An experimental study on the effect of fluorocarbon perfusion for the preservation of an amputated limb

We assessed the ability of oxygenated fluorocarbon (chilled Fluosol-DA) to minimize the effect of ischemia on devascularized skeletal muscle of dog. 1) Short term follow-up study: Perfusion of the limb with Fluosol-DA solution significantly minimized edema formation and leakage of skeletal muscle enzymes (GOT, CPK) into the serum 5.5 hours after revascularization as compared to Collins sol., lactated Ringer's sol. or merely cooling. Histological finding showed that the Fluosol-DA group had significantly less hyaline degeneration and separation of the muscle fiber from the endomysium than the groups receiving Collins sol., lactated Ringer's sol. or merely cooling group. 2) Long term follow-up study: The Fluosol-DA group showed less enzyme elevation than the Collins sol. and lactated Ringer's sol. groups. Histologically, the Collins sol. and lactated Ringer's sol. group showed significant scarring compared to the Fluosol-DA group. It was concluded that perfusion with chilled Fluosol-DA was effective for preservation of devascularized skeletal muscle.     

An experimental study on "replantation toxemia". The effect of hypothermia on an amputated limb.

Hind legs of dogs were amputated at the middle of the thigh and preserved in three different conditions: in ice water, in a refrigerator, and at room temperature. After 6 or 12 hours of ischemia, recirculation was established. The survival rate of the animals was observed and measurement of limb edema, potassium, pH, and lactate in the blood was performed to study the effects of hypothermia on prevention of "replantation toxemia." Cooling of the amputated limb was effective for prevention of toxemia, and the cooling effect was greater in ice water than in a refrigerator. However, when cooled in ice water, some animals died due to toxemia when the time of ischemia was prolonged to 12 hours. In the dead animals, a close relationship was observed between the developement of toxemia and metabolic acidosis due to the increase in lactate.     

Effect of fluorocarbon perfusion upon the preservation of amputated limbs. An experimental study

The present study was undertaken to investigate the effect of fluorocarbon on the preservation of an amputated limb. The hind limbs of dogs were completely amputated through the mid-thigh; some were perfused with fluorocarbon, others with lactated Ringer's solution and some were not perfused at all. After six hours of ischaemia, all the limbs were replanted. Perfusion with fluorocarbon had an inhibitory effect on the anaerobic metabolism of an amputated limb, thus increasing the survival rate. Leakage of creatine phosphokinase from the replanted limb also was inhibited by perfusion with fluorocarbon. These effects were more striking when the amputated limb was perfused continuously rather than intermittently and when it was preserved in iced water rather than at room temperature; these measures helped to prevent replantation toxaemia and to preserve muscle function.     

The experimental study on long-term cardiac preservation: the efficacy of low-flow continuous perfusion with fluorocarbon

We compared the effect of simple immersion and continuous perfusion on long-term cardiac preservation, and evaluated the effectiveness of perfusion with oxygenated fluorocarbon solution. The isolated rabbit hearts were preserved for 24 hours at 4 degrees C using the following five preservation techniques: (1) simple immersion with Collins M solution (Group I), (2) perfusion with oxygenated Collins M solution at a flow rate of 10 ml/hr (Group II), (3) perfusion with the same solution as in Group II at a flow rate of 20 ml/hr (Group III), (4) perfusion with oxygenated Collins M solution containing 10% fluorocarbon at a flow rate of 10 ml/hr (Group IV), (5) perfusion with the same solution as in Group IV at a flow rate of 20 ml/hr (Group V). The hearts of Group I showed a significant decrease of myocardial ATP and an increase of myocardial lactate during preservation compared to the hearts of perfusion groups. Assessment of isovolumic left ventricular function following 24-hour preservation using a support animal showed a significant decrease of Max dp/dt and increase of end-diastolic pressure in the hearts of Group I. Perfusion with fluorocarbon (Group IV and V) significantly increased oxygen consumption compared to Group II and III in association with minimum accumulation of myocardial lactate, indicating that aerobic metabolism during preservation is better maintained in the fluorocarbon-perfused hearts. Moreover, CPK release and myocardial water gain during preservation were significantly less, and left ventricular function following preservation was significantly better in these hearts. Increasing the flow rate from 10 ml/hr to 20 ml/hr resulted in sustained increase in perfusion pressure (1.80 +/- 0.53 to 3.70 +/- 0.34 mmHg) and myocardial water content (79.2 +/- 0.4 to 87.2 +/- 0.3%) during preservation in the hearts of Group III, but it did not further improve left ventricular function despite significant enhancement of myocardial oxygen uptake in both Group III and V. These results suggest that hypothermic low-flow continuous perfusion with oxygenated Collins M solution is superior to simple immersion with the same solution for long-term cardiac preservation, and that the addition of fluorocarbon to the perfusate enhances the efficacy of such a perfusion.     

颈交感神经网切除对肢体痉挛骨骼肌肌电图、酶及肌纤维结构的影响

目的　探讨交感神经切除后 ,痉挛肌肉内肌电图、酶及肌纤维结构的改变。方法　将2 0只Wistar大白鼠作成痉挛性模型 ,分成两组 ,随机选择 1组行颈总动脉周交感神经网剥脱切除及颈上节交感神经切除术 ,另 1组作为对照 ,于术后第 8天用Dantec肌电图仪观察肱三头肌F波幅度。术后第 2 0天切取两组大鼠部分肱三头肌组织 ,采用 (Ellman)爱尔蒙法测定肌组织内乙酰胆碱酯酶活性 ,在IBAS图像分析仪上观察两组肌组织内快收缩肌纤维与慢收缩肌纤维的改变情况。结果　交感神经切除后 ,F波幅度及乙酰胆碱酯酶活性明显下降 ,分别从 (0 .3 778± 0 .160 0 )mm降至 (0 .15 5 2± 0 .0 80 0 )mm (P <0 .0 1) ;(3 .3 7± 1.0 1)U / g降至(0 .84± 0 .65 )U / g(P <0 .0 1) ,快肌纤维明显减少 ,从 (2 75 72 7.3 1± 982 40 .2 3 )U/m2 降至 (8814 8.2 2± 3 5 111.18)U /m2 (P <0 .0 1) ,慢肌纤维显著增加 ,由 (4 2 710 .78± 2 885 8.3 7)U/m2 增至 (179184.73± 870 44 .5 9)U/m2(P <0 .0 1)。结论　交感神经切除后痉挛肌肉兴奋性下降     

The effect of fluorocarbon emulsion on 24-hour canine heart preservation by coronary perfusion

This study was designed to evaluate the effects of fluorocarbon emulsion as an oxygen carrier in myocardial preservation. The hearts were preserved for 24 hours by coronary perfusion with either oxygenated crystalloid cardioplegic solution (group C) or crystalloid cardioplegic solution with fluorocarbon added (group FC). The perfusion pressure was kept at 20 mm Hg, and myocardial temperature was maintained at 4 degrees C. Group FC demonstrated better recovery in developed pressure and maximum rate of rise of left ventricular pressure compared with group C. The postpreservation end-diastolic pressure in group C increased significantly compared with the baseline value (value obtained before preservation). On the other hand, group FC showed no significant increase of end-diastolic pressure after preservation. Group FC released a significantly lower level of creatine kinase into its perfusate than did group C. Ultrastructural changes after preservation in group C showed severe ischemic injury, but there was no evidence of ischemic injury in group FC. The use of fluorocarbon emulsion proved beneficial to myocardial protection in long-term preservation of canine hearts.     

Improved preservation of skeletal muscle in amputated limbs using pulsatile hypothermic perfusion with University of Wisconsin solution. A preliminary study.

To determine whether pulsatile hypothermic perfusion with University of Wisconsin preservation solution is superior to topical cooling as a method for the preservation of amputated limbs, six pairs of amputated canine limbs were preserved for twelve to fifteen hours. One limb of each pair was subjected to topical cooling and the other, to pulsatile hypothermic perfusion with University of Wisconsin solution. The bioenergetic status of the limbs was monitored by 31phosphorus magnetic-resonance spectroscopy, and histological evaluation was performed to assess ischemic changes in the preserved tissue. The pH and tissue levels of adenosine triphosphate declined three times more slowly in the limbs that were preserved by pulsatile hypothermic perfusion than in the topically cooled limbs. Consistent with these findings, the perfused limbs also had less histological evidence of ischemic injury. The data from this in vitro study show that pulsatile hypothermic perfusion with University of Wisconsin solution, in combination with an optimum degree of topical cooling, is superior to topical cooling alone as a method of preserving the bioenergetic status of amputated limbs.     

Investigation of metabolism and perfusion in arterialized venous replantation: experimental study in rabbits.

Replantation of amputated digits at distal levels is difficult because the vessels are either too small or absent in the amputated part. Clinically unconventional anastomoses have been tried in such instances. Arterialization of the venous system in the amputated part was also utilized for this purpose. In this study, a digital replantation model utilizing the amputated rabbit ear was used to evaluate the survival, metabolism, and perfusion of unconventionally replanted parts. Mean survival areas were measured and perfusion studies with technetium 99m, blood gases, pH, blood glucose and lactate levels, tissue glucose and lactate levels, and histological evaluations were performed. Surviving areas did not reveal a significant difference. Perfusion of the arterialized venous replants were not as good as the conventionally replanted ears. The blood glucose and lactate levels in the afferent vein were initially near normal arterial levels but almost reached normal venous levels in the samples taken 24 hours after the arterialized venous replantation. Tissue glucose levels were lower than the conventionally perfused tissue, and the lactate levels were higher, but these two metabolite levels were normalized in later samples. The results of the metabolic and perfusion studies are interpreted to elucidate the survival mechanisms in the arterialized venous replants. As a result, arterialized venous replantation may be supported by the favorable results of metabolic and perfusion studies in our experimental model.     

Comparative study of electric excitability and metabolism in the assessment of vitality of the ischemized muscles of the amputated limb before replantation

The authors have studied the dynamic changes of metabolic processes and the functional and the structural preservation of the muscles under the influence of heat ischemia (18-21 degrees C) on 29 amputated posterior extremities of 21 adult dogs of the both sexes. It has been shown that direct muscle excitability disappears 5 hours after the influence of heat ischemia, although after 6 hours of observation the values of the metabolic processes in the muscles made 35-40% of the basic level. The character of the changes in the muscle excitability gives an idea of the duration of action of heat ischemia and the preservation of vitality of the amputated extremity only up to the moment of its complete disappearance. The final irreversible ultrastructural lesions of the overwhelming majority of the myocytes appear during the period of 6 to 8 hours of action of heat ischemia.     

An experimental study on the significance of muscle tissue interposition on fracture healing.

Ten ulnae of mongrel dogs were osteotomized and a 3 to 5 mm cylindrical segment of bone removed from each shaft. In 10 ulnae of the opposite forelimb, living muscle tissue was placed in the resection site. The control ulnae (Set 1, Set 2) revealed 60 per cent unions, while the experimental ulnae revealed 100 per cent non-unions as determined by radiological and histological examination. Although necrotic muscle will be invaded by primitive bone, healing is certainly delayed or does not occur. A large muscle mass contributes a barrier too large to be successfully incorporated into the healing process of a fracture of the shaft of a long bone. On the basis of this experiment fractures should be opened and obstructing tissue removed whenever conservative measures fail to remove muscle interposition.     

Single lung allotransplantation in pigs. A morphologic study of tissue preservation with modified Euro-Collins and fluorocarbon solutions

In the present study the functional and morphologic effects of two pulmoplegic solutions are evaluated. Single left-lung allotransplantation with ligation of the right pulmonary artery was performed in 15 piglets (13-20 kg). The lungs were preserved after donor prostaglandin E-1 treatment with single pulmonary artery flush with either modified Euro-Collins solution (mECS) (9 pigs) or oxygenated fluorocarbon emulsion (FC-43) (6 pigs) and transplanted after 6-hr storage in cold Physiosol solution. Tidal volumes of 15 ml/kg x fr (18) with 40% inspired oxygen were used for ventilation during reperfusion. Function of the transplanted lung was monitored for 4 hr postoperatively by determining pa CO2 and pa O2 levels from arterial samples and by noninvasive monitoring of end-tidal CO2 values and arterial oxygen saturations. Sequential morphologic changes in pulmonary artery flow surface and lung tissue were studied after 6-hr storage and 4-hr reperfusion, using light, scanning, and transmission electron microscopy (LM, SEM, TEM). There was no mortality. After transplantation the mECS group experienced significant hypoxia and hypercarbia and had low end-tidal CO2 values as signs of defective oxygenation and gas exchange, whereas the FC-43 group was normoxic and normoventilated without disturbed elimination of carbon dioxide. After storage and reperfusion, LM showed signs of increased vascular permeability and reperfusion damage--more evident in the mECS group compared with the FC-43 group--while the lymphoid cell population was more intensely activated in the latter group. Electron microscopy after storage showed good overall preservation of structures in both groups. After reperfusion preservation of pulmonary artery flow surface and lung tissue was estimated to be moderate in the mECS group, whereas it was good-to-moderate in the FC-43 group by SEM (NS). TEM of lung tissue, however, showed significantly better-preserved alveolar epithelial lining in the FC-43 group compared with the mECS group. In conclusion, oxygenated fluorocarbon (FC-43) pulmoplegia gave better functional and morphologic preservation of lung grafts compared with modified Euro-Collins solution.     

Effect of addition of magnesium to potassium-containing fluorocarbon cardioplegic preparations. An experimental study

The effect of magnesium on potassium fluorocarbon cardioplegia was studied in 20 rabbits. Isolated hearts with ascending aortas were perfused with oxygenated and cooled (+4 degrees C) fluorocarbon solutions. In 10 experiments, the solution contained 20 mmol/l of potassium, in 10 further experiments, 20 mmol/l of potassium and 15 mmol/l of magnesium. Ultrastructural preservation of the myocardium was studied using transmission electron microscopy. In addition, the cation contents of the myocardium were studied by flame atomic absorption spectrophotometry (calcium, magnesium) or electrothermal atomic absorption spectrophotometry (copper). After 120 minutes of ischaemia, areas of moderate or severe ultrastructural myocardial cell damage were observed in both groups but severe damage was more extensive and commoner in the group with no access to magnesium. Preservation of the capillary endothelium was also worse in that group. There was a highly significant increase in magnesium content, a decrease in calcium content and an increase in the magnesium/calcium ratio following magnesium-potassium fluorocarbon infusion. The copper content remained unchanged. A protective effect of magnesium was observed. The electrolyte content of cardioplegic solutions also alters the electrolyte content of the myocardium. This may be important in reperfusion.     

Retrograde flush perfusion with low-potassium solutions for improvement of experimental pulmonary preservation.

BACKGROUND: Optimal preservation of post-ischemic organ function is a continuing challenge in clinical lung transplantation. Retrograde instillation of preservation solutions has the theoretic advantage of achieving homogeneous distribution in the lung because of perfusing both the pulmonary and the bronchial circulation. So far, we have seen no experimental studies that include stereologic analysis of intrapulmonary edema concerning the influence of retrograde preservation on post-ischemic lung function after preservation with Perfadex and Celsior. METHODS: In an extracorporeal rat model, we perfused 8 lungs, each, using either antegrade or retrograde perfusion technique with Celsior (CE(ant)/CE(ret)) and Perfadex (PER(ant)/PER(ret)). Results were compared with low-potassium Euro-Collins. Post-ischemic lungs were reventilated and reperfused mechanically. We continuously monitored relative oxygenation capacity (ROC), pulmonary artery pressure, flush time, and wet/dry ratio. Furthermore, we used stereologic means to evaluate edema formation. Statistics comprised different analysis of variance models. RESULTS: Relative oxygen capacity of CE(ant)-protected lungs was superior to that of PER(ant) preservation (p = 0.05). Use of PER(ret) resulted in significantly higher ROC as compared with PER(ant) (p < 0.001) and was comparable to results obtained with CE-preservation, which was not further improved with retrograde application. CONCLUSIONS: Celsior provides better lung preservation than does Perfadex when administered antegradely. Retrograde application of Perfadex results in significant functional improvement as compared with antegrade perfusion, which reaches the standard of Celsior-protected organs. Additional in vivo experiments in combination with ultrastructural analysis are warranted to further evaluate retrograde delivery of preservation solutions, which could be used in clinical lung transplantation to further optimize current results.     

Experimental study on myocardial preservation by perfluorochemical "comparison of myocardial preservation effect between continuous perfusion and intermittent perfusion"

The effect of myocardial preservation with perfluorochemical as cardioplegic solution was studied with isolated canine hearts which was compared between intermittent coronary perfusion and continuous coronary perfusion. Intermittent perfusion group (group I) was infused every 30 minutes during 5 hours ischemia with oxygenated perfluorochemical at the amount of 10 ml/kg. Continuous perfusion group (group II) was infused continuously at the amount of 10 ml/kg/30 minutes. After 5 hours of ischemic time, total perfusion volume of both group were same 100 ml/kg. The comparison of myocardial preservation effect between group I and group II was examined with biochemical study, hemodynamic study and histological study. As a result, biochemical study such as GOT, CPK, and Lactate showed higher in group II than in group I, and value of catecholamine and adenylate levels in myocardial tissue showed higher in group I than in group II. In hemodynamic study, LVSW and LVEDP showed excellent value in group I, but never showed adequate function in group II at late working phase. On the other hand, LVmax dp/dt was recovered excellently in group I but in group II was not recovered at early working phase. In histological findings with electronic microscopy, there were some limited ischemic lesion in group II, which was suggested disturbance of micro circulation. It may be attributable to low perfusion pressure of continuous perfusion method. Finally, with regard to SOD (Super oxide dismutase) consumption, group I took higher than group II, and also oxygen consumption. It shows that in group I there is an effective activity of aerobic metabolism during ischemia, which explain not only the improved functional recovery but also generation of free radical, caused by super oxide etc. It is concluded from these results that intermittent perfusion has provided excellent preservation against myocardial ischemia, and also has possibility of danger to set up reperfusion injury.