining, we carried on the statistics of neuron numbers in CA1 and CA4 regions. The result showed that at the fourth day, the number of neurons in this two regions significantly reduced, however, at the seventh day, they began to recover, suggesting LPS damaged neurons in the CA1 and CA4 region, while it didn't damage other cells like glial cells or cells in other regions of hippocampus, moreover, this morphological damage might be substantially fixed within three days. At the fourth day after injecting LPS, Nestin expression was significantly up-regulated, while immunofluorescence staining results suggested that neural stem cells were activated, meaning progenitor cells started to participate in repairing the injury. At the same time, tubular structure consisting of neural precursor cells appeared in the Slm region of hippocampal CA1, exactly the nerve rosette. The appearance of this structure indicated that nerve tissue stayed in the early stage of repair. At the forth day after injecting LPS, Nestin-positive staining newborn neural precursor cells appeared at the outer edge of hippocampal DG region, while a small amount of neural precursor cells appeared in the hippocampal CA1, CA4 and other regions. All these results suggested that after injection of LPS to cause acute injury in rat hippocampus, neural stem cells existing in DG region could be activated and showed significant migration signs.Western Blotting results showed that, at the fourth day after injecting LPS, the expression of p-STAT3 significantly increased in the hippocampus, suggesting that STAT3 came into the nucleus, started downstream transcriptional regulation, activated the differentiation of neural progenitor cells to repair damage.Conclusions:Intraperitoneal injection of LPS mainly resulted in acute injury of neurons in the CA1 and CA4 region, and this damage could be repaired in morphology.The Second PartPhagocytosis mediated by Junctophilin-3/4 during injury process induced by intraperitoneal injection of LPS in rat hippocampusObjects:To explore phagocytosis mediated by Junctophilin-3/4 during injury process induced by intraperitoneal injection of LPS in rat hippocampus.Methods:After modeling by intraperitoneal injection of saline or LPS (2 mg/kg) to male Sprage-Dawley rats respectively, we collected brain tissue at the first, fourth, seventh, tenth, thirteenth day respectively. Using Western Blotting to detect the protein expression of JP3, L-type calcium channels, SK, NMDA receptors, p-CaMKII and RyRl, RyR, RyR3. Using co-immunoprecipitation to detect protein interaction of JP3 with L-Type Ca2+ Channels, SK, NMDARs, RyR1,RyR2 and RyR3 respectively. Using Immuno-fluorescence staining to observe expression of JP3 in glial cells. Also, calculating the co-infection ratio of JP3 with astrocyte marker GFAP and microglia marker Iba-1 in the hippocampus.Using co-immunoprecipitation to detect protein interaction of JP4 with L-Type Ca2+ Channels, NMDARs and RyR3 respectively. Using Western Blotting to detect the protein expression of JP3, JP4, phagocytosis receptors Megf10, glial cells GFAP and Iba-1 in rat hippocampus. Using immunofluorescence staining to observe co-localization of JP3 with Megf10, GFAP, Iba-1 respectively. Using Co-immunoprecipitation to detect protein interaction between JP3 and Megf10, JP4 and Megf10.Results:Western Blotting results showed that at the fourth day after injecting LPS, the expression of L-Type Ca2+ channel,RyR3 and NMDAR was up-regulated, while the level of CaMKII phosphorylation decreased, suggesting the reduced level of intracellular calcium. This paradox indicated that neural inflammation may damage the calcium channel, thereby affecting calcium homeostasis. Co-immunoprecipitation showed, at the fourth day after injecting LPS, the protein interaction of JP3 with NMDAR and RyR3 reduced, suggesting that channels extracellular calcium ions flowing into calcium store were inhibited, thereby affecting the homeostasis of intracellular calcium ions, while up-regulation of L-Type Ca2+ Channel and NMDAR was most likely compensatory increase. Using immunofluorescence staining of JP3 in glial cells, the results showed that at the fourth day after injecting LPS, the expression of JP3 was significantly up-regulated in astrocytes and microglial cells, suggesting up-regulated JP3 activated glial cells for phagocytosis. What's more, Co-immunopreci-pitation showed that there was no interaction of JP3 with NMDAR and RyR3.At the fourth day after injecting LPS, the JP3 and JP4 expression in the hippocampus was significantly up-regulated, and the phagocytic receptor Megf10 expression also significantly increased, meaning that their changes were similar. Immunofluorescence staining showed that, in CA1 region, JP3, JP4 and Megf10 increased, moreover, they showed good co-localization, suggesting that the cells expressing Megf10 also expressed JP3. However, co-immunoprecipitation showed JP3 and Megf10 didn't have protein interactions, suggesting that JP3 did not directly connect with Megf10 to participate in phagocytosis, it might be involved in phagocytosis through other ways such as regulating calcium iron. Meanwhile, co-immunoprecipitation showed JP4 and Megf10 have protein interactions, suggesting that JP4 directly connect with Megf10 to participate in phagocytosis. At the the fourth day after injecting LPS, in the hippocampal CA1 region, the expression of astrocyte marker GFAP and microglia marker Iba-1 was significantly up-regulated. Meanwhile, immunofluorescence staining results were consistent with the results of Western Blotting, showing glial cells were activated in the CA1 region, indicating the phagocytosis of apoptotic cells. In the hippocampal CA1 and CA4 region, JP3, Megf10 and astrocyte marker GFAP were up-regulated, while GFAP with JP3 and Megf10 had good co-localization respectively, suggesting that at astrocytes surface Megf10 was activated, triggering phagocytosis, and JP3 participated in this process. Similar phenomenon appeared in microglia cells. On the other hand, astrocytes, and microglia cells showed different distribution in the CA1 region. Microglial cells were mainly distributed in pyramidal neurons soma zone, while astrocytes were mainly distributed in pyramidal neurons axon zone, suggesting that two types of glial cells might carry on different duties in the phagocytic process.Conclusions:JP3 and JP4 were involved in the activation of astrocytes and microglial cells for phagocytosis. |
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