| Spinal cord injury(SCI)is continuously improved the incidence with the development of transportation and construction.SCI is mainly caused by mechanical violenc.The continuous occurrence of cell and molecular chain reaction of SCI induce secondary injury.In addition,SCI can lead to many serious complications,such as,urologic complications,the complications of respiratory system,digestive system complications,deep vein thrombosis and pulmonary embolism,pain,autonomic hyperreflexia and so on.Those complications can lead to higher mortality.Although the clinical and basic research of SCI for many years,due to the mechanism of SCI is very complex,involving inflammatory reaction,cell apoptosis and necrosis,ion mediated injury,free radical injury and other injury mechanism,there is no effective treatment method so far.Inflammation is the main cause of the secondary injury of SCI,which is the center of the development process in the pathophysiology of SCI.Therefore,it is a focus to take effective interventions to control secondary injury and reduce inflammation in the treatment of SCI.The complications of Spinal cord injury also affect SCI treatment,especially pain.Pain persists throughout the spinal cord injury and develop to the chronic central pain.It need a long-term treatment,but because of the side effects and tolerance of drugs,lead to drug addiction,depression,drug tolerance and other adverse consequences,and affect the prognosis and rehabilitation of patients.Therefore,how to reduce pain is also the focus of SCI treatment.Methylprednisolone(MP)has been used in a large number of basic experiments and clinical trials in the treatment of SCI.MP is effective to treat SCI,but recently been questioned because of its adverse reactions.Lappaconitine(LA)is a non-addictive analgesic discovered firstly in China.It also has extensive biological activities,such as anti-inflammatory,local anesthesia,anti-tumor,immune-regulation,anti-pyretic and so on.LA used widely in analgesic,anti-inflammatory and anti-arrhythmia in clinical field.Because the research focus of SCI treatment is related to the characteristics of LA,such as analgesia,anti-inflammation,immune regulation and local anesthesia.As the focus of SCI treatment match LA's properties like analgesic,anti-inflammatory swelling,immunomodulation,local anesthesia and so on.Therefore,we take LA as an intervention,and observe its effect on the treatment of SCI.By setting up SCI models of rats,and comparing the changes in model groups of treatment with LA,treatment with MP and single SCI by locomotor function,pain behavior determination,spinal cord water content determination,ELISA test of inflammatory factors,pathology,immunohistochemistry,Western blot.We evaluate the effect of LA on SCI,and explore the effect of LA on spinal cord injury in rats and its possible mechanism to provide experimental ground for the therapy of SCI.Objective:By setting up SCI models of rats,and comparing the changes in groups by locomotor function,pain behavior determination,spinal cord water content determination,ELISA test,histomorphology,immunohistochemistry,Western blot,we evaluate the effect and mechanism of LA on spinal cord injury in rats and to provide theoretical and experimental evidence for the application of LA in spinal cord injury.Methods:720 SPF male Sprague-Dawley(SD)rats were randomly divided into 5 groups: sham operation group(sham group),SCI group,SCI+saline group(saline group),SCI+LA group(LA group),and SCI+MP group(MP group).We use the aneurysm clip(the force of 110g)to construct the T10 of spinal cord injury models for clamping 1min.We test the pain behavior,behavior determination(BBB score and inclined plane test),water content in spinal cord,the expression of TNF-alpha,IL-1 beta,IL-10 after SCI by ELISA,pathology changes of spinal cord tissue by HE staining,the expression of P2X3 receptor protein by immunohistochemistry and Western blot at all the time points after SCI in all the group.Result:1.BBB score: All rats had BBB motor function score of 21 before operation.The BBB score of the rats in sham group were decreased slightly,but during 1W after SCI,the score of sham group was close to normal.SCI group,saline group,LA group and MP group all showed complete hind-limb functional impairment in the 1d after SCI.0 points say that the model make success.During 1W after SCI,the hind-limb motor function of four groups of rats get improved with time(P>0.05).Form 2W after SCI,the hind-limb motor function of rats were gradually increased,then the score of LA group and MP group were higher than that of SCI group and saline group,with significant difference(P<0.05),there was no significant difference between LA group and MP group score at the 14 d and 21 d after injury(P>0.05).2.Inclined plate test: Normal rats are up to 65oin the Inclined plate test.The plate angle of the rats in sham group has declined,the extent of the decline is relatively small,but during 1W after SCI,the plate angle of the rats in the sham group was close to normal.The plate angle of the rats in the sham group were significantly higher than those of the rats in the SCI,saline,LA and MP group each time point after operation(P<0.05).The plate angle of the rats in the LA group and the MP group at each time point of the spinal cord injury have no significant difference(P>0.05).The plate angle of the rats in the SCI group and the saline group at each time point of the spinal cord injury have no significant difference(P>0.05).During 7d after SCI,the critical angles of SCI,saline,LA and MP groups increased with time,but the plate angle of the rats in each group have no significant difference(P>0.05).We found that in the LA group,the angle of oblique plate was higher than that of SCI group and saline group at the 7d,14 d and 21 d after injury(P<0.05).The oblique plate angle in the MP group were higher than those of SCI and saline groups in the 7d,14 d and 21 d after injury(P<0.05).3.Pain behavior measurement:(1)the mechanical withdrawal threshold of rats in sham group had no significant change after SCI.The mechanical withdrawal threshold of rats decreased significantly at the all time points after injury.The mechanical withdrawal threshold in SCI group and saline group showed no significant difference at the 7d,14 d and 21 d after SCI injury(P>0.05);There is no significant difference compared with the mechanical withdrawal threshold in SCI group and saline group and MP group at the 7d,14 d and 21 d after SCI(P>0.05);the mechanical withdrawal threshold in LA group was significantly higher than those of SCI and saline groups,with significant difference(P<0.05);the mechanical withdrawal threshold in the LA group was significantly higher than that of the MP group at the 7d,14 d and 21 d after injury,with significant difference(P<0.05).(2)the thermal withdrawal latency of rats in sham group had no significant change after SCI.The thermal withdrawal latency of rats decreased significantly at the all time points after SCI.The thermal withdrawal latency in SCI group and saline group showed no significant difference at the all time points after SCI injury(P>0.05).The thermal withdrawal latency in the MP was significantly higher than those of SCI and saline groups at the 14 d and 21 d after SCI,with significant difference(P<0.05).There is no significant difference compared with the thermal withdrawal latency in SCI group and saline group and MP group at the 1d,3d,5d and 7d after injury(P>0.05).The thermal withdrawal latency in LA group was significantly higher than those of SCI and saline groups at the all time points after injury,with significant difference(P<0.05).The thermal withdrawal latency in LA group was significantly higher than that of MP group at 1d,3d and 5d after injury,with significant difference(P<0.05).4.The water content of the spinal cord: The water content of spinal cord in normal rats was about 72%.There was no significant difference between the sham group and the normal rats in the water content of the spinal cord.The water content of the spinal cord in the SCI,saline,LA and MP groups were significantly higher than those in the sham group at the 1d,3d,5d,7d and 14 d after operation(P<0.05).The water content of spinal cord in each group at 21 d after operation have no significant difference(P>0.05).The water content in the LA group and the MP group at each time point of the spinal cord injury have no significant difference(P>0.05).The water content in the SCI group and the saline group at all time points of the spinal cord injury have no significant difference(P>0.05).We found that there has no significant difference in the SCI group,saline group,LA group and MP group of the water content of the spinal cord at 1d,3d,5d after SCI.The spinal cord's water content the in the LA,MP groups were higher than those of SCI,saline groups at the 7d,14 d after SCI(P<0.05)5.ELISA:(1)The IL-1 beta expression of spinal cord in the SCI,saline,LA,MP groups were significantly higher than those in sham group at all time points after SCI and the difference has significant difference(P<0.05).The IL-1 beta expression of spinal cord in the LA group and the MP group at all time points after injury have no significant difference(P>0.05),and The IL-1 beta expression of spinal cord in the SCI group and the saline group at all time points after injury have no deference(P>0.05).The IL-1 beta expression of spinal cord in the LA group were lower than those of SCI group and saline group at all time points after SCI(P<0.05).The IL-1 beta expression of spinal cord in the MP group were lower than those of SCI group and saline group at the 1d,3d,5d,7d after SCI operative(P<0.05).But in the 14 d and 21 d after SCI,we found that there has no statistically significant difference in the SCI group,saline group,MP group of the IL-1 beta expression of spinal cord(P>0.05).(2)The TNF-? expression of spinal cord in the SCI,saline,LA and MP group were significantly higher than those in sham group at all time points after SCI(P<0.05).The TNF-? expression of spinal cord in the LA group and the MP group at all time points after operation have no significant difference(P>0.05),and the TNF-? expression of spinal cord in the SCI group and the saline group at all time points after operation have no significant difference(P>0.05).We found that the TNF-?expression of spinal cord in the LA group and MP group were lower than those of SCI group and saline group at the 1d,3d,5d,7d,14 d after operation(P<0.05).But at the 21 d after SCI,there has no statistically significant difference in the SCI group,saline group,MP group of the TNF-? expression of spinal cord(P>0.05).(3)The IL-10 expression of spinal cord in the SCI,saline,LA and MP groups were higher than those in sham group at all time points after SCI(P<0.05).The IL-10 expression of spinal cord in the SCI group and the saline group at all time points after operation have no significant difference(P>0.05).The IL-10 expression of spinal cord in the LA group and MP group were higher than those of SCI group and saline group at the 5d,7d,14 d and 21 d after SCI operative(P<0.05).But there has no statistically significant difference in the SCI group,saline group,LA group,MP group of the IL-10 expression of spinal cord at 1d,3d after operative(P>0.05).We found that the IL-10 expression of spinal cord in the LA group were higher than those of MP group at the 5d,7d,14 d and 21 d after SCI(P<0.05).But The IL-10 expression of spinal cord in the LA group and MP group have no significant difference at 1d,3d after SCI(P>0.05).6.HE staining: There have same trend of the spinal cord morphological changes in the SCI group,saline group,LA group and MP group at all time points,while the morphological changes have no significant difference in the sham group at each time point.In this study,the morphological changes at different time points were analyzed.The morphology of spinal cord was not different from that of normal rats in sham group.The morphology of the spinal cord was normal,the structure was complete,and the gray matter was clear.No hemorrhage,edema,necrosis,cavity and inflammatory cell infiltration were found;In the SCI group,saline group,LA group and MP group,the morphology of the spinal cord was normal,the structure was complete,and there was a small amount of hemorrhage and edema at 1d after SCI;The structure of the spinal cord is still intact,and the bleeding area is enlarged,Some gray matter and white matter are not clear at 3d after spinal cord injury;The contour of the spinal cord remained intact,but lost its typical form,and the structure was disordered at 5d;The outline of the spinal cord is not clear,the typical form of the loss,the structure of the disorder,a large area of necrosis of the injured area off,there are many visible vacuoles in the spinal nerve fibers at 7d after SCI;The normal structure of the spinal cord was lost,and the density of long spindle fibroblast cells increased at 14 d,21d after SCI.7 immunohistochemical staining: The expression of P2X3 receptor in the spinal cord mainly distributed in the dorsal horn of the spinal cord.The sham group had almost no P2X3 receptor expression.The P2X3 receptor were expressed in the SCI group,saline group,LA group and MP group at all time points,and the expression of P2X3 receptor in the LA group was lower than that of SCI group,saline group at each time point.The expression of P2X3 receptor in the LA group was lower than that of MP group at the 14 d after SCI.8.Western blot: The results showed that sham group had no P2X3 receptor expression.The expression of P2X3 receptor in the spinal cord of SCI group,saline group,MP group and LA group were all increased at all the time points after operation.The expression of P2X3 receptor in the LA group was lower than that of SCI group,saline group at each time point.The expression of P2X3 receptor in the MP group was lower than that of SCI group,saline group at 1d,5d,14 d,21d after SCI.At the 5d and 14 d after injury,the expression of P2X3 receptor in the LA group was lower than that of MP group.Conclusion:1.LA can promote hind-limb motor function recovery after SCI.LA can reduce the water content and down-regulation the edema.LA can inhibit the inflammatory factor the expression of IL-1?,TNF-? and upregulation the expression of IL-10,inhibit inflammatory reaction,reduce the degree of secondary injury to protect the nerve cells after SCI.The anti-inflammatory effect of LA in SCI was not significantly different from that of MP.2.LA can increase the mechanical withdrawal threshold and the thermal withdrawal latency of rats induced by SCI,and decrease the expression of P2X3 receptor after SCI.In addition,the mechanical withdrawal threshold and the thermal withdrawal latency of LA group were higher than those of MP group.The expression of P2X3 receptor in the LA group was lower than that of MP group.It showed that the analgesic effect of LA was better than MP. |
PDF Full Text Request