Successful five-day perfusion preservation of the canine kidney.

Over 20 years ago, successful 3-day-perfusion preservation of canine kidneys was obtained. Since then, consistent 5-day preservation has not been reported. In this study, we investigated how the perfusate calcium concentration affected both mitochondrial function and posttransplant viability in dog kidneys preserved for 5 days. Dog kidneys were preserved by machine perfusion (5 degrees C) using a hydroxyethyl starch-gluconate solution that contained either 0.0, 0.5, 1.5, or 5.0 mM calcium. Mitochondria isolated from preserved kidneys has a loss of respiratory control when either 0.0, 1.5, or 5.0 mM calcium were present. However, the use of a perfusate with 0.5 mM calcium preserved the mitochondrial function at levels equivalent to controls for 5 days. Transplantation of kidneys preserved for 5 days with 0.0 or 1.5 mM calcium yielded poor survival (0% and 17%, respectively). The use of a 0.5-mM calcium perfusate increased posttransplant survival to 63% (5 of 8 transplanted). Donor pretreatment of kidneys with chlorpromazine (2.5 mg/kg i.v.) did not improve the function of mitochondria isolated from preserved kidneys but did increase survival in the 1.5-mM calcium group to 67% (4 of 6 transplanted) and in the 0.5 mM calcium group to 100% (7 of 7 transplanted). This is the first report to document consistently successful 5-day preservation of canine kidneys and clearly shows the importance of the perfusate calcium concentration in long-term kidney preservation. The specific mechanism by which calcium or chlorpromazine exert their effect is not known, but it is apparent that excessively high or low concentrations of calcium are damaging to the preserved organ, and an optimal calcium concentration combined with metabolic inhibition of calcium-dependent pathways can significantly improve the function of organs preserved for extended time periods.

Successful 72-hour cold storage of dog kidneys with UW solution.

Effects of three cold-storage solutions on kidney function in dogs were examined with the isolated perfused (IPK) kidney model and the autotransplant model. EuroCollins' (EC) solution, phosphate-buffered sucrose solution, and a new solution developed at the University of Wisconsin (UW) were studied. Kidneys were cold-stored for 48 hr or 72 hr. With the IPK model, cold storage for 48 hr or 72 hr in each of the three solutions caused creatinine clearance to decrease by 80%-90%. More protein was excreted by kidneys stored for 48 hr in PBS solution than by kidneys stored in EC or UW solution; protein excretion after 72 hr of storage was similar for kidneys stored in EC or UW solution. Sodium reabsorption decreased after 48 hr or 72 hr of storage, but was higher in kidneys stored in UW solution (83% and 56%, respectively) than in EC solution (52% and 22%, respectively). With the autotransplant model, 40% of the kidneys were viable after 48-hr storage in PBS solution, but 80% viable when stored in EC solution and 100% were viable when stored in UW solution. All kidneys were viable when stored for 72 hr in UW solution; none were viable when stored for 72 hr in EC solution. These results suggest that UW solution effectively preserves kidneys for 72 hr. We previously reported successful 72-hr pancreas preservation. Recently UW solution was able to preserve canine livers for 30 hr. Thus, this single solution appears to be effective for preserving all intraabdominal organs and may simplify cold storage of organs for transplantation.

Preservation of dog liver, kidney, and pancreas using the Belzer-UW solution with a high-sodium and low-potassium content.

The UW solution developed for cold storage of the liver, pancreas, and kidney was used in a modified form in this study and tested in the orthotopic transplantation of dog livers, kidneys, and pancreases preserved for 48 hr. The modification was the alteration of the concentrations of potassium and sodium. The original UW solution contained 120 mM K+ and 30 mM Na+. In this study the Na+ was 140 mM and the K+ only 9 mM, all other agents were identical to the original UW solution. Six of 11 dogs survived with livers preserved for 48 hr. The five deaths were due to technical complications and unrelated to preservation failure. Postoperative AST and partial thromboplastin time (PTT) values were lower (statistically significant on days 1, 3, and 4) in livers preserved in the high Na+ UW solution than as previously shown in the high-k+ UW solution. Other measures of liver function (bilirubin and fibrinogen) were similar between the high-Na+ and high-K+ groups. Six dogs survived with kidneys preserved for 48 hr in the high-Na+ UW solution. The results were comparable to those obtained with the high K+ solution. Four of six dogs survived for up to 28 days with pancreases preserved for 48 hr. The two deaths were due to technical complications unrelated to preservation failure. Three of the four dogs had normal blood glucose values for one month, and intravenous glucose tolerances test on day 7 and 28 were identical to those obtained in pancreases preserved with the high-K+ UW solution. The high-Na+ version of the UW solution appears equally or slightly more effective for 48-hr organ preservation than the original high-K+ UW solution. The use of a high-Na+ UW solution reduces the problems of hyperkalemic cardiac arrest in in situ flushing of the donor for multiple organ harvesting and in transplantation of the liver. Thus, with this solution livers do not need to be flushed with a low K+-containing solution prior to transplantation.

Prevention of postoperative intestinal intussusception by prophylactic morphine administration in dogs used for organ transplantation research.

In this report 149 kidney transplants were performed as part of experiments on renal perfusion preservation, and the effect of morphine treatment on the incidence of postoperative intussusception was concurrently evaluated. Intestinal intussusception developed in 17% (14 of 83 dogs transplanted) of untreated dogs after transplantation. Intraoperative intravenous administration of morphine (0.5 mg/kg) resulted in a reduction in the rate of intussusception to 3.3% (2 of 61 dogs transplanted). Postoperative administration of morphine was ineffective in reducing the incidence of intussusception. The results indicate that intraoperative administration of morphine is effective in preventing postoperative intussusception in dogs used for experimental transplantation and may reduce animal losses and research costs in experimental surgical laboratories.

Oxygen-derived free radical damage in organ preservation: activity of superoxide dismutase and xanthine oxidase.

It has been suggested that the generation of oxygen-derived free radicals during the reperfusion of injured tissue causes organ dysfunction. The mechanism apparently involves the xanthine oxidase (XOD) enzyme system, which becomes activated during reperfusion and generates oxygen-derived free radicals faster than their dismutation by naturally occurring superoxide dismutase (SOD). In this study, we measured the XOD and SOD activities of liver and kidney tissue in species used for organ preservation studies (rats, dogs, and human beings). We also measured the effect that up to 5 days of hypothermic preservation has on the activity of SOD and XOD in canine kidneys and expressed activities as the ratio of SOD to XOD; large ratios suggest lower susceptibility to oxygen-derived free radical damage. The SOD:XOD ratios for rat tissue were consistently lower (kidney = 2.7 X 10(4), liver = 8.9 X 10(3)) than for canine tissue (kidney = 7.0 X 10(5), liver = 4.1 X 10(4)) and human tissue (kidney = 1.1 X 10(6), liver = 6.4 X 10(5)). Canine kidneys perfused for 3 days showed no change in SOD:XOD ratio. After 5 days of perfusion, the SOD:XOD ratio decreased by 50% but was still quite large (5 X 10(5]. This high SOD:XOD ratio in canine and human tissues suggests that they may be less sensitive to oxygen-derived free radical damage than rat tissues.

Effect of pharmacologic agents on the function of the hypothermically preserved dog kidney during normothermic reperfusion.

We examined how a combination of pharmacologic agents ("rescue" agents) affect the function of hypothermically preserved dog kidneys at the time of reperfusion. Dog kidneys were preserved either by simple cold storage in EuroCollins' solution for 24 or 48 hours or by continuous perfusion at 5 degrees C in Belzer's gluconate-hydroxyethyl starch solution for as long as 5 days. After preservation, renal functions were measured with the isolated perfused kidney model. Kidneys were reperfused at normothermia either with or without the addition of a combination of rescue agents to the reperfusion medium. The rescue agents studied were allopurinol (1 mmol/L); superoxide dismutase (32,000 U/L); catalase (137,500 U/L); dimethylthiourea (3 mmol/L); glutathione (3 mmol/L); desferrioxamine (0.2 gm/L), for protection against O2 free radical injury and lipid peroxidation injury; verapamil (25 mg/L), as a Ca channel blocker; and ATP-MgCl2 (0.3 mmol/L), to stimulate energy metabolism. The renal functions we measured were glomerular filtration rate (GFR) (creatinine clearance), urine production, perfusate flow, urinary protein concentration, Na reabsorptive capacity, and tissue concentrations of ATP, K, and total tissue water. GFR was reduced by 75% to 90% after all periods of preservation, and the rescue agents had no effect on GFR. Sodium reabsorption was reduced from 98% to a range of 40% to 50% after 48 hours of cold storage or 5 days of machine perfusion and was not increased by rescue agents. There was a time-dependent increase in the amount of urine protein that was not affected by rescue agents. The addition of rescue agents did not affect total tissue water or concentrations of ATP or K in kidneys after normothermic reperfusion. These results demonstrate that pharmacologic agents previously suggested to suppress reperfusion damage in kidneys are not effective in this model. Therefore it is likely that kidneys damage occurs primarily during preservation, which suggest that optimal function on reperfusion calls for the development of better methods of preservation.

Effect of various short-term storage methods on viability of cancellous bone fragments.

OBJECTIVE: To determine effects of various storage methods on ex vivo viability of cancellous bone fragments. SAMPLE POPULATION: Cancellous bone fragments obtained from 4 New Zealand White rabbits. PROCEDURE: Cancellous bone fragments were stored for 3 hours on ice in 1 of 5 preservation solutions or in 0.9% NaCl. Fragments were then reperfused (37 C) in oxygenated physiologic buffer solution for 1 hour. Cellular viability in fragments was assessed by ethidium monoazide labeling and fluorescence microscopy. RESULTS: Viability in fresh cancellous bone ranged from 85 to 100% (mean+/-SEM, 88+/-7%). Storage of fragments significantly reduced viability. Viability in bone fragments stored at 22 C in blood-soaked sponges or 0.9% NaCl solution was 63.8+/-3 and 65.2+/-7%, respectively. Use of cold 0.9% NaCI solution reduced viability to 53.6+/-3%. Viability was significantly better for fragments stored in cold phosphate-buffered sucrose (70.4+/-2%) and EuroCollins (71.4+/-3%) solutions. After warm reperfusion, viability was best for fragments stored in cold phosphate-buffered sucrose (70.2+/-4%), EuroCollins (72.6+/-3%), or UW lactobionate solutions (69.6+/-3%), compared with those stored in cold 0.9% NaCl (47.6+/-3%) or hypertonic citrate (53.0+/-3%) solutions or blood-soaked sponges (57.2+/-3%). CONCLUSIONS: Hypothermic storage in solutions designed to prevent temperature-dependent cell injury were best for maintaining cancellous bone fragment viability. CLINICAL RELEVANCE: Hypothermia may be advantageous for use in storing cancellous bone fragments during procedures that dictate a prolonged period between harvest and placement of graft fragments.

Thrombocytopenia associated with vaccination of a dog with a modified-live paramyxovirus vaccine.

Thrombocytopenia (10,000/mm3), with hematochezia and melena, appeared in a dog 8 days after it was given modified-live canine distemper, virus vaccine and persisted for approximately 5 days. Clinical investigation discounted other possible causes of thrombocytopenia; the condition was considered to be associated with vaccination. The problem spontaneously resolved. The appearance of thrombocytopenia after modified-live canine distemper virus vaccination is not unknown and may assume a severe form. This condition may be mistaken for idiopathic thrombocytopenia of immune origin, and in other instances, it may contribute significantly to surgical risk if concurrent coagulation disorders are present. Administration of levamisole HCl may alleviate the decrease in platelet count in affected animals.

Repair of pectus excavatum by percutaneous suturing and temporary external coaptation in a kitten.

Severe pectus excavatum sternal deformity in a 3-month-old kitten was repaired by use of percutaneous circumcostal and circumsternal sutures to coapt the sternal deformity to an external splint made from thermoplastic material. One year after treatment, the kitten remained free of clinical signs of the disease or complications from the repair. The technique represents a conservative method of repair of pectus excavatum deformities in immature animals.

Nephron sparing by partial median nephrectomy for treatment of renal hemangioma in a dog.

A 6-year-old neutered male Golden Retriever was admitted for evaluation of intermittent hematuria of 2 months' duration. A 3-cm heterogeneous mass causing distortion of the caudomedial aspect of the left kidney was detected via ultrasonography. Histologic examination of a renal tissue sample obtained by ultrasound-guided biopsy revealed a telangiectatic vascular plexus of unknown origin. Low glomerular filtration rate was identified by a modified exogenous creatinine clearance test. Excretory urography revealed a filling defect in the medial aspect of the caudal pole of the kidney, near the hilus. Because total renal function was low, a decision was made to perform nephron-sparing surgery involving resection of centrally located renal parenchymal and pelvic tissue by en bloc resection in the median plane, instead of radical nephrectomy. After surgery, the hematuria resolved and further decrease in renal function was not evident. Nephron-sparing surgery is a viable option for dogs with compromised renal function when there is concern that radical nephrectomy may precipitate uremia.