Abstract: BACKGROUND: The effect of protective agent penetration is crucial in organ cryopreservation. Quantitative analysis of the effect of cryoprotectant dimethyl sulfoxide introduction can provide a theoretical basis for the successful cryopreservation of organs. 
OBJECTIVE: To study the effect of dimethyl sulfoxide penetration on the cryopreservation of rabbits’ severed hindlimb. 
METHODS: Fifty New Zealand white rabbits were randomly divided into group A1 (n=8), group A2 (n=8), group B1 (n=8), group B2 (n=8), group C1 (n=6), group C2 (n=6), and group C3 (n=6) by random number table method. The severed hind limb cryoprotectant perfusion model was established in all groups. Groups A1 and A2 were perfused with 10% and 20% dimethyl sulfoxide solution through the femoral artery for 50 minutes, respectively. The concentration of dimethyl sulfoxide in muscle tissue was detected by microdialysis-freezing osmometer. Group B1 and group B2 were perfused with 10% and 20% dimethyl sulfoxide solution through the femoral artery for 30 and 20 minutes, respectively. The concentration of dimethyl sulfoxide in perivascular, muscle and subcutaneous tissue was detected by nuclear magnetic resonance spectroscopy. Group C1, group C2, and group C3 were immersed in 50%, 35%, and 20% dimethyl sulfoxide solution for 30 minutes, respectively. Nuclear magnetic resonance spectroscopy was used to detect the concentration of dimethyl sulfoxide in perivascular, muscle and subcutaneous tissues.
RESULTS AND CONCLUSION: (1) The concentration of dimethyl sulfoxide in the muscle tissue of groups A1 and A2 increased with the extension of perfusion time. The concentration of group A1 stabilized at about 5% after 30 minutes of perfusion, and the concentration of group A2 stabilized at about 12% after 20 minutes of perfusion. The concentration of dimethyl sulfoxide in the muscle tissue of group A2 at each perfusion time point was higher than that of group A1 (P < 0.05). (2) The concentrations of dimethyl sulfoxide in the muscle, perivascular and subcutaneous tissue of group B2 were 12%, 20%, and 8.6%, respectively. The concentrations of dimethyl sulfoxide in the perivascular, muscle tissue and subcutaneous tissue of group B1 were 10.9%, 6.9%, and 1%, respectively. There were significant differences in the concentrations of dimethyl sulfoxide in the same tissues between the two groups (P < 0.05). (3) The presence of dimethyl sulfoxide was not detected in the muscle and perivascular tissue of groups C1, C2, and C3. The concentrations of dimethyl sulfoxide in the subcutaneous tissue of groups C1, C2, and C3 were 6.5%, 2.3%, and 1.85%, respectively, and the difference between the groups was significant (P < 0.05). (4) These results suggest that for the rabbits’ severed hindlimb model, the dimethyl sulfoxide penetration is ineffective by traditional immersion method, while 20% dimethyl sulfoxide can reach or approach effective vitrification concentration in most tissues after being introduced into the model through arterial perfusion. 

Key words: severed limb replantation, cryopreservation, dimethyl sulfoxide, microdialysis-freezing osmometer, nuclear magnetic resonance spectroscopy