Abstract:

Objective To investigate the effects of application of cryoprotectants (CPA), dimethyl sulfoxide (DMSO) and/or ethylene glycol (EG), on the vitrification preservation of sciatic nerve. Methods Bilateral sciatic nerves of 30 3-month-old male SD rats were harvested and prepared into nerve segments of 15 mm. Nerve segments were preserved in the vitrification solution of different cryoprotectants at -80 ℃ for 4 weeks and divided into the blank control group (no cryoprotants), 10% DMSO group, 20% EG group, and 10% DMSO+20% EG group (n=15). The ultrastructure of sciatic nerves was observed by TEM. The biological activity of sciatic nerves was detected by LSCM after Calcein-AM and PI double staining. Expressions of Caspase-3 and Caspase-8 in sciatic nerves were tested by Western blotting. The sciatic nerves preserved for 4 weeks were cultured by DMEM (containing 10% fetal bovine serum) in an incubator with 5% CO₂ at 37 ℃ for 7 d. Expressions of NGF and GDNF were detected by RT-PCR and Western blotting. Results After sciatic nerves were preserved for 4 weeks, the results of TEM showed that demyelination and axoplasmic atrophy of the blank control group were severe and those of other groups were mild (those of the 10% DMSO+20% EG group were the mildest). The results of LSCM showed that there were weak green fluorescent and strong red fluorescent in the blank control group, strong green fluorescent and weak red fluorescent in the 10% DMSO group and 20% EG group, and the strongest green fluorescent and the weakest red fluorescent in 10% DMSO+20% EG group. The results of Western blotting indicated that the differences of expressions of Caspase-3 and Caspase-8 of sciatic nerves being preserved for 4 weeks between the blank control group and other groups, and the 10% DMSO+20% EG group and other groups (except the blank control group) were statistically significant (P<0.05). But the difference between the 10% DMSO group and 20% EG group was not statistically significant (P>0.05). Results of RT-PCR and Western blotting showed that the differences of expressions of NGF and GDNF of cultured nerves between the blank control group and other groups, and the 10% DMSO+20% EG group and other groups (except the blank control group) were statistically significant (P<0.05). But the difference between the 10% DMSO group and 20% EG group was not statistically significant (P>0.05). Conclusion The 10% DMSO or 20% EG can protect the sciatic nerves preserved by vitrification preservation at -80 ℃ and improve the biological activity of the sciatic nerves after vitrification preservation. The combination of 10% DMSO and 20% EG has the synergistic effect.

Key words: sciatic nerve, vitrification preservation, cryoprotectant, synergistic effect