EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Key words : Kidney transplant, Ureterocystoanastomosis, Ureterocystostomy, Ureteroneocystostomy, Urologic complications

Some of the most common problems after kidney transplant are urologic complications, which occur in 2.5% to 30.0% of cases.^1,2 Urine leakage is the most common early urologic complication after renal transplant, with an incidence of 1.2% to 8.9%.³ Early urologic complications include neoureterocysto-anastomosis (NUCA) leakage and stenosis with the development of severe complications, and a strategy is required for the prevention of these complications.^4,5 The NUCA leakage can be caused by insufficient blood supply to the donor ureter, and damage of the so-called golden triangle structures can lead to necrosis of the ureteral distal portion in up to 70% of cases.^2,5,6 Isolated plasma contains active substances that cause the activation of various growth factors for the processes of tissue repair or regeneration, has an anti-inflammatory effect, activates local angiogenesis, and reduces the risk of local infectious complications.^7-9 Platelet-rich plasma (PRP) is actively used for the following purposes: to stimulate bone regeneration, heal wounds and skin ulcers, enhance the reconstruction of the larynx and trachea, treat musculoskeletal and nerve injuries, promote the engraftment of various types of skin flaps, support gastrointestinal anastomoses, and ameliorate urethral damage, among other purposes.^10-16 This study was developed to evaluate the positive effect of PRP on the healing process of an anasto-motic wound in a model of ureterocystostomy (UCS) in rats.

Materials and Methods

Animals and ethics statement

We randomized 14 Wistar albino male rats, weighing 360 to 536 g, into 2 groups: group 1 consisted of 7 rats that received PRP during UCS; group 2 was the control group of 7 rats that underwent UCS without PRP (Table 1). The rats of both groups were kept in the same conditions before and after the surgery, in compliance with the necessary regimens for temperature, light, and food. This study was approved by the Baskent University Ethical Committee for Experimental Research on Animals (project No. E-94603339-604.01.02-38157).

Platelet-rich plasma preparation

Blood was obtained by whole body blood collection from 3 donor rats with ketamine anesthesia (60 mg/kg; VetaKetam, Vet-Agro) through cardiac puncture. A volume of 58 mL of blood contained 276 × 10⁹ platelets/L. All collected blood was placed into tubes with 3.8% sodium citrate. Tubes were centrifuged twice, as described by Tobita and colleagues: 10 minutes at 2400 rpm and 15 minutes at 3600 rpm.¹⁷ After the first centrifugation, a long needle was used to collect the middle layer with leukocytes and thrombocytes and transfer into another tube. After the second centrifugation, the platelet count was 942 × 10⁹ platelets/L, which was 3.4-fold greater than the initial amount. Before PRP in group 1 was used, plasma with concentrated platelets was activated by 10% calcium chloride solution, and a gel-like mass was obtained (Figure 1).

Surgical procedure

All the rats in each group underwent surgery on the same day and by the same surgeon for technical uniformity. After an overnight fast, the rats were anesthetized by intramuscular injection of ketamine (60 mg/kg; VetaKetam) and xylazine (10 mg/kg; VetaXyl, Vet-Agro). The rats were on spontaneous respiration during the surgery. The abdominal wall of each rat was shaved; under aseptic conditions, a 4-cm incision was made along the midline of the abdomen. In both groups, the left ureter was crossed at the level of the confluence with the bladder, and then an anastomosis was performed with single-layer interrupted 8-0 absorbable sutures. In group 1, activated PRP gel was applied to the ureterocys-toanastomosis line. The laparotomy incision was closed with a continuous absorbable 3-0 suture (Figure 2). Postoperative conditions were the same in both groups, with feeding started the next day after surgery. No postoperative analgesics or antibiotics were use. On postoperative day 7, all animals were exsanguinated through percutaneous cardiac puncture under ketamine anesthesia by the method described by Donovan and Brown.¹⁸ The rat abdominal wound was reopened, and the anastomosis zone was carefully dissected and the anastomosis was located. The anastomosis area was resected with a margin of at least 2 cm proximally and distally for determination of the tissue hydroxyproline (THP) levels and histopathology examination.

Tissue hydroxyproline measurement

The excised and purified anastomosis line tissue was cooled, neutralized in potassium hydroxide, and oxidized with alanine and potassium borate buffers with the addition of potassium chloride. A solution of chloramine-T, thiosulfate, and toluene was added and left at room temperature for 20 minutes. After the sample was stained with hematoxylin-eosin, the color and intensity were evaluated spectrophoto-metrically at a wavelength of 560 nm.

Histopathology analysis

For histopathology examination of the resected anastomosis area, tissue was fixed in 10% formalin and embedded in paraffin, and 4-?m sections were stained with hematoxylin-eosin. Epithelial damage, acute inflammation, and fibrosis were determined as parameters of inflammation in the tissue samples. Each parameter was evaluated on a scale of 0 to 3 points: 0 = absence, 1 = mild, 2 = moderate, and 3 = severe.

Statistical analyses

We used SPSS software (version 20.0 for Windows) for statistical analyses; the Mann-Whitney U test was used to compare the variables of the 2 groups with median values. Statistical differences between the 2 groups were considered significant at values of P < .05.

The levels of THP in both groups are shown in Table 2. The mean THP levels were 767 ± 62.9 ?g/g in group 1 and 256 ± 28.0 ?g/g in group 2 (control), and this difference was statistically significant (P < .05). These data indicated a positive effect of PRP application on the NUCA. The results of the histology evaluations are shown in Table 3. There were no significant differences in epithelial damage, acute inflammation, or fibrosis between the tissue samples of group 1 and group 2 (Figure 3).

Ureteral anastomosis leakage remains a serious problem, especially in kidney transplant, and is associated with a high risk of graft failure and recipient mortality.^2,19 Previous studies have investigated and described the risk factors that can affect the healing of the UCS.^20,21 To date, various methods of UCS are in use for kidney transplantation, the most common of which is the Lich-Gregoir method. Some authors claim that the Lich-Gregoir method has an advantage versus other methods by preventing urologic complications in UCS.²² In a similar manner, Szabo-Pap and colleagues did not find a significant difference between the types of urinary anastomosis and the frequency of leakage in kidney transplant recipients.²³ Also, Peng and colleagues have reported that there is no evidence that ureteral stents could prevent the occurrence of ureteral anastomosis leakage.²⁴ The local use of PRP has been mostly limited to studies of intestinal anastomoses and the positive effect of PRP in the healing of intestinal anastomoses.^15,25-28 In the available literature, we did not find any report of local use of PRP to strengthen the urinary or ureteral anastomosis. We found a single experimental study by Tavukcu and colleagues on the use of PRP in urethral trauma.¹⁶ We used double centrifugation in the preparation of PRP, which facilitated an increase in the initial platelet concentration by 3.4-fold, to 942 × 10⁹ platelets/L, and this corresponds to the recommen-dations of Tambella and colleagues.²⁹ In our study, we euthanized the rats and obtained tissue samples for examination on postoperative day 7. Usually in clinical practice, anastomosis leakages are diagnosed during the period from postoperative day 5 to day 7.³⁰ After euthanasia, we did not find any signs of anastomosis leakage or ureteral dilation in the surgical field in the rats. In this study, we found no differences between the 2 groups in terms of histology parameters. However, THP levels in the PRP group showed significantly better results than in the control group. There were some limitations in our study. We used homologous blood from 3 donor rats for PRP preparation. Theoretically, these homologous blood cells could cause the processes of immunologic inflammation in the area of application of PRP. Another limitation is the reimplant of the ureter without prior nephroureterectomy. That is, we did not use the kidney transplant model in these rats, which undoubtedly carries risk factors against the healing of urinary anastomosis in clinical practice. To exclude such influences, in the future we plan to use autologous PRP for ureteral anastomoses in big models. In conclusion, the results of this study showed that the use of PRP during UCS could have a positive effect. Platelet-rich plasma has potential usefulness to prevent leakage of ureterocystoanastomosis after kidney transplant. To confirm this fact, further studies with big experimental models and the use of autologous PRP are necessary.

Volume: 21 Issue: 1 January 2023

FULL TEXT