Experimental Study On A Broad Spectrum Caspase Inhibitor Q－VD－OPH Treatment Of Rabbit Degenerative Disc Disease

Part I Culturing and identification of rabbit anulus fibrosus cells andnucleus pulposus cellsObjective: To investigate the isolating and culturing method of annulus fibrosus cellsand nucleus pulposus cells from rabbit intervertebral discs in vitro. To identify theirphenotype. Methods: The annulus fibrosus cells and nucleus pulposus cells were isolatedwith enzyme digestion and grew as monolayer. Morphology, structure and biologicalcharacteristics of cells were detected by an inverted phase contrast microscope. Thephenotype was identified with the toluiding blue staining and immunocytochemistry.Results: The annulus fibrosus cells and nucleus pulposus cells of rabbit were isolatedand cultured in monolayer. The original annulus fibrosus cells were fusiform or trianglemorphology, the cells displayed intense toluiding blue metachromasia, theimmunohistochemical test revealed that the positive expression of type I and II collagen onthe cells. The original nucleus pulposus cells were round, triangle or short fusiformmorphology, the cells displayed intense toluiding blue metachromasia, theimmunohistochemical test revealed that the positive expression of type II collagen on thecells. Conclusion: The0.25%trypsinase and collagenase digestion can successfullyisolated the annulus fibrosus cells and nucleus pulposus cells from rabbit intervertebraldiscs. Annulus fibrosus cells demonstrated the chondrocyte-like phenotype in terms ofproteoglycan and collagen I and II expressions. Nucleus pulposus cells demonstrated thechondrocyte-like phenotype in terms of proteoglycan and collagen II expressions. Part Ⅱ Experimental study on apoptotic pathway of anulus fibrosuscells and nucleus pulposus cellsObjective: To investigate the apoptotic pathway of anulus fibrosus cells and nucleuspulposus cells. Methods: Using the original generation of anulus fibrosus or nucleuspulposus cells. Two experimental groups were designed as follows: anulus fibrosus cells ornucleus pulposus cells+10%fetal bovine serum, anulus fibrosus cells or nucleus pulposuscells+0%fetal bovine serum. After48hours,the percentage of apoptosis of anulus fibrosusand nucleus pulposus cells was detected by flow cytometry. The changes of caspase3,8,9and cytochrome C, Bid were determined by kit or western blot. Results: The percentage ofapoptosis in anulus fibrosus and nucleus pulposus cells incubated in0%fetal bovine serumwas increased compared with those in the cells incubated in10%fetal bovine serum(bothP＜0.01). In the apoptotic process of anulus fibrosus cells, the amount of caspase3,8wassignificantly increased(P＜0.01), but the amount of caspase9, cytochrome C and Bid didnot change significantly. On the other hand, In the process of nucleus pulposus cellsapoptosis, the amount of caspase3,9and cytochrome C, Bid was significantly increased(P＜0.01), but the amount of caspase8did not change significantly. Conclusions: Theapoptotic pathways of anulus fibrosus and nucleus pulposus cells are different. Anulusfibrosus cells are Fas Type-I cells, which undergo apoptosis through the death-inducingsignaling complex. Nucleus pulposus cells are Type-II cells, which undergo apoptotic celldeath through mitochondrial involvement. Part Ⅲ The anti-apoptotic effects of Q－VD－OPH on anulus fibrosusand nucleus pulposus cells in vitroObjective: To investigate the anti-apoptotic effects of broad spectrum caspaseinhibitor Q－VD－OPH on anulus fibrosus and nucleus pulposus cells in vitro. And toinvestigate the effects of Q－VD－OPH on caspase3,8and9. Methods: Using theoriginal generation of anulus fibrosus or nucleus pulposus cells. Three experimental groupswere designed as follows: anulus fibrosus cells or nucleus pulposus cells+10%fetal bovineserum, anulus fibrosus cells or nucleus pulposus cells+1%fetal bovine serum, anulusfibrosus cells or nucleus pulposus cells+1%fetal bovine serum+50μM Q-VD-OPH. After24,48and72hours, the percentage of apoptosis of anulus fibrosus or nucleus pulposuscells was detected by flow cytometry, the ratio of Bcl-2/Bax was determined by Westernblot. The changes of caspase3,8and9were determined by kit. Results: The percentage ofapoptosis in anulus fibrosus and nucleus pulposus cells incubated in1%fetal bovine serumwas increased compared with those in the cells incubated in10%fetal bovine serum (bothP<0.01). After anulus fibrosus and nucleus pulposus cells were incubated in1%fetalbovine serum+Q-VD-OPH, the percentage of apoptosis was decreased (both P<0.01), andthe amount of caspase3,8and9was significantly decreased(both P<0.01). Conclusions:Apoptosis of both anulus fibrosus cells and nucleus pulposus cells can be attenuated bybroad spectrum caspase inhibitor Q－VD－OPH. Part Ⅳ The anti-apoptotic effects of Q－VD－OPH on rabbitintervertebral disc cells in vivoObjective: To establish a rabbit model of degenerative disc disease by puncturing theannulus with needles of defined gauge. To investigate the anti-apoptotic effects of broadspectrum caspase inhibitor Q－VD－OPH on rabbit intervertebral disc cells in vivo. Methods: The L3/4, L4/5and L5/6lumbar intervertebral discs of32New Zealand Whiterabbits were stabbed by18-gauge hypodermic needle to a depth of5mm in the annulusfibrosus. Four experimental groups were designed as follows:(1) Normal discs;(2) Stabbeddiscs;(3)5ul normal saline was infiltrated into discs after stab;(4)5ul Q－VD－OPH wasinfiltrated into discs after stab.The rabbits were killed at2,4,6,8weeks post operation, thepercentage of apoptosis of intervertebral disc cells was detected by flow cytometry, thechanges of caspase3were determined by kit. Results: At2,4,6,8weeks post stab, thepercentage of apoptosis in stabbed discs, normal saline groups and Q-VD-OPH groups wasincreased compared with those of normal discs, but the percentage of apoptosis inQ-VD-OPH groups was decreased compared with those of the other two groups(both P＜0.01). The amount of caspase3in Q-VD-OPH groups was significantly decreasedcompared with those of the other two groups(both P＜0.01). Conclusions: Caspase3inrabbit discs can be inhibited by Q－VD－OPH, and apoptosis of rabbit lumbarintervertebral disc cells can be attenuated by broad spectrum caspase inhibitor Q－VD－OPH in vivo.