| Background: For the past few years, with the development and application ofmany new types and efficient immunosuppressant, the long-term survival ratio afterthe transplantation of many kinds of organs has been greatly improved. Organtransplantation has been not only the measure for curing final stage diseases of theimportant organs, but also one of the methods for the satisfaction of improving thelife quality, such as: transplantation of cornea, transplantation of limbs, "changingface" and so on. However, organs can not be kept for a long time, so it confines thedevelopment of organ transplantation and makes the contradiction of supplier lackingmore and more outstanding, then lots of people die in the process of waiting fortransplantation. In addition, important organs are always damaged accompanied byisolated limbs, and doctors try to save the life and have to give up the transplantationfor the time being, as well as giving up the transplantation temporarily for the reasonof psychology, economy or society. When they are in need of transplantation, theisolated limbs have been necrosis. This case is very often in the clinical case. Nowone important task facing the medical staff, especially for organ transplantationworkers is how to solve the problem of long-term preservation of organs, then to solve the "area difference" and "time difference" between organ donor and receptors,and to reduce the waste of organ resource. The donated organs which can bepreserved for long time enable the doctors to have more time to find the well-matchedreceptor, and make the operation on the best condition between themselves andpatients.At present, the cryopreservation is considered as the most effective method oflong-term preservation of tissues and organs. Success of cryopreserved limbs orother organs offers a wider prospect for autologous transplantation and homogenoustransplantation. Now, body marrow stem cell, blood, tendon, spermatozoa, embryo,pancreatic islet, cornea, skin and animal ovaries have been cryopreservedsuccessfully. In Dec, 2002, Wang Zengtao transplanted an isolated fingersuccessfully which was kept in liquid nitrogen for eighty-one days. And itconfirmed the feasibility of the long-term clinical cryopreservation. This finger hadrecovered well after the operation, but the tip shrinked. Moreover, it can be oftenobserved in the animal experiment that, on the process of anastomosis of bloodvessels, and after recanalization of blood vessels, transplanted organ orretransplanted limb body circulation block appeared. But anastomotic stoma wasnormal. It is considered that it is close related to second injury after transplantation(mainly because of ischemia reperfusion injury). It showed by some research that,though cryopreserved issues or organs can keep a certain biological activity, acertain injury of tissue or organ could occur in the process of freezing, i.e.hypothermia injury. And with the recovery of blood flow, tissue or organ (limbs)would suffer from heavier injury, i.e. ischemia reperfusion injury. In the aboveinjuries, the former can lead to destroy of cell membrane protein compound anddecomposition of membrane; the latter can lead to lipid peroxidation of cellmembrane, thus destroy unsaturated fatty acid of cell membrane and damage the cell. In a sense, ischemia reperfusion injury is "overprinting and reinforcement" ofhypothermia injury. And it will directly influence the survival of transplanted organor limbs and influence its function.Skeletal muscle in transplanted limbs is the most sensitive to ischemiareperfusion injury, and the injury after reperfusion is heaviest. Therefore, the researchon ischemia reperfusion injury of skeletal muscle has been hot of limb ischemia ofischemia reperfusion injury. Now, the research on ischemia reperfusion injury aftertransplantation of cryopreserved limbs is little. What is more, the limbs are complextissues; the research on ischemia reperfusion injury of cryopreserved limbs has notbeen reported. Therefore, on the basis of the study on mechanism of ischemiareperfusion injury, together with anatomical characteristics of blood vessel of ratposterior limb, we have established the model of transplanted (autologous) skeletalmuscle with ischemia reperfusion injury after cryopreservation of rat isolated limb.This model can minimize the interference of immunoreactions and other factors.Through the detection of relevant biochemical parameters and morphologicalobservation, the mechanism and prevention measure of ischemia reperfusion injury ofcryopreserved limb can be better studied, so as to provide the theoretical basis onclinical practice. The content of this study is divided into four parts:Part One: Study on histological pathology of cryopreserved rat limbsObjective: To provide morphological basis for the feasibility of transplantation oflimbs after long-term cryopreservation.Method: Eight healthy male and adult Wistar rats (weighing 500～600g) wereselected in the study. Left posterior limb was regarded as control group, and rightposterior limb as freezing group. Anesthesia was performed with injection of 3%pentobarbital (30mg/kg.b.w) into abdominal cavity. Both two posterior limbs werecut off on knee joint level, and then right posterior limb was processed with profound hypothermia freezing, and reduced the temperature gradually, then put it into liquidnitrogen. In the experiment, took the isolated limb out, and put it into constant warmwater tank to rewarm (6 minutes). Then, collected the skin of inside thigh, femoralartery and vein, femoral nerves, gastrocnemius and other issues, respectively formaking histological slice to make morphological contrast analysis with eachcorresponding issue slice of left posterior limb.Result: (1) The general morphological observation: the color of each issue offreezing limb after the recovery was slightly light and it was slightly edema. Therewas no significant difference of flexibility and elasticity compared with that ofcontrol group. (2) Histological observation: each issue after freezing showed:①Skin: Nucleuses of epidermic cell shrunk partly;, parts of cutinized layer desquamated;dennis was appeared with edema.②Blood vessel: Internal membrane wasuncompleted; part of inner elastic membrane was broken; nucleuses of smoothmuscle cell dissepiment shrunk partly and retorted; mesenchymal was appeared withedema.③Nerve: Edema was appeared in the nerve tracts and between the nervefibers.④Skeletal muscle: Muscle fiber and nucleuses shrunk partly; Mesenchymalwas edema, and transverse striation was obscured.Conclusion: (1) Skin, blood vessels, and nerves, skeletal muscle issue after perf-usionwith freezing protection liquid and programmed cooling, cryoperservation can stillkeep certain integrity of cell structure. Once the condition of recovering cellularmetabolism is given, life activity can be expected to recover. (2) Through observationof the various tissues of the cryopreservated rat limes, it provides morphological basisfor the feasibility of transplantation of cryopreserved rat limbs.Part two: Establishment of reperfusion injury model of skeletal muscle of thecryopreserved rat limbsObjective: To establish a reperfusion injury model of skeletal muscle of the cryopreserved rat limbs in order to study the ischemia reperfusion injury of skeletalmuscle of the cryopreserved rat limbs after transplantation.Method: (1) Experiment one: twelve healthy male and adult Wistar rats (weighing500～600g) were selected, and anesthesia was performed by injection into theabdominal cavity. First, a fragment of femoral artery and vein about 0.5cm long werecut off, and were fixed by the 10% formaldehyde, buried by olefin, sliced, dyed byHE, then the structure and the thickness of the wall of the vein were observed, and themethod of the foam rubber perfusion through the thoracic aorta were adopted, theoutside diameter and the length of the thigh artery and vein were measured,meanwhile, its branch, trend, and the interval were also observed, so as to provide thetheoretical basis for the option of the amputation level and the freezing process andthe retransplantation of the isolated lambs. (2) Experiment two: 72 healthy male adultWistar rats were selected, weighing 500～600g, and were divided into 6 groupsrandomly: A, B, C, D, E, F, 12 in each group. According to the data provided byexperiment one, an incision was make from the bottom of the right groin along withthe femoral vessel to show the femoral artery and vein and the branches. After thefemoral artery and vein were ligated at the level of the lower rim of the superficialiliac circumflex branches, the limb was cut off quickly on this level; The isolatedlimbs were treated respectively according to different groups, then transplanted to theright posterior limb (the skin of the right isolated were sewn up and fixed with theskin of the left side, An end to end anastomosis of right femoral artery of isolatedlime with the left femoral artery was made directly, an end to end anastomosis of rightfemoral vein with the left femoral was made by bushing method). The parameterswere detected after the recovery of blood supply for 4h. Group A (normal controlgroup): only put on the line on the femoral artery and vein without ligation, sampleswere collected directly; Group B (group with ischemia reperfusion on the normal temperature): the right isolated lime was put in the room temperature for 2.5h, thenthe samples were collected; Group C (cryopreservation group): the right isolatedlimbs were dealt with pre-frozen, preserved in the liquid nitrogen container for onemonth, and then they were picked up, rewarmed, perfused with perfusate,transplanted and then the samples were collected; Group D, E, F (low, middle andhigh dose of edaravone groups in turn) was all cryopreserved, the process was all thesame to group C except that the perfusate was not normal ones but contained low,middle and high dose of Edaravone, respectively.Result: The anatomic data of the femoral artery, vein and the branches showed thatthe femoral artery of rat is the continuity of the external iliac artery (usually referringto the fragment of the vessel that begins from inguinal ligament and ends at poplitealartery, which is (19.39±0.72)mm in length, (0.96±0.13) mm. In outside diameter ofmiddle piece, (235.83±19.87)μm in thickness of the wall; the branches from the topto the bottom is: superficial iliac circumflex branches, muscular branches, superficialepigastric branches, the top branches of the knee, the hidden branches, the deepfemoral branches, and the perforating branches. Among these, the last three branchessupply blood to the distal part to knee; muscular branches originate at the one third ofthe femoral artery, and the proximal end is located in bottom of the groin ligament(9.07±0.52) mm; the superficial epigastric branch is located in the bottom of thegroin ligament (11.46±0.55) mm; the top branch of knee is located in the bottom ofthe groin ligament (13.72±0.71) mm; popliteal artery is direct continuity of femoralartery. The length from top knee branch to muscular branches is (4.65±0.37) mm; thelength from muscular branch to superficial iliac circumflex branch is (2.39±0.32) mm.The femoral vein accompanies the femoral artery, and the vein wall of rats is verythin, soft and little elastic. The dividing line of three layers of the vein wall is notclear, smooth muscle of middle membrane is less thick than artery, muscle fiber is sparse; inner and outer elastic membrane is not obvious and easy to collapse. Theouter diameter of femoral vein is (1.10±0.12) mm, and the thickness of the wall is(107.50±19.25)μm.Conclusion: (1) In the process of making isolated limb model, amputation level is atleast 0.6 cm above the top artery of knee, in order to keep enough length of femoralartery, vein of isolated limb. (2) In order to keep the same ischemia time in eachgroups, the ischemia time of the isolated limes in normal temperature in group B is2.5 hours before transplantation. (3) Because of the anatomic characteristics, theanastomosis of the femoral vein was made by bushing method, which is easy to make,time needed is less, and easy to keep persistent blood flow. (4) Because thisexperimental model is only an acute one which only lasts 4 hours, so it needn't tosuture the muscle and fix the bone. (5) The reperfusion injury model of skeletalmuscle of the cryopreserved rat limbs is very easy to made, easy to nurse in shorttime. The model is stable, safe and practical, which is a fairly ideal acute animalmodel for the study of ischemia reperfusion injury of skeletal muscle. Because thismodel is autologous transplantation, it can avoid the immunity factors, minimizevarious interfered influence, and it can be used for the study of the mechanism ofischemia reperfusion injury of the cryopreserved skeletal muscle and its protectivemeasure.Part Three: Study of reperfusion injury of the skeletal muscle of the cryopreservedrat limbsObjective:Using the reperfusion injury model of skeletal muscle of the cryopreservedrat limbs to compare the pathological changes of the skeletal muscles between thegroup with cryopreserved rat limbs and the group with isolated limes exposed tonormal temperature, in order to explore the mechanism of the reperfusion injury tothe skeletal muscle of the cryopreserved rat limbs and to provide the theoretical basis for the clinical prevention and treatment.Method: 36 healthy male adult Wistar rats, weighing 500～600g, were randomlydivided into three groups: Group A (normal control group), group B (ischemiareperfusion in normal temperature), and group C (cryopreserved ischemiareperfusion), 12 in each group. The animal models were made according to part 2: ingroup A: only put on the thread on the femoral artery and vein without ligation,samples (venous blood and the tissue of the gastrocnemius muscle in right leg) werecollected directly; the right posterior limbs were cut off at the superficial iliaccircumflex artery level in group B and C. In group B, the right isolated limbs wereput in the room temperature for 2.5h, the anastomosis of the vessels of the isolatedlimbs with femoral artery and vein in left limbs were made. 4 hours after the recoveryof blood flow, the samples were collected(the same to group A); In group C, the rightisolated limbs were dealt with pre-frozen, preserved in the liquid nitrogen containerfor one month, and then they were picked up, rewarmed, perfused with perfusate,transplanted. 4 hours after the recovery of blood flow, the samples were collected (thesame to group A). In the three groups, LDH, CPK, the W/D ratio, enzymatic activityof the Na+-k+-ATPase and the Ca2+-ATPase, MDA, SOD and MPO were detected andthe morphological change of skeletal muscle was observed under microscope toexplore the differences between the group of ischemia reperfusion in normaltemperature and the group of cryopreserved ischemia reperfusion.Result: (1) Compared with group A, plasma LDH, CPK and W/D, MDA and MPO inskeletal muscle in group B and C were significantly higher than those in group A(P＜0.05); the enzymatic activity of SOD in gastroenemius muscle and theNa+-K+-ATPase, Ca2+-ATPase in the muscular membrane in group B and C weresignificantly lower than those in group A; (2) Compared with group B, plasm LDH,CPK and W/D, MDA and MPO in skeletal muscle in group C were significantly higher than those in group B (P＜0.05); The enzymatic activity of the Na+-K+-ATPase,Ca2+-ATPase in the muscle cell membrane was significantly lower than those in groupB; There was no significant difference of SOD in gastrocnemius muscle betweengroup B and C (P＞0.05). (3) For the histological section, in group A, the muscularfiber was normal, there was no edema in the stroma, and there was no inflammatorycell infiltration; in group B, the blood vessel was dilated and congestive, there wasedema in the stroma; There was inflammatory cell infiltration; the stria was chaotic;In group C, the muscular fiber was chaotic and ruptured, interstitial substance wasdropsical and there was infiltration with many inflammatory cells; The stria wasblurred. (4) Observations from transmission electron microscope: the arrangement ofmyofibril in group A was relatively regular; Z line was clear; mitochondrial crista wascompact; membrane was intact and without swelling. In group B, the arrangement ofmyofibril was relatively regular, Z line was a little obscured; Mitochondria wereswollen and mitochondrial crista was in chaos, parts had been vacuoles. Parts ofmyofibril in group C were broken; Z line was unclear or disappeared, mitochondriawas highly swollen and mitochondrial crista was in chaos, and parts had beenvacuoles.Conclusion: (1) Ischemia reperfusion injury of skeletal muscle is a multi-factorinvolved process and multiple mechanism injury. The pathological process ofischemia reperfusion injury of the limbs is a synthetic injury process which is relatedlimbs and other organs. (2) Profound hypothermia can keep the activity of issue andcell in a certain condition, but at same time, it can also develop a certain degree injuryto issue and cell. After the recovery of blood flow of the replanted limbs, more severeischemia reperfusion injury usually occurs. It is considered that it is because ofhypothermic damage together with ischemia reperfusion injury. The double injuriesresult in the severe damage of vascular inner cell, blockage of the capillary of the ischemia tissue, reduction of the blood flow, and more severe ischemia of the limbs,which we call severe ischemia reperfusion injury. However, the detailed mechanismis needed to be investigated.Part Four: Experimental study of the intervention of Edaravone on ischemiareperfusion injury of the cryopreserved rat limbsObjective: Using the reperfusion injury model of skeletal muscle of thecryopreserved rat limbs, to investigate the influence of ischemia reperfusion injuryupon cell membrane mitochondrion of skeletal muscle in rat limbs aftercryopreservation, to evaluate protective effect and mechanism of Edaravone upon cellmembrane mitochondrion of skeletal muscle with ischemia reperfusion injury.Method: Sixty healthy adult and male rats, weighing 500～600g, were divided into 5groups randomly: normal controlled group (A), regular profound hypothermiafreezing group(C), low dose Edaravone group(D), middle dose Edaravone group (E),high dose Edaravone group(F), 12 rats in each group. The animal models were madeaccording to part 2: In Group A: only put on the thread on the femoral artery and veinwithout ligation, samples (the tissue of the gastroenemius muscle in right leg) werecollected directly; The right posterior limbs were cut off at the superficial iliaecircumflex artery level in group C, D, E, F. in group C, the right isolated limbs weredealt with pre-frozen, preserved in the liquid nitrogen container for one month, andthen they were picked up, rewarmed, perfused with perfusate; among them,Edaravone group (D, E, and F) was perfused with perfusate containing edaravone0.1mg/kg.b.w, 0.3mg/kg.b.w, 0.5mg/kg.b.w respectively; At last, the isolated limbswere transplanted to its original rats. 4 hours after the recovery of blood flow, thesamples were collected (the same to group A). In all groups, the content of MDA,SOD, MPO, fluorescence polarization, activity of Na+-K+-ATPase and Ca2+-ATPasewere detected; Mitochondria of skeletal muscle were separated and content of MDA, SOD and RCR are tested. Structure change of skeletal muscle in each group wasobserved by light microscope and ultrastructure of skeletal muscle was observed bytransmission electron microscope.Result: (1) Compared with group A, the content of MDA in group C, D and E weresignificantly higher than those in group A(P＜0.05); The activity of SOD and RCRwere lower than those in group A (P＜0.05); There were no significant differences ofthe content of MDA, activity of SOD and RCR between group F and group A(P＞0.05); Fluorescence degree of polarization in group C and D were significantlyhigher than those in group A (P＜0.05); There were no significant differences offluorescence degree of polarization in group E and F; MPO, mitochondrial MDA ingroup C, D, E and F were significantly higher than those in group A(P＜0.05);Na+-K+-ATPase, Ca2+-ATPase of cell membrane of skeletal muscle and mitochondrialSOD in each groups were significantly lower than those in group A(P＜0.05). (2)Compared with group C: SOD activity of skeletal muscle in group E and F,mitochondrial SOD and RCR were significantly higher than those in group C(P＜0.05), but there were no significant differences of the above parameters betweengroup D and group C(P＞0.05); The activity of Na+-K+-ATPase and Ca2+-ATPase ofskeletal muscle cell membrane in group D, E and F were significantly higher thanthose in group C (P＜0.05); The content of MDA, mitochondrial MDA, MPO andfluorescence degree of polarization in group D, E, F were significantly lower thanthose in group C (P＜0.05). (3) Compared with group D, SOD activity of skeletalmuscle, mitochondrial SOD and Na+-K+-ATPase of cell membrane in group F weresignificantly higher than those in group D (P＜0.05), and the content of MPO andmitochondria MDA were significantly lower than those in group D (P＜0.05); Theactivity of Ca2+-ATPase of skeletal muscle cell membrane in group E and F wassignificantly higher than those in group D(P＜0.05); The content of MDA in skeletal muscle was significantly lower than that in group D (P＜0.05); But there were nosignificant differences of fluorescence degree of polarization and RCR between groupE, F and D, respectively (P＞0.05). (4) Compared with group E, Na+-K+-ATPase ofskeletal muscle cell membrane only in group F was significantly higher than that ingroup E (P＜0.05), and there were no significant differences among the othergroups(P＞0.05). (5) From the histological slice of skeletal muscle: In group A, themuscular stria was clear and there was no interstitial edema; In group C, structure ofmuscular fiber was in chaos and ruptured, and intermuscular gap was widen; Therewas mesenchymal edema and lots of inflammatory cell infiltration. Muscular fiber ingroup D was mildly broken; Mesenchymal was in edema and inflammatory cellsinfiltration. In group E skeletal muscle mesenchymal was in a little edema, and themuscular stria was blurred; There was infiltration with a few inflammatory cellsinfiltration; In group F skeletal muscle mesenchymal was in mild edema; There wasinfiltration of sparse inflammatory cells. (6) From the observation of electronmicroscope: In group A, the arrangement of myofibril was relatively regular, Z linewas clear; mitochondrial crista was compact; Membrane was intact and withoutswelling. In group C: Parts of myofibril were broken; Z line was unclear ordisappeared, mitochondria was highly swollen and mitochondrial crista was in chaos,and parts had been vacuoles; There was mesenchymal edema; But the injury degreeof skeletal muscle in group F was milder than that group C: myofibril arrangementwas relatively in order; Z line was clear, mitochondia wais slightly swollen;Membrane was relatively intact; crista existed, but part was in chaos, and parts hadbeen vacuoles.Conclusion: (1) The isolated limb after cryopreservation perfused with Edaravonebefore transplantation can reduce the reperfusion injury of skeletal muscle. Theprobable mechanism is that Edaravone can refraine membrane lipid peroxidation, remove the free radicals, so it can protect skeletal muscle cell membrane andmitochondria. (2) The best dose of edaravone to prevent the ischemia reperfusioninjury for the cryopreserved limbs is still unclear and need further investigation. |
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