Measurement Of Cryoprotectant Permeation In Amputated Rabbit Limb By Microdialysis: Preliminary Studies

Objective:Using microdialysis to detect the change of osmolality in typical tissue under body temperature and blood pressure. To work out a method to understand the concentration of cryoprotectant which could perfuse into amputated rabbit limb via blood vessels. To work out if the concentration is enough to protect amputated limb in cryopreservation. Provide a basis for cryoprotectant applying to cryopreservation of amputated limb. Methods:Condition: room temperature kept at 20-24℃ and kept solution in reactor or amputated limb at 38℃. The flow rate of Microdialysis pump is 2.2 μL/min. Get the animal test’s relative recovery through calibration test in vitro and in vivo. Fifteen New Zealand white rabbits, finished skin preparing before surgery day. The rabbits were anesthetized with a solution of chloral hydrate through the posterior auricular vein. Skin and muscle of the lateral thigh were sutured together. Cut the skin and muscle at the proximal end of suture. Arteria femoralis and femoral vein were kept from hurts. Femur and skeletal muscles were dissociated out up to hip and opened joint capsule. Took out the femoral head. All tests were in physiological condition in terms of both body temperature and blood pressure. NS containing heparin was perfused into the amputated limb through arteria femoralis until backflow liquids was clear and colorless. Two MD probes were inserted into subcutaneous tissues near fascia lata and at the back of shanks, respectively. Then the amputated rabbit thigh was perfused with cryoprotectant or NS through arteria femoralis for 40 mins. Cryoprotectant contain 10%v/v Me2 SO or 20%v/v Me2 SO was used in the study. Dialysate samples were Collected every 15 mins. Backflow liquids from the femoral vein were collected at 1, 3, 5, 10, 20, 30, 40 or 50 mins. All samples were analyzed by osmometer and analyzed statistically. Results:Relative recovery is 14.07% in the animal test. The results revealed that there is no change of osmolality in MD sample in 10%Me2SO group. But 5.746%v/v Me2 SO was detected in backflow liquids sample; In 20% Me2 SO group, Me2SO’s concentration reached 8.932%v/v and 9.996%v/v in subcutaneous and skeletal muscle tissues, respectively. And 18.360%v/v Me2 SO was detected in backflow liquids sample. Conclusions:Condition: room temperature kept at 20-24℃ and kept amputated limb at 38℃. CMA 30 Linear MD 4/pkg could be applied in Me2 SO detecting. The animal test’s relative recovery is 14.07%. In physiological condition, Concentration of Me2 SO perfused into Amputated Rabbit Limb via Blood Vessel could reach or almost reach the ideal concentration within 30 mins.