The Investigation Of Apoptosis After Spinal Cord Injury And Correlative Iatreusiology In Rats

BACKGROUND AND PURPOSE: With the development of modern traffic andconstruction industry, the incidence rate of spinal cord injury (SCI) increase year byyear. Although mortality rate of acute SCI has reduced to 6%, it results in highmutilation rate and brings heavy burden to family and society. How to adopt measuresafter SCI to protect spinal cord tissue effectively and promote restoration of structureand function is hotspot and difficulty all over the world. The mechanism of cell deathand regeneration after SCI is not yet well known. In 1998, good gymnastics athleteSang Lan suffered suddenness that result in perch paraplegia to her, which remindneuroscientist of finding mechanism and repair following SCI. The final result isdetermined by original trauma and secondary injury together. Secondary cells damageand death results from pathology process started up by many cytokines followingprimary mechanical insult, and it progresses days to months. Controlling of it is responsiblefor the last outcome. Recent study deems that apoptosis as a active death takes aimportant role in secondary SCI as well as passive cells death caused by mechanicaltrauma and inflammation and that demyelization following SCI is due to apoptosis ofoligodendrocytes. Intervention on apoptosis following SCI has been a cut-in point forSCI studies. As studies on nitric oxide (NO) being extensive and intensive, its action onsecondary SCI catches researchers' attention. Physiological behavior of NO is tomaintain blood circulation in tissue. When it increases abnormally, it causes oxidizingdamage and cells apoptosis. NO is catalyzedly-generated by nitric oxide synthase(NOS), NOS is widespread in body including center nerve system. NOS have threeisoforms, among them, inducible nitric oxide synthase (iNOS) generate NO abundantlyand last long time, then cause oxidative injury directly or introduce apoptosis as signalmedium. The specific inhibitor of iNOS, aminoguanidine (AG), improved rats hindlimb function significantly. Study characteristic of apoptosis and expression of iNOS,especially relation between them after SCI in rats is important for disclosing mechanismof SCI and directing clinical therapy, which is also the objective of our experiment.Secondary nerve cells death affects prognosis severely, and how to protect neurons andpromote functional restoration is important content which has been being explored byscientist. Methylprednisolone (MP) has versatile protective effect on neurons, and it hasbeen using to treat brain and SCI in clinic and produce effect to some extent, but themechanism of its action is not yet well clear. Glucocorticoids are reported can inhibitthe induction of iNOS by down-regulating cytokine-induced activity of transcriptionfactor nuclear factor-kappa B. Extracts of Ginkgo biloba (EGb) can protect neurons byagainst toxic effect of glutaminic acid, and is used in clinic to treat variouscerebrovascular disease, but there is no reports about its application on SCI yet. Thesource of EGb is widespread, and its price is low, so it has practical value. So thepresent experiment observed effects of MP and EGb on iNOS expression and apoptosisafter SCI, and discussed the potential mechanism.Part.1. correlation between Apoptosis and expression of iNOS after spinal cordinjury in ratsObjective: To study characteristic of apoptosis and expression of iNOS, as well asrelation between them after spinal cord injury (SCI) in rats. Methods: Thirty-two adultSD rats were divided at random into eight groups: sham-operated controls group andacute SCI models groups that were sacrificed at 4h, 8h, 1d, 3d, 7d, 14d and 21d aftersurgery respectively, then the sections were stained with immunohistochemistry andterminal deoxynucleotidyl teansferase-mediated dUTP nick end lebeling(TUNEL)method. Other thirty-two adult SD rats were divided at random into eight groupssimilarly and detect iNOS expression in the injuried spinal cord tissue with Western blotanalysis. Results: Immunohistochemistry results demonstrate that a little iNOSimmunoreactivity was detected in sham-operated controls particularly in the regionssurrounding the central cannal and in the blood vessels. INOS-IR was localized widely ingray matter neurons, white matter glial cells, ependymal cells and endothelial cells at theepicenter and adjacent to the region of spinal impact and injury, increasing at 8 hr withthe most intense immunoreactivity observed in the white matter, around the centralcannal and in the blood vessels on day 7 postinjury. The expression of P53 and the ratesof TUNEL-positive cells corresponded temporally to iNOS expression after SCI. TheP53 immunoreactivity and TUNEL-positive cells located mostly in white matter glialcells at day 7 postinjury. The gray degree value-time curve of iNOS expression detectedby Western blot analysis corresponded to iNOS-positive cells rates-time curve. There ispositive correlation between iNOS expression and apoptosis index(r=0.854,P<0.01),aswell as between iNOS expression and p53 expression(r=0.951,P<0.01). Conclusion:Expression of iNOS and p53 was increased and a large number of TUNEL-positive cellspresented after SCI in rats. There is positive correlation between iNOS expression andapoptosis index.Key words: Spinal cord injuries;Apoptosis;Nitric oxide synthase;Gene expressionPart.2. Effect of high dose methylprednisolone on expression of iNOS and apoptosisafter spinal cord injury in ratsObjective: To investigate the effect of high dose methylprednisolone on expression ofiNOS and apoptosis after spinal cord injury (SCI) in rats. Methods: Adult SD rats weredivided at random into two groups: with Allen's weight drop to cause spinal cord injuryand use high dose methylprednisolone group (group A), and use normal saline group(group B). After operation the rats were observed Tarlov's scores on neurofunctionrecovery of dual behind limbs at 4h, 8h, 1d, 3d, 7d, 14d and 21d after surgery, and weresacrificed, then the tissue sections were stained with hematoxylin and eosin(HE),immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nickend lebeling(TUNEL) method, and the injuried spinal cord tissue was evaluated.Results: The histopathology changes in spinal cord tissue observed with HE stainingmicroscopic examination in group B is worse than group A. Expression of iNOS andapoptotic cells were observed in two groups, and the apoptotic index and expressionof iNOS in group B is higher that in group A(P<0.01). Conclusion: This studydemonstrated that high dose methylprednisolone could inhibit expression of iNOS andapoptosis after SCI in rats.Key words: Spinal cord injuries;Apoptosis;Inducible nitric oxide synthase;MethylprednisolonePart.3. Protective effect of extract of leave ginkgo biloba on neurons after spinalcord injury in ratsObjective To investigate the effect of extract of ginkgo biloba (EGb) on apoptosis ofnerve cells and its mechanism after spinal cord injury (SCI) in rats . Methods Fortyeight adult SD rats were divided randomly into two groups, i.e. EGb group and normalsaline (NS) group. After having been made spinal cord hemitransection at ninth thoracicvertebrae level, rats of EGb group were given 20mg extract of ginkgo biloba daily, andrats of NS group were given 2ml NS daily as control. Respectively at 1d, 7d, 14d and21d after surgery, six rats in each group were sacrificed, and tissue sections were stainedwith Nissl's staining, myelin sheath staining, and inducible nitric oxide synthase (iNOS)immunohistochemistry as well as terminal deoxynucleotidyl transferase-mediated dUTPnick end lebeling (TUNEL) were performed, then the injuried spinal cord tissue wasevaluated. Results Nissl's staining manifested nerve cells swelled near the injuryepicenter (rostral and caudal), Nissl bodies stained light and neurons diminished withtime passing. Ratio of bilateral anterior horn neurons (i.e. transection side/uninjuredside) in EGb-group was higher than that in NS-group (P<0.01). Myelin staining showedsignificant demyelination in the zone adjacent to injuried site after SCI in both groups,while the cavity and demyelinated area in EGb-group was smaller than that in NS-group.Expression of iNOS and apoptotic cells were observed in both groups, and the apoptoticindex (AI, i.e. the number of apoptotic cells/the number of total cells) and expression ofiNOS in NS group was higher than that in EGb group (P<0.05). Conclusions This studydemonstrated that EGb could prevent nerve cells from apoptosis after SCI in rats,probably by inhibiting the expression of iNOS.