| Part 1 The effects of vitreous cryopreservation with tetramethylpyrazine on apoptosis of Schwann cells and the expression of regulation gensObjective: To investigate the effects of vitreous cryopreservation with tetramethylpyrazine on apoptosis of schwann cell and the expression of regulating gens under different temperature.Methods: 48 sciatic nerves obtained from 24 SD rats were randomly divided into 4 groups by vitrification solution containing tetramethylpyrazine ( 0mg/L—A group, 100mg/L—B group, 200mg/L—C group, 400mg/L—D group ) and cryo-preserved at -196℃. After 3 weeks, all the nerves were obtained for tests: HE staining for light-microscope test, apoptosis of Schwann cell quantitatived by the terminal transferase UTP nick end labeling (TUNEL) method, the expression of Bcl-2 and Bax by immunohistochemisty technique.Results: The number of Schwann cell apoptosis of C and D groups was significantly smaller than A and B groups ( P <0.05 ); the expression of Bcl-2 were significantly increased in C and D groups compared with that in A and B group and the expression of Bax decreased significantly ( P <0.05 ). There was no significant difference between group A and group B.Part 2 The effects of vitreous cryopreservation with tetramethylpyrazine on immunological reaction of sciatic nerve allograftsObjective: To observe the change of immunity in vivo after vitreous cryopreservation with tetramethylpyrazine nerve allograft was implanted into the recipient so as to judge its immunogenicity.Methods: 48 Sciatic nerves of 24 Sprague-Dawley rats were resected and randomly divided into 4 groups by vitrification solution containing tetramethylpyrazine, then those nerves were cryo-preserved at -196℃by vitreous cryopreservation with tetramethylpyrazin. After 3 weeks, the nerves from Sprague-Dawley rats were implanted into the dorsal subcutis of Wistar rats in experimental group. Fresh nerves were implanted into the dorsal subcutis of Wistar rats as control group. T-lymphoeytes infiltraion in isograft nerve was investigated by immunohistochemical methods,staining CD4 and CD8 positive T-lymphocytes with anti-CD4 and CD8 monoclonal antibodies (MoAb). Results: 200mg/L and 400mg/L tetramethylpyrazine combined vitreous cryopreservation stainings at -196℃after 3 weeks showed that the number of CD4 and CD8 positive T-lymphocytes infiltrated into the nerve mplants of the experimental group is much lower than that other tetramethylpyrazine combined vitreous cryopreservation in the control group. But the 200mg/L and 400mg/L tetramethylpyrazine combined vitreous cryopreservation stainings at -196℃after 3 weeks nerves could be preserved after implantation in the experimental group.Part 3: The effects of vitreous cryopreservation with tetramethylpyrazine on peripheral nerve allografts regenerationObjective: To investigate the effect of tetramethylpyrazine with a certain concentration added to vitrification solution on peripheral nerve allografts regeneration.Methods: 48 male SD rats were randomly divided into four groups equally. A 15 mm sciatic nerve segment was removed and applied vitrification solution containing tetramethylpyrazine ( 0mg/L—A group, 100mg/L—B group, 200mg/L—C group, 400mg/L—D group ) cryo-preserved at -196℃for 3 weeks. Nerve defect 10mm in length were made in the sciatic nerves of 96 Wistar rats as donors. After the storage,the allografts were transplanted to the corresponding rats. The gross appearance,morphological and electrophysiological changes,the image analysis of axons were detected at postoperative week 4,8,16.Results: The regeneration of group A and B was the best among four groups.That of the group C was better than group D. The best storage time was 3 weeks. There were significantly statistical difference in the results of electrophysiological examination, the image analysis of axons and motor end-plate (P<0.01).Part 4 Regeneration of sciatic nerve allografts preserved in vitrification solution containing tetramethylpyrazine at -20℃Objective: To study the effect of tetramethylpyrazine added to vitrification solution on rats sciatic nerve allografts regeneration at different times on -20℃.Methods: Male SD rats sciatic nerve were stored in vitrification solution containing 200mg/L tetramethylpyrazine for 1, 2 weeks respectively, holding temperature of -20℃(Group A).Wistar rats were donors. Afer the storage, the allografts were transplanted to the corresponding rats. The gross appearance , morphological ,electrophysiological changes and counts of motor end-plate were detected at postoperative week 4,8,16. The comparison of allografts were performed between Group A (vitrification solution containing tetramethylpyrazine at -20℃) and Group B (vitrification solution at-20℃) or Group C (vitrification solution containing tetramethylpyrazine at -196℃).Results: There were not significantly statiscal diffierence in the results of Group A and Group C holding 1 week ( P>0.05 ) . There were significantly statiscal diffierence in the results of Group A and Group B holding 1 week ( P<0.01 ) . The results show that the holding temperature of -196℃the regenerration of the nerves were the best after 2 weeks.Conclusion:1. These findings suggest that vitreous cryopreservation with a certain concentration tetramethylpyrazine can depress apoptosis of Schwann cell in peripheral nerve of rats by regulating the expression of Bcl-2 and Bax.2. The nerve treated by 200mg/L and 400mg/L tetramethylpyrazine combined vitreous cryopreservation at -196℃after 3 weeks methods had lower immunogenicity after implantation,and there as no significant rejection response after transplantation in the rat.3. The tetramethylpyrazine with a certain concentration in vitrification solution has protective effect on regeneration of peripheral nerve allografts.4. The tetramethylpyrazine with a certain concentration in vitrification solution can raise holding temperature of peripheral nerve allografts. |
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