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Study on the Development of Glial Scar and the Role of Acid-sensing Ion Channels in Spinal Cord Injury

Author: HuRong
Tutor: FengHua;XuTianLe
School: Third Military Medical University
Course: Surgery
Keywords: Spinal cord injury Secondary injury Glial scar Cysts Damage to the surrounding area Acid-sensitive ion channels Organization acidification Gray matter White matter Oligodendrocytes Ca2 permeability Ca2 imaging Patch clamp Functional recovery
CLC: R651.2
Type: PhD thesis
Year: 2008
Downloads: 339
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Important factor as the country's economic development, transportation, construction and mine accidents caused by spinal cord injury (spinal cordinjury, SCI) has become a threat to people's health, the study has resulted in a high degree of attention from Chinese scholars on the SCI. Unfortunately, to date, no effective treatment of SCI methods and drugs, prompting people to re-examine the pathophysiological process in SCI and pathogenic mechanisms. Mainly through two pathophysiological stages of acute and chronic phase after SCI, acute phase lesions mainly in the primary injury to start a series of secondary injury, resulting in damage to the expansion of the scope and degree of injury aggravated, so to study the acute phase of SCI secondary injury mechanisms, how to protect the primary damage to surrounding normal spinal cord from secondary injury spread as much as possible, the strategy of the SCI treatment of primary. The SCI chronic phase Zeyi astrocyte activation, reactive proliferation of glial scar (glial scar) and cysts formation of its main pathological features, they become an important barrier to block nerve regeneration, so far there is no effective treatment method . Therefore, the study glial scar formation process, the thickness of the scar and nerve fibers, in-depth understanding of the characteristics of the glial scar and re-examine its role in spinal cord injury repair has important meaning and value. The study found that many of the acute phase of the pathogenic mechanisms involved in secondary injury after SCI, including ischemia, excitotoxicity, inflammation, oxidative stress, metabolic energy barriers. In recent years, many basic and clinical research for the secondary injury mechanisms of drug, but so far no clinical efficacy, suggesting that there are other unknown mechanism of secondary injury. Acid-sensitive ion channels (acid-sensing ion channel, ASICs) reported recently brought new inspiration for SCI research, especially ASICs subunit of ASIC1a. Because of a variety of factors can lead to acidification of the local tissue microenvironment after SCI, such as ischemia and hypoxia can cause CO 2 and other acidic metabolites can not be taken away, and the release of excitatory amino acids and ATP (both acidic substance), metabolic disorders caused by lactic acid accumulation, inflammation can also lead to acidification. So after SCI pH decreased (tissue acidification) may be more serious, it will activate ASIC1a channels, leading to a series of pathological processes. Spinal cord expression of ASIC1a of in distribution and its changes after SCI is unclear. Therefore, this topic will be dynamic observation of changes in the characteristics of ASIC1a after SCI, to explore the role of secondary injury in SCI and its possible mechanism. Glial scar formation of cysts is important pathological changes of the chronic phase of SCI, is important reasons that lead to nerve regeneration after SCI failed, the study found that even through the nerve graft bridging the cavity of necrotic area, but still can not graft and nerve fibers crossing dense glial scar. Therefore, the reduction or removal of the glial scar is a hope for the chronic phase of SCI treatment strategies, but there are two important factors to determine the effect of this strategy: first, how the characteristics of the distribution of the scar? Remove scar thickness should be? cysts around the scar thickness? removal of excessive scar will damage the normal spinal cord tissue causing new damage. The second glial scar removal time window should be used? Too early or too late may have problems. Therefore, this study is the dynamic observation of glial scar formation process, the thickness of the scar and nerve fibers, and to provide experimental evidence for the answer to the above. Objective: dynamic observation after SCI glial scar formation of the law and its relationship with the nerve fibers, and quantitative measurement of the thickness of the glial scar. 2. Observed ASIC1a expression changes after SCI characteristics study ASIC1a SCI secondary injury and possible molecular mechanisms: 1. Using the rat spinal cord contusion model, histopathology, behavior scores, evoked potentials, immunofluorescence technique and nerve tract tracing methods, observation SCI histopathological changes in the process of axon regeneration and glial scar formation law relationship, and measurements of scar thickness. Applications western blotting, immunofluorescence and laser confocal microscopy, RT-PCR methods such as multi-side after SCI ASIC1a expression variation of its changes in meaning. Vivo, external damage model, using the TUNEL staining, electrophysiological patch clamp recording, calcium imaging, intrathecal catheter administration, antisense oligonucleotide technology to explore the role of ASIC1a in SCI secondary injury and its possible mechanism. Results: In this study, first produced four different level spinal cord contusion injury model, histopathology, behavior function score, evoked potential, immunofluorescence technique and nerve tract tracing methods, observed their pathology changes in nerve function recovery results found 10g × 50mm heavy injury group damage, the animal functional recovery more difficult, 10g × 5mm injury group then the damage too light, animal basic own return to near-normal function, 10g × 25mm The injury group functional recovery curve shows early and 10g × 50mm similar hind limb paralysis, post similar 10g × 10mm injury group. 10g × 10mm injured animals spontaneous functional recovery curve stable, unique, with the obvious difference between the other three groups. Further, this study compared 10g × 25mm and 10g × 10mm group differences in pathology, electrophysiology, results show both injury level tissue damage caused by the extent and scope of the movement / somatosensory evoked potential recovery and so there is a significant difference. (2) The experiment found a significant decline in motor function after SCI animals gradually recovered after SCI the 4w (week) after about recovery up to the platform. Accordingly, neurophysiological examination showed movement / sensory evoked potentials latency was significantly longer after the injury, then gradually recovered, also 4 w after SCI is relatively stable. The pathology observed that about 2 w cysts after SCI began to 4 w cysts formed and more stable. Dynamic observation glial scar formation after SCI astrocytes activation, cellular hypertrophy, projections thicker and cross-linked with each other, and gradually form a glial scar formed around the cavity around the obvious glue around 4 w after SCI the quality of the scar with. These results prompted rat SCI 4 w about to enter the chronic phase. 3 Application of Neural tract tracing, double immunofluorescent labeling methods such as observed nerve fibers after SCI has the ability to regenerate most of the running in the glial scar, no penetrate the scar area reaches the cavity edge or penetration cysts, but also some axons extending into the scar lateral part of a certain depth, prompted the glial scar axonal regeneration barrier. Provide the basic data for the future removal of the scar, the research is still measuring the thickness of 4 w glial scar after SCI, the results show that there is a certain difference in the cavity of the head and tail side scar thickness with bilateral wall thickness of the thickness of the head and tail side 107.00 ± 20.12μm, the bilateral edges scar thickness of 69.92 ± 15.12μm. 4.Western blotting and immunofluorescence observations indicate that damage to the surrounding area ASIC1a expression was significantly upregulated after SCI, in gray and white matter were increased, and reached a peak at 12-24 h, and then gradually falling back, basically restored to about 1 w after SCI original level, continuously observed no changes were observed in the 6 w. Different ASIC1a expression of the damage zone after SCI decline that is not detected, the application Nissl and NeuN staining found severe loss of neurons in the damage zone may damage zone ASIC1a expression decreased about 1 w after SCI. The double immunofluorescence display gray matter neurons express ASIC1a, white matter oligodendrocytes expression of ASIC1a. Interestingly, Western blotting observed normal to spinal cord ASIC2a express very low levels, but the expression of the damage area and damage to the surrounding area after SCI significantly increased, especially the injury surrounding area ASIC2a of the elevated expression continued to SCI after about 4w restored to its original level. Also may be due to cell death, damage zone ASIC2a the elevated expression of early and rapid decline after 24 h, until undetectable. RT-PCR results prompted SCI ASIC1a mRNA showed no change, while ASIC2a mRNA levels were significantly increased. 5 by TUNEL labeling methods, found that most of the TUNEL-positive cells ASIC1a positive, suggesting that ASIC1a may be involved in delayed cell death occurred after SCI. In contrast, TUNEL-positive cells was virtually no ASIC2a positive, on the one hand, prompt secondary cell death, on the other hand, in turn Description the ASIC1a and TUNEL Total marked specific ASIC2a may not participate in SCI. Further use of the body, external damage model, combined with TUNEL, PI / FDA staining method, found that cell death specificity were given ASIC1a the blocker PcTx1 and non-specific blocker Amiloride may reduce injury. ASIC1a specific antisense oligonucleotide knock downASIC1a in expression can also achieve the same protective effect. 6 application electrophysiological patch clamp recording and Ca 2 imaging technology found in acid (pH 6.0) stimulation of spinal cord neurons can induce a large inward current (acid-current), can also cause intracellular Ca 2 concentration increased rapidly this current and Ca , 2 flow can be ASIC1a channel-specific blockers blocking. Ischemia and hypoxia secondary to pathological conditions in the simulation after SCI, the acid of ASIC1a mediated currents and Ca 2 influx showed enhanced effects. 7 Further experiments showed that pathological conditions ASIC1a channel function enhancements might and its phosphorylation Co-IP experiments did find ASIC1a phosphorylation levels were significantly increased after SCI, and calcium / calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ ) may be involved in this process. Western blotting showed CaMK Ⅱ expression after SCI significantly increased its expression changes in the spatial and temporal characteristics, and ASIC1a have a good correlation, the application of a specific inhibitor of CaMK Ⅱ the KN93 significant ASIC1a-mediated inhibition of ischemia and hypoxia-induced acid current and Ca 2 flow enhancement effect and reduce cell damage. Finally, to verify the role of ASIC1a in the SCI in the overall level of applications intrathecal catheter technology were given SCI rat ASIC1a-specific the blockers PcTx1 and non-specific blocker amiloride, found that both can be reduce tissue damage and promote recovery of motor function in animals. ASIC1a specific antisense nucleic acid also has a similar protective effect. Conclusion: the Institute SCI model not only to the different levels of damage zone separately and animal behavior, electrophysiological and histopathological changes want to be consistent. The model used in this study has better objectivity, stability, and repeatability. 2.10g × 10mm injured animals spontaneous functional recovery curve, a unique, distinct with different levels of injury can be more objectively reflect the different the SCI treatment measures, and the effect of drugs, the study of this injury level. Comprehensive behavior science, electrophysiology, pathology and glial scar formation observed many evidence prompted rat SCI 4 w into the chronic phase, so important for people to understand and study chronic SCI. 4.SCI nerve fiber regeneration capacity, but rarely penetrate the glial scar glial scar barrier axons, suggesting an important strategy to remove the glial scar may be the treatment of SCI, the The study measured the thickness of the scar, provides important space-time reference data for the future removal of scar. 5.SCI ASIC1a expression raised significantly, and were higher in the gray and white matter and reached a peak in the 12-24 h and back down to the original level of about 1 w after SCI. Expression of of ASIC1a the cell type is immune double standard in the identification of white matter oligodendrocytes. ASIC1a upregulation may be related to the transcriptional level has nothing to do with the translation and / or metabolic. 6 vivo experiments showed that the ASIC1a indeed involved in the secondary injury after SCI, which may process: after SCI appears tissue acidification can activate ASIC1a channel, causing within the flow of a divalent cation, especially Ca 2 influx, leading to intracellular Ca 2 accumulation, leading to cell damage, increased aggravate the injury process of ASIC1a expression of SCI after. 7. Secondary to ischemia and hypoxia after SCI pathological conditions ASIC1a channel function enhancement effect, this effect may be related calcium / calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) mediated ASIC1a the phosphorylation levels. Activation of CaMK Ⅱ may ASIC1a channels mediated Ca 2 influx. 8 This study shows that the organization after SCI acidification and activation of ASIC1a channels is a new pathogenic mechanism of secondary injury after SCI, future design ASIC1a to the intervention target specific SCI treatment of new drugs provides important experimental basis. In summary, tissue acidification and ASIC1a channels from the secondary injury in SCI important role in the possible mechanisms: SCI tissue acidification and is accompanied by an increase in ASIC1a expression, H activate ASIC1a channels, causing Ca 2 , flow the tissue acidification persistence led to the accumulation of intracellular Ca 2 , activate CaMK Ⅱ, and CaMK Ⅱ reacts on the of ASIC1a increase its phosphorylation caused ASIC1a channel enhancements , the pass of Ca 2 increase intracellular Ca 2 increase further activate CaMK II, creating a vicious cycle, leading to cell Ca 2 is overloaded, causing cell damage, ASIC1a expression after SCI elevated aggravated the injury in the process. The subject for the first time revealed the ASIC1a mediated acid poisoning an important role in SCI secondary injury, deepen the understanding of the people on the SCI secondary injury mechanisms, design ASIC1a as intervention targets, specific the SCI treatment of new drugs and new strategies to provide the experimental basis, has important theoretical significance and clinical value.

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CLC: > Medicine, health > Surgery > Of surgery > Head and Neurosurgery > Spinal cord
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