| The treatment of paraplegina is a substantial topic in the clinic after spinal cord injury. There are many complicating causes which can affect efficient regeneration and reparation. Such as, at the site damaged the myeloid tissue could form glial scar, deficient neurotrophic factors, exit molecules which could inhibit the regeneration of axis or obstruct the axis to find correct target, excessively express the genes which could enhance spinal cord injury or deficiently express the genes which could enhance the regeneration or prevent the injury of axis. There were many ways to promote the reparation after spinal cord injury:①To promote the regeneration of axis, such as, we can give varied neurotrophic factors or enhance its expression, provide bridges and pipe lines for the axis, provide Schell cell and extracellular matrix which could support and lead the regeneration of axis.②To eliminate the factor which could inhibit the regeneration of axis and these factors principally concerned with the myelin of central nervous system and the glial scar and cavity formed after spinal cord injury.IN-1 is the antibody of the myelin-associated neurite growth inhibitory proteins, internal and abroad scholars are prone to study the faction which opposite its corresponding antigen at the present. If the mechanisms exit or not that IN-1 can enhance the nerve regeneration? For this reason, we give IN-1 or use neurotrophic-3(NT-3) associated to study if they can effect the expression of the immediate early genes—c-fos and c-jun in another point of view. We also evaluate the recovery after spinal cord injury from multitude directions.Chapter one: The change of expression of c-fos and c-jun when used IN-1 or NT-3 associatedObjective: To study the change of expression of c-fos and c-jun when used IN- 1 or NT-3 associated in the level of gene and albumen after spinal cord injury.Materials and methods: Two hundred and forty adult health Sprague-Dawley (SD) rats were randomly three groups:(1) control group(n=80), (2) IN-1 group(n=80), and (3)IN-1 and NT-3 group(n=80). In each group, the rats were randomly divided into eight groups at the time of 15,30minates and 1,2,4,6,8,12hours after spinal cord injury, there were eight rats in one group. The laminectomy was performed on all animals at spinal levels of T8 and the dorsal 2/3 of the spinal cord were cut with a pair of microscissors to sever the dorsal parts of the corticospinal tract. At the injured sites of corresponding group, we pumped NS,IN-1 or IN-1 and NT-3 at the injured sites. All rats were killed at the corresponding time and its myeloid tissue was take out. In each group, we examined the expression of c-fos and c-jun gene utilized reverse transcription- polymerase chain reaction technique (RT-PCR) of five rats and the expression of c-fos and c-jun albumen utilized western blot of another rats. All the results were analyzed by one-way variance and there was a significant statistically difference when P＜0.05.Result: (1) The transcriptional levels of c-fos gene and albumen raised up at the first time and broke down later after spinal cord injury. The transcriptional levels of c-fos mRNA achieved the peak at the time lhour after spinal cord injury, but c-fos albumen expressed its peak at the time 4hour later than c-fos mRNA; (2) The expression c-fos gene and albumen broke down when used IN-1 and apparently when associated with NT-3. There was a significant statistically difference between them (P＜0.05) expect the rats killed at the time 15minates; (3) The transcriptional levels of c-jun gene and albumen also raised up at the first time and broke down later. The transcriptional levels of c-jun mRNA achieved the peak at the time 4hour after spinal cord injury, but c-jun albumen expressed its peak at the time 6hour later than c-jun mRNA; (4) The expression of c-jun gene and albumen raised up when used IN-1 and apparently when associated with NT-3. There was a significant statistically difference between them (P＜0.05) expect the rats killed at the time 15 and 30minates. Conclusion: One of the mechanisms of IN-1 and NT-3 which protect the spinal cord maybe through inhibiting the expression of c-fos and enhancing the expression of c-jun.Chapter two: The changes of the expression of the genes which could enhance nerve regeneration when used IN-1 and NT-3 associatedObjective: To study the changes of the transcriptional levels of GAP-43,NGF and bFGF when used IN-1 and NT-3 associated.Materials and methods: Ninety adult health Sprague-Dawley(SD) rats were randomly divided into three groups:(1) control group(n=30), (2) IN-1 group(n=30), and (3)IN-I and NT-3 group(n=30). In each group, the rats were randomly divided into six groups at the time of 1,4,7,14,21 and 28days after spinal cord injury, there were five rats in one group. First we placed tubes at all rats' intrathecal, then the laminectomy was performed on all animals at spinal levels of T8 and the dorsal 2/3 of the spinal cord were cut with a pair of microscissors to sever the dorsal parts of the corticospinal tract. We given NS(30μl) for control group,IN-1 (24μl) for IN-1 group or IN-1(24μl) and NT-3(3μl) for IN-1 and NT-3 group through the tube every day. The most longest time is one week, and we given the drug at three time every day, in the morning, at moon and at night. All rats were killed at the corresponding time and its myeloid tissue was take out. We examined the expression of GAP-43,NGF and bFGF gene utilized reverse transcription- polymerase chain reaction technique (RT-PCR) of all rats. All the results were analyzed by one-way variance and there was a significant statistically difference when P＜0.05.Results: (1) The transcriptional levels of GAP-43,NGF and bFGF gene raised up at the first time and broke down later after spinal cord injury. The peak of the expression of GAP-43,NGF and bFGF mRNA differently appeared at the time 7days, 14days and 14days. (2) The transcriptional levels of GAP-43,NGF and bFGF gene raised up when used IN-1 and apparently when associated with NT-3. There was a significant statistically difference between them (P＜0.05).Conclusion: IN-1 and NT-3 can promote the nerve regeneration by enhancing the expression of GAP-43, NGF and bFGF mRNA.Chapter three: Construction of the recovery of the spinal cord function after spinal cord injuryObjective: To construction the recovery of the spinal cord after spinal cord injury by given IN-1 or combined with neurotrophic-3 from several angles.Materials and methods: Eighteen adult health Sprague-Dawley(SD) rats were randomly divided into three groups:(1) control group(n=6), (2) IN-1 group(n=6), and (3)IN-1 and NT-3 group(n=6). How to make the animal model,the way and time to give drug was same to chapter two. But after 14 days, BDA was injected into the right sensorimotor cortex to anterogradely track the CST fibers. Behavior test was carried out on the 5 rats from each group at 1 hour before spinal cord surgery, 1day, 1week, 2weeks, 3weeks, and 4weeks after surgery using the BBB(Basso, Beattle, Bresnahan) scores. Then, we cut out the specimeins to perform HE and BDA staining and examine the expression of the neurofilaments(NF) and glial fibrillry acidic protein(GFAP) by using the immunohistochemecal technigue. All the results were analyzed by one-way variance and there was a significant statistically difference when p＜0.05.Results: (1) All animals in the injury group showed a paralysis on their hindlimbs. The BBB scores were highest in the IN-1 and NT-3 group than the other two groups at the time points of 14 days, 21 days and 28 days. There was a significant statistically difference between them (P<0.05). (2) The meangray of NF was the highest and GFAP was the smallest in the IN-1 and NT-3 group, There was a significant statistically difference between them (P<0.05). (3) Regenerating axons grew through the lesion site in IN-1 and NT-3 group after spinal cord injury, and there were several regenerating axons grew into the cicatricial tissue but couldn't pass through. In the control group, there were many positive axons of BDA staining in the side of head of the cicatricial tissue.Conclusion: By given IN-1 or combined with neurotrophic-3 can enhance the regeneration of axons and recovery of the behaviors.SummaryIn our study, we utilized the animal model which intersected the corticospinal tract, researched the effect of the expression of the immediate early genes—c-fos and c-jun mRNA and albumen when used IN-1 or NT-3 associated first, and then we studied GAP-43,NGF and bFGF gene which were the objective of c-fos and c-jun gene, we also evaluate the recovery after spinal cord injury from multitude directions at last. All results have collectively shown that IN-1 and NT-3 inhibited the expression of c-fos gene and albumen and promoted the expression of c-jun gene and albumen, and enhanced expression of its objective gene further such as GAP-43,NGF and bFGF gene, promoted the regeneration of axis and recovery of spinal cord function. The evidence in our study indicated that it was an effective way by giving IN-1 to treat the spinal cord injury and it was more effectively by using NT-3 combined, it can promote the regeneration of axis by many ways. |
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