| Secondary injury after primary spinal cord damage plays an important role in thefinal outcome of the ailment. Vasculature pathological changes and the subsequent eventsare crucial for the development of the secondary injury. Destruction of blood spinal cordbarrier leads to massive leakage of blood components, which initiate inflammation andgliosis. Post-traumatic ischemia resulted from tissue compression, thrombosis andvasospasm causes progressive neuronal death, and shows a direct linear relationship withthe severity of injury. Our previous study has shown that there were two ischemic zones atthe front of the expanding secondary injury. In the zone adjacent to the injury, most of theneurons were degenerating or disappeared, whereas in the farther zone, although therewere clear signs of ischemia, the morphology of the Nissl bodies appeared not muchchanged, indicating a possibility of those neurons being rescued via measures ofimproving blood flow. Thus, enhance of blood flow in the spinal cord could be an efficientstrategy for decreasing its secondary injury.Fibrinogen is a340-kDa protein secreted by the hepatocytes in liver and present inblood at a high concentration. Upon activation of coagulation cascade, both α and β chains of fibrinogen are cleaved by thrombin to form fibrin polymer. The latter, via interactingwith platelets, accomplishes the process of blood clotting. Furthermore, recent studieshave reported that the fibrinogen leaked from the ruptured blood vessel triggers theactivation of astrocytes and microglia, suggesting its involvement in the development ofsecondary spinal cord injury.Batroxobin is a thrombin-like serine protease produced from venom of the snakeBothrops moojeni. In contrast to thrombin, Batroxobin only cleaves the α chain offibrinogen, resulting in the production of the fibrin monomer, which has a poorer linkingability than the fibrin polymer, and can thus decrease the blood fibrinogen and promotethe blood flow. Therefore, it has been successfully used in various ischemic disorders,such as stroke, deep-vein thrombosis, myocardial infarction, peripheral arterial thrombosisand sudden deafness. Its effect on SCI, however, has not been studied. Given theimportant role ischemia plays in the pathology of SCI, the present study was aimed toinvestigate whether Batroxobin could have a beneficial effect in SCI of the rats and itspossible clinical application. This study is composed of the following three parts.Experiment1: Effects of Batroxobin on coagulation factors, blood viscosity andspinal cord blood flow after SCIIn this part of experiment, we studied the effects of Batroxobin on coagulation factors,blood viscosity and spinal cord blood flow through a crush model of spinal cord injury inrat. Two hours after the last injection, peripheral blood samples were collected to examinethe effects of Batroxobin on coagulation factors by using an Automated CoagulationAnalysis Instrument, blood viscosity by application of a cone-plate viscometer and fordetection of spinal cord blood flow, laser Doppler scanning was performed at the lesionsite. The results showed that Batroxobin decreased fibrinogen in blood and lowered bloodviscosity dose-dependently.The coagulation results showed that2BU/kg of BX had no effect on coagulationparameters while4BU/kg of BX could significantly prolong prothrombin time (PT) andactivated partial thromboplastin time (APTT). Results of Laser Doppler measurementshowed that, compared with control group,2BU/kg of BX significantly improved spinal cord blood flow, and there is no significant improvement of blood flow in the4BU/kggroup.Experiment2: Effects of Batroxobin on neuronal survival, lesion size and locomotionrecovery after SCIIn the second part of the experiment, we studied the effects of batroxobin on theneuronal survival, lesion size and locomotion recovery after SCI by using the same SCImodel. Seven days after SCI, immunohistochemistry of NeuN and GFAP showed that2BU/kg of BX significantly increased the survival of neurons next to the lesion site by theincrease of NeuN positive cells and reduced the lesion area, while4BU/kg of BX did notpromote the survival of neurons adjacent to the lesion or reduce the lesion sizesignificantly. To test whether Batroxobin administration could be beneficial for thefunctional recovery after SCI, we evaluated the locomotion of the spinal cord injured ratsby BBB scoring and RHI assay at1d,4d and7d after SCI, and footprint analysis at7dafter SCI. The results showed that at4and7d after SCI,2BU/kg of BX-treated ratsshowed significant improvement. However,4BU/kg of BX did not show any significanteffects on the BBB scores, RHI and SLF as2BU/kg of BX did.Experiment3: Effects of Batroxobin on astrocyte and microglia activationLastly, to examine the effects of Batroxobin on the activation of astrocyte andmicroglia, immunostaining of GFAP, Neurocan and Iba-1was performed at7daypost-SCI.2BU/kg of BX significantly decreased the Immune-Fluorence-Intensity (IFI) ofGFAP，Neurocan and Iba-1. Taken together, these data suggested that2BU/kg of BXtreatment can alleviate the astrocyte and microglia activation.Based on the above results, the present study have demonstrated that2BU/kg of BXcan reduce blood viscosity by reducing fibrinogen in blood and improve the local bloodflow after SCI. Besides,2BU/kg of BX can promote neuronal survival, alleviate theactivation of astrocyte and microglia, and improve locomotion recovery. That the use of4BU/kg of BX did not result in any effect, good or bad, and the mechanism of lacking anyeffect remains to be elucidated. Spinal cord injury can inevitably lead to the disruption of blood-spinal cord barrier,which plays a key role in triggering the secondary spinal cord injury. Permeability changesin BSCB result in massive leakage of blood components followed by vasogenic edema,which involves in the SSCI. Studies have showed that promoting the reconstruction ofBSCB after SCI could alleviate the SSCI and improve functional recovery. However, themolecular mechanisms underlying the changes of BSCB after SCI are still unclear.After SCI, several development associated proteins reexpressed in spinal cord, andtook part in the pathological changes of the SSCI. As an important morphogenic signal,the role of sonic hedgehog/Gli1(Shh/Gli1) signaling pathway in embryonic developmenthas been extensively studied. During the development of spinal cord, sonic hedgehogsignaling specifies the ventral neural tube through autocrining or paracrining Shh proteins.Recent studies found that the Hedgehog signal is essential for maintenance of integrity ofembryonic and adult blood-brain barrier. After SCI, whether Shh/Gli1signaling could be activated and participate in SSCI has not been reported.In this study, we explored the roles of sonic hedgehog/Gli1(Shh/Gli1) signaling in thepathological changes and locomotion recovery after SCI in mice. Adult male wild type,Gli1lzand Gli1lz/lzmice were adopted for visualizing Shh/Gli1signaling activation,changes in BSCB and locomotion recovery after SCI. This study includes the followingthree parts.Experiment1: Activation of Shh/Gli1signaling after SCIGli1lzmice, in which the exon2of Gli1gene was replaced by LacZ, were adopted fordetecting the Shh/Gli1signaling activation after SCI. The mice were subjected to spinalcord contusion or sham operation. X-gal staining was conducted3d post-injury. Dark bluestaining was observed in the lesion site, while no positive X-gal staining in the spinal cordof sham-operated mice. These results indicated that the Shh/Gli1signal could be activatedafter SCI.Then, we explored the expression of Shhh and the idendity of Shh responsive cellsafter SCI using the same mice and contusion model. Double-immunostaining ofShh/PDGFr-α, Shh/GFAP and LacZ/GFAP were performed at7d post-injury. The resultsshowed that majority of Shh positive cells were in the epicenter and PDGFr-α positive,and were closely surrounded by GFAP positive astrocytes. Double-staining of LacZ andGFAP at7d after SCI showed that majority of LacZ positive cells located around thelesion area and were GFAP positive. These results suggested that pericytes in the lesioncenter might express Shh, which in turn activated the Shh/Gli1signaling in thesurrounding astrocytes.Experiment2: Effects of Gli1mutation on the expression of GFAP and blood spinalcord barrier after SCIGiven responsiveness of astrocytes to Shh/Gli1signaling and the important role ofastrocyte in BSCB, we studied the effects of Gli1mutation on expression of GFAP andBSCB after SCI. Wild type and Gli1lz/lzof mice were adopted and subjected to spinal cordcontusion. Immunostaining and real-time RT-PCR of GFAP were performed7d after SCIand the results showed that there was no significant difference in mRNA of GFAP between wild type and Gli1lz/lzmice. The results indicated that Gli1mutation did not affect theexpression of GFAP.Evans blue (EB) staining was performed to exmine the integrity of BSCB14d afterSCI. In the lesion site of Gli1lz/lzmice, the spinal cord was heavily stained, while in thewild-type mice, there was no positive staining. Quantification of Evens Blue in theparenchymal of injured spinal cord showed that the amount of EB was significantly higherin Gli1lz/lzmice than that of wild-type mice. The above results indicated that Gli1might beinvolved in the permeability changes of BSCB after SCI.Experiment3: Effects of Gli1mutation on the locomotion recovery after SCIConsidering the fact that changes in BSCB are important for the development ofSSCI and locomotion recovery,, we then explored the roles of Gli1in locomotionrecovery after SCI in mice. Locomotion recovery was assessed by the BMS scoring at1,3,5d and7d post-injury in wild type and Gli1lz/lzmice. The results showed that thelocomotion recovery of Gli1lz/lzmice was much poorer than that of wild type. |
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