Improvement Of Perfusion In Cryopreservation Of Rabbit Hind Limbs

ObjectiveThe purpose of this paper was to further improve the dimethylsulfoxide(DMSO)perfusion pretreatment methods used rabbit taken off hind limbs,to test the effectiveness of the pretreatment and the protective effect in cryopreservation of composite tissues.To provide theoretical basis of successful replantation after cryopreservation.MethodsThe experiment was divided into two parts.First part: 19% DMSO-30 min + 10% DMSO-10 min modified pretreatment of rabbit hind limb perfusion model validation.Nine healthy New Zealand white rabbits were selected,18 rabbits hind limb models.Randomly divided into group1,group2 and group3,each group had 6 hind limb models.Group1 as modified perfusion group,which was perfused with 19% DMSO via femoral artery for 30 min,followed by 10% DMSO perfusion for 10 min;group2 as perfusion group,perfused with 19% DMSO via femoral artery for 40 min;group 3 as control group,perfused with normal saline(NS)via femoral artery for 40 min and used for baseline adjustment of nuclear magnetic resonance.In each group,magnetic resonance spectroscopy was used to quantitatively measure the specific concentration of DMSO in the blood and muscle tissue of the same site.The data of each group obtained in groups 1 and 2 were compared and statistically analyzed.Part two: The observation of microscopic morphological changes after freezing-thawing with different pretreatments in rabbit hind limb model.Twenty-four healthy New Zealand white rabbits were randomly divided into group A,group B,group C and group D,with 6 in each group.Group A as the modified perfusion group,perfused with 19% DMSO via femoral artery for 30 minutes and then with 10% DMSO for 10 minutes.Group B as perfusion group,perfused with 19% DMSO via femoral artery for 40 minutes.Group C as the control group,infused with normal saline(NS)via femoral artery for 40 minutes.Group D as blank group,and the samples were directly taken without freezing replantation.The right hind limbs of rabbits in group A,group B and group C were severed first,slow freezing rapid thawing after preconditioning.further replanted and taken samples at last.Group D received fresh samples directly.Each sample was subjected to HE Stained,observed and compared between groups of blood vessel injury,muscle injury and nerve injury.ResultsPart 1,the concentration of DMSO around the vessels in group 1 and group 2 was significantly different,P =0.0000004,the average concentration of perivascular DMSO in group 1 was 6.8550±1.97581%,the average concentration of perivascular DMSO in group 2 was 18.6367±0.35359%.There was no significant difference in concentration of DMSO around muscle between Group 1 and Group 2,P =0.784 the average concentration of DMSO around the muscles in Group 1 was 9.4633±2.48194%,and the average concentration of DMSO around the muscle in Group 2 was 9.7517±1.54743%.The second part: the degree of vascular tissue injury in each sample: group D <group A <group B <group C,the degree of muscle injury and nerve tissue injury in each group: The damage degree in group A and group B is quite good,which is obviously better than that in group C,but there is still a big gap compared with fresh group D.ConclusionsThis experiment demonstrated that the perfusion with 19% DMSO for 30 minutes and then with 10% DMSO for 10 minutes in rabbit limb model can significantly reduce the concentration of DMSO in blood vessels and ensured the concentration close to effective protective concentration 10% in muscle,significantly reduced the DMSO toxicity damage to the blood vessels.