The Expression Variety And Distribution Of Slit2 In Adult SCI Rats And Molecular Mechanism Of Growth Inhibition Of Regenerative Axonon With Rho GTPases Unbalance

PartⅠ: Slit2 Expression Variety And Distribution In Adult SD Rats During Acute Phase of Spinal Cord InjuryObjective:To observe the Slit-2 expression distribution and expressive variety during SCI of the rats,then investigate the molecular biological reason of the growth cone guidance.Methods :Make the model of SCI with Allen's method, 72 adult SD rats were randomly divided into SCI 2, 4, 7, 14d groups, sham operation group and normal control group. After the spinal cords of rat were injured, hemi-quantitative Reverse transcription polymerase chain reaction was applied to detect the expressive variety of Slit2 mRNA. On the other hand, Immunocytochemistry was done to detect the cell location of Slit2 protein expression, immunofluorescence was done to investigate the relation of Slit2 protein expression hoist and the reactive astrocytes hyperplasiaResults :There was little detectable Slit2 mRNA expression in normal control group and sham operation group spinal cord tissue samples. However,the mRNA expression of Slit2 had began to be upregulated just 2 days after spinal cord injury(on day 2) of the experimental rats(p<0.05), and it increased on day 2 to day 4 . The expression of Slit2 mRNA reached to a peak level on day 7 after SCI, and the signal intensity for Slit2 mRNA decreased from day 7 to day 14; On the other hand,immunohistochemistry showed that there was little detectable expression of Slit2 protein in normal control group tissue. However, Slit2 was positive in the spinal cord gray astrocytes of all SCI rats, microscope demonstrated many secretive brown-yellow color granules with a high density in cells. The irregular granules with a high density were Slit2, they appeared in the astrocytes of spinal cord gray on day 2 of SCI, the Slit2 protein was expressed in the astrocytes and neurocytes cytoplasm on day 4. The expression of Slit2 protein distributed diffusely in the spinal cord gray of SCI on day 7, but the granules density of central canal and anterior motor column was significantly higher than the other region. Our study showed that on day 7, the expression of Slit2 protein of SCI group had a 28.92% higher level than control tissue(x2=0.019,P<0.05 ); The Slit2 immunofluorescence positive signal also appeared at the extensive area of spinal cord gray, located in the astrocyte.Conclusion:This study demonstrates that Slit2 mRNA was expressed in the damaged local and vicinity part of gray matter, Slit2 proteins were excreted from astrocytes and transported to plasma membrane of growth cone of the regenerative axon, it is likely to do something importantly with axon regeneration after SCIPartⅡ: Expression Variety and Distribution of Netrin-1 and Slit2 In Adult SD Rats During Acute Phase of Spinal Cord Injury through immunofluorescence techniques by Confocal Laser MicroscopyObjective:To observe the Netrin and Slit expression distribution and expressive variety during posttraumatic acute phase of SCI and investigate the molecular biological mechanism of the regenerative growth cone guidance.Methods:40 SD rats were randomly divided into 5 groups( SCI2, 4, 7, 14d groups and one normal control group),the model of SCI was made by Allen's method. The expression and distribution of Netrin-1 and Slit2 were detected through immunofluorescence techniques, the co-expression of then were observed by confocal laser microscopy.Results:significant expressions of Netrin and Slit were showed at injured location at 2nd day post SC(IP<0.05), extensive green fluorescence and manipulus scattered red fluorescence were showed at 3 fields microscopically;on day 4, the expressions of Netrin and Slit were intensified significantly(P<0.05), the former expressed more extensively with greater amplitude in contrast with the latter, the co-expression on the cell membrane of neuron was obvious;on day 7, Netrin persistently high-expressed, Slit peaked its expression, the co-focalization of Netrin and Slit was presented microscopically, but a circle orange fluorescence was emerged from the cell membrane of neuron. Slit expression was significantly intensified at spinal cord posterior horn;both's expressions were weakened after 2 weeks, but the down-regulation of Slit was more significant compared with Netrin. hinted the expressions of Netrin and Slit post SCI was time-dependent;Netrin fluorescence intensity had no significant variation at same time of spinal cord damage at different regions(P>0.05),but the fluorescence of Slit anterior angle was stronger than that of central canal and posterior horn(P<0.05).Conclusion:High level expressions and co-expression on plasma membrane of Netrin and Slit indicate that they may play some important roles in the progression of axon regeneration. PartⅢ: Expression Unbalance of The Rho GTPases: Rac,Cdc42 and Rho In Adult SD Rats During Acute Phase of Spinal Cord Injury and Growth Inhibition of Regenerative AxonObjective:Traumatic spinal cord injury(TSCI) is characterized by a progressive cell loss and a lack of axonal regeneration. In the central nervous system(CNS), the Rho GTPases: Rac, Cdc42 and RhoA are considered the molecule switch of the F-actin cytoskeleton recombination, having affinity with axon retractation.To observe the expression of the Rho-GTPases Rac,Cdc42 , Rho and the phosphorylation levels of myosin light chain(MLC) during posttraumatic acute phase of SCI ,we attempt to investigate the mechanism of the growth cones collapse of regenerative axon.Methods:36 SD adult rats were randomly divided into 6 groups( SCI4, 7, 14, 21d groups ,sham operation group and control group),the model of SCI was made by Allen's method. The expressive variety of the Rho GTPases: Rac1,Cdc42, RhoA protein and the phosphorylation levels of MLC were detected through Western Blotting technique.On the other hand, GST Pull down assay was used to determine the level of GTP-RhoA.Results:The expressions of Rac1 and Cdc42 were manifestly increased at 4 day post SCI(P<0.05)and peaked at 7 day followed by a rapid decrease, both expressions fundamentally regressed to the initial levels at 3 weeks; On the contrast,RhoA total protein expression had no apparent variations post SCI, which compares unfavorably with Rac1 and Cdc42,RhoA expression levels had merely tenuous elevations compared with sham-operated groups at 4,7,14,21d post SCI, but with no statistical significance(P>0.05).On the other hand, Damaged spinal cord MLC phosphorylated level had a gradually elevated tendency with a time-lapse mode compared with sham-operated groups(P<0.05),nonetheless, no apprarant changes were detected in the expressions of myosin B chain total protein(P>0.05),and GST Pull down assay displayed in vivo regenerating axon GTP-RhoA expression had a gradually elevated tendency with a time-lapse mode post SCI(P<0.05).Conclusion:The expression unbalance of the GTPases Rac,Cdc42 and Rho may be important for the collapse of the growth cones of regenertative axon. we confirmed that the activation of Rho had increased notwithstanding its total protein had no apparent changes to switch on Rho-ROK pathway. Rho GTPases activations are regulated by in vitro cellular targeting signalling, on these grounds, we consider that Rho over activation in vivo regenerating axon post SCI plays a negative role for growth cone elongation, especially as the attractive signaling is attenuated at 3 weeks post SCI, we presume that the episode of disequilibrium was occurred in the attractive and repellent signals of SCI microenvironment at 3 weeks post SCI in rats ,this sort of disequilibrium is likely induced by astrocyte persistent hyperplasy, which directly leading to unbalanced expressions among in vivo regenerating axons Rac,Cdc42 and Rho, resulting in uncontrollable enhanced Rho activation followed by GTP-Rho activating ROK,after ROK phosphorylation Ca2+ non-dependent myosin B chain phosphatase myosin binding with subunit to inactivate it, thus cut down the dephosphorylation function on phosphorylational, causing MLC over-phosphorylation, MLC over-phosphorylation is likely one of the chief causes leading to growth cone ultimate detelectasis, which make albumen cytoskeleton calcium–sensitized, accordingly contributing on the polymerization and depolymerization of actin-globulin, ultimately leading to growth cone detelectasis,axon recovery. PartⅣ: Protection Effect of The Inhibition of Rho Association Kinase During N2a cell Mimic Ischemia and Ischemia Reperfusion Injury in VitroObjective:To observe the reorganization of F-actin of neuron during mimic ischemia and ischemia reperfusion injury in vitro,we attempt to investigate the mechanism of the growth cones collapse of regenerative axon and the protection effect of the inhibition of Rho-ROK.Methods:After N2a cells induced by ischemia and ischemia-reperfusion were treated with different dilute Y-27632, a specific inhibitor of Rho association kinase, cell damage was analyzed by cell proliferation assay(MTT assay); on the other hand , N2a cells were then prepared for routine scanning observation through Immunofluorescence techniques by Confocal Laser Microscopy which stained with Fitc-phalloidin for F-actin visualization.Results:Ischemia induced a striking reorganization of actin cytoskeleton with a weakening of fluorescent intensity of the peripheral filament actin bands and formation of the long and thick stress fibers, lamellipodia and filopodia, but Pretreatment of Y-27632 could reversed the changes of ultrasturcture on the cellular surface, the stress fibers were diminished by pretreatment of it, and the protection of Y-27632 has related with its concentration in determinate bound. MTT assay show that Y-27632 could prolong the survival time of the N2a cells after mimic ischemia-reperfusion for 24h.Conclusion:The activation of Rho had a exceptional hoist after SCI. The activation of Rho association kinase(ROK) is likely to induce the collapse of the growth cone . Suppression of Rho association kinase activity could promote axonal growth on inhibitory spinal cord through ischemia and ischemia-reperfusion.