The Protective Effect Of Kidney During The Early Warm Ischemic Time Using Different Hypothermal Methods

ObjectiveLaparoscopic kidney surgery are usually performed under the condition of renal pedicle occluded,because of warm ischemic injury,the renal function is decreased inevitably. So it is important to use protection in operation process , such as hypothermia, to decline the injury. Although many kinds of hypothermia way are in use,there still lack a large number of clinlical cases and expenrimental data.so we can not evaluate the hypothermia way definitely. The objective of our experiment is to investigate and compare the effect of the temperature,enzyme activity and histopathology in renal tissues under different kinds of hypothermia way.To do so, a pedicle occluded rats model in vitro was constructed. We can find the appropriate way and time during the early warm ischemic time and give a right choice in clinic use.Material and methods1. Rats model preparation120 adult male Sprague-Dawley rats weighing 300-400g randomized to four treatment groups,named A,B,C,D, were evaluated. The animals were anesthetizedby phenobarbital sodium 50mg/kg by intraperitoneal injection.A "T" shape abdominal incision was made. The left ureter,renal pedicle and renal itself were isolated. After intravenous heparin injection,the four different treatment were performed: simply occluded the renal pedicle, renal surface cooling retrograde ureteral access perfusion and incontinuous renal artery perfusion. We divided into 5 groups,each of them include 6 rats. Removed the same left kidney.2.Assessment of renal temperatureThe renal temperature, all of three kinds of hypothermia methods and control were detected by a spiculate thermocouple thermometer, both parenchyma (3mm underneath the surface) and core medulla. 3 points and 3 times were randomized detected, using general average to evaluate the hypothermia effect.3.Measurement of enzyme activityWe turned a small piece of renal tissue into 10% homogenate in a glass homogenizer, using the kit from Nanjing Jiancheng Bioengineering Institute. According to the step of the instruction,we measured the activity of Na+-K+ ATPase^ Ca2+ ATPase> LDH and GSH-px in renal tissues. To quantify the protein level in renal tissue(Coomassie Brilliant Blue),we use another kit bought from the same company.4.Observation of histopathological changeThe sample of different time points from renal parenchyma in 4 groups were made into tissue slice.After ordinary HE dyeing,we used light microscope to observe the pathological change of renal tissue and give a Paller's score of renal tubule and compared.Result1. Effect of renal temperatureRenal surface cooling is the best hypothermia way on renal cortex,otherwise, retrograde ureteral access perfusion is best on medulla, renal artery perfusion can uniformly reduce the whole renal temperature. 2.Result of enzyme activity changeThe enzyme activity changed obviously after 15min simple warm ischemia.The activity of Na+-K+ATPase > Ca2+ ATPase and GSH-px reduced gradually,while LDH increased with the time continue. Compared with simple warm ischemia rats model(A group), the trend of activity change in all treament groups were the same as A group,but decrease the trend. At 30min,the artery perfusion team (D group) show no statistics difference with the beginning while other two teams changed significantly. At the 45th minute point, all enzyme except Ca2+ ATPase in group D show notable statistics difference with A, B or C group(p<0.05).Any enzyme between B and C group show no statistics differenence.If exceed 45min,all treament groups changed significantly and show no statistics differenence among them. 3.Result of histopathological changeUnder the optical microscope,we find whatever hyperaemia of tissue, protein cast and endothelial cell degeneration,all the treament groups are more serious than control group,and grow serious with time continue.At the same time, we find that all hypothermia groups are better than A group at the same time pointD group is better than B or C group after 30min.At 60thminute,group D is better than B or C on tissue change,but show no statistics difference on Paller's score of renal tubule, compared with other controlled group.Conclusion:1. The activity of Na+-K+ ATPase, Ca2+ ATPase ^ LDH and GSH-px can reflect the function change in the renal tissue and can be evaluated the factor contribute tokidney.2. The renal tissue begin to injury 15min after renal pedicle occluded. The main manifestation is the activity change of tissue enzyme and histopathology. The injury gradually become worse with the time continue.3. The three hypothermia methods can reduce the tissue injury because of warm ischemia and anoxemia ,which can protect renal function within 30min perfectly.4. The renal hypothermia achieved by renal artery perfusion is much better than ice slush or retrograde ureteral access,which can be effectively and enduringly applied to protect the renal function in surgery. But if exceed 60min,all treament groups changed significantly.5. The shortage of this experiment need to go step to compare the change of renal tissue after reperfusion because the clinical surgery be done in vivo and the clamp occluded renal pedicle must be released at last, the reperfusion is inevitable.So simply compare the change of early warm ischemia time is not complete and the compare index is unilateral.The conclusion could be entirely accepted if we transplant the reperfusion kidney into other rat, observe and compare the long-term effect.