Adenovirus-mediated tissue inhibitor of metalloproteinase-3 gene transfection inhibits rabbit intervertebral disc degeneration in vivo

The aim of this study was to investigate the inhibitory effects of recombinant adenovirus vector carrying tissue inhibitor of metalloproteinase-3(RAd-TIMP-3)against degeneration of rabbit intervertebral disc.Thirty Japanese white rabbits of 4 months old were randomly divided into 5 groups.Mild or moderate rabbit lumbar disc degeneration model was constructed with the controllable axial loading device by imposing 98 N pressure at the discs for 2 weeks.Various doses of virus were injected into the degenerated discs as follows:20μL of normal saline in group 1;20μL of RAd66(an empty adenovirus vector,1.0�1010 OPU/mL)in group 2;and 20,10,and 5μL of RAdTIMP-3(1.0�1010 OPU/mL)in groups 3,4,and 5,respectively.Two weeks after the injection,the discs were collected for investigations,including assessment of degeneration degrees according to the Thompson’s grading system,reverse-transcription polymerase chain reaction(RT-PCR)assay for TIMP-3 gene,Safranin O-Fast green staining,and immunohisto-chemical staining for TIMP-3 and type II collagen.According to Thompson’s criteria,the degeneration of groups 3,4,and 5,especially group 3,was alleviated as compared with groups 1 and 2.RT-PCR revealed that the expression of TIMP-3 in groups 3,4,and 5,especially in group 3,was significantly enhanced as compared with group 1(P<0.01).Both Safranin O-Fast green staining and type II collagen staining demonstrated better reserved integrity of disc matrix in groups 3,4,and 5 than in groups 1 and 2.TIMP-3 staining exhibited an obvious increase of positive-staining rate in groups 3,4,and 5 as compared with group 1.The positive-staining rate in group 3(79.42%�1.35%)was about 3 times that of group 1(25.47%�5.46%,P<0.01).RAdTIMP-3 can effectively protect the matrix of rabbit intervertebral disc against overloading-induced degeneration in a dose-dependent manner,resulting in the alleviation of disc degeneration.