| Objectives:To study the role of inducible nitric oxide synthase in articular tissue with limbs IR injury and introduce into tissue microarray, we designed to observe the pathologic changes, the expression of iNOS and the number of apoptotic cells in articular tissue in rabbits with limbs IR injury and apply TMA technique.Methods:54 adult New Zealand white rabbits(2500±100g)were divided into 9 groups randomly(Normal, Ischemia, IR2h, IR4h,IR8h, IR24h, IR3d, IR7d, IR14d,6 per group). Femoral arteries and femoral veins of right hind limbs were clamped by vessel serrefines. Tourniquets were used simultaneously (except Normal group). The left hind limbs were treated as self-control which performed same operation only. After 4 hours of ischemia, the right limbs were reperfused (except Normal and Ischemia group). Then the rabbits were killed at corresponding time. Synovia was smeared; synovial membrane, muscle, menisci, and cartilage were excised from the knee of both sides respectively. They were embedded into paraffin. Expression of iNOS was evaluated using immunohistochem-istry and immunofluorescence; Apoptotic cells were identified using the TUNNEL (terminal dUTP nick end labelling) assay; Collagen type II was evaluated using immunohistochemistry; Proteoglycan was evaluated using haematoxylin-brilliantgreen-safranine staining. Manual TMA technique was designed to simplify the experiment.1.The articular tissue in experimental groups showed a time-dependent injury, though that in self-control groups changed insignificantly.2. The expression of iNOS in articular muscle of right limb changed time dependently. The expression of iNOS was detected during the initial stages (IS, IR2h) and it located in capillary endothelial cells only; during the following stages, such as IR4h group and IR8h group, it located in skeleton muscle cells at large scale; Then the iNOS expression. in skeleton muscle cells declined to an undetectable level in IR3d group. Accordingly, the number of Apoptotic cells in skeleton muscle changed in the homologous way. A correlation analysis was made between the expression of iNOS and the number of apoptotic cells. The correlation (r=0.682,p=0.043) was significant at the 0.05 level (2-tailed).3. INOS was labeled using SABC-cy-3, and the apoptotic cells were identified using the TUNNEL. Both iNOS and apoptotic cells changed time-dependently in the experimental groups. INOS which located in menisci chondrocytes cytochylema increased in IR2h group, culminated in IR8h group, and began to decline in IR24h group. The number of apoptotic cells in menisci increased from IR2h group and reach maximum in IR24h group, then decreased slightly and sustained at lower level compared with control side. It was significant at the 0.05 level (2-tailed). Correlation analysis result suggested that there was a positive correlation between the expression of iNOS and the number of apoptotic cells(r=0.754, p=0.031). It was significant at the 0.05 level (2-tailed).4. The same methods were used to examine iNOS and apoptotic cells in cartilage. In the experimental groups iNOS located in cytochylema, which increased in IR2h group, reach maximum in IR8h group, and decreased in IR24 group. Compared with control side, it was significant at 0.01 level (two tailed). The number of apoptotic cells of cartilage appeared in IR2h group, culminated in IR24h group, then declined slightly and maintained a lower level even in IR7d group. Compared with control side, it was significant at 0.01 level(two tailed). Both iNOS and apoptotic cells changed time-dependently in the experimental groups. A correlation analysis was made between the two groups of enumeration data and its outcome (r=0.828,p=0.006) indicated that there was a positive correlation which was significant at 0.01 (at two-tailed).5. Proteoglycan was evaluated using haematoxylin-brilliantgreen-safranine staining and collagen typell was evaluated using immunohistochemistry. There was no difference of PG and collagen typell between the control and experimental group during the initial time. Both PG and collagen typell decreased significantly after 3d and sustained a lower level to 14d in the experimental group.. It was significant at 0.05 level (at two-tailed) compared with the control side.6. Designed an manual method to prepare TMA and use them to examine the expression of iNOS, apoptotic cells in both experimental and control group. The results were no significant compared with that examined by usual paraffin section.1. The injury of articular tissue aggravated time-dependently after reperfusion, as obviously suggests that there was a IR injury in knee joint in rabbits with hind limbs IR.2. Both the expression of iNOS and the number of apoptotic cells in muscle, menisci and cartilage changed time-dependently in the experimental group. There was positive correlation between the two signs. PG and collagen typeⅡdecreased significantly after 3d and sustained a lower level to 14d in the experimental group. It was significant compared with those in control side (p<0.05). INOS is considered to be one of the mechanisms responsible for the death of cells during reperfusion time. Go further and say; it may be one of the mechanisms responsible for the articular tissue IR injury in rabbits.3. Self-designed TMA preparation method possess the following properties:①imple;②practical;③economical;④reliable;⑤efficient;⑥easy to be popularized.
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