Indian Journal of Surgery Medknow Publications on behalf of Association of Surgeons of India ISSN: 0972-2068 Vol. 65, Num. 6, 2003, pp. 500-503

Indian Journal of Surgery, Vol. 65, No. 6, November-December, 2003, pp. 500-503

Evaluation of wistar rat model with pre-transplant subcutaneous transposition of spleen (sts) for possible role in improving results of orthotopic liver transplantation

Amit Bhandari, Sunil Kumar, Geeta Dev,* R. L. Gupta

Department of Surgery and *Pathology, UCMS & GTB Hospital, Shahdara, New Delhi, India.
Address for correspondence: Dr Sunil Kumar, D-5, Dilshad Colony. New Delhi - 110095, India.

Paper Received: August 1999. Paper Accepted: July 2002. Source of Support: Nil.

How to cite this article: Bhandari A, Kumar S, Dev G, Gupta RL, Evaluation of wistar rat model with pre-transplant subcutaneous transposition of spleen (sts) for possible role in improving results of orthotopic liver transplantation. Indian J Surg 2003;65:500-3.

Code Number: is03108

ABSTRACT

Orthotopic liver transplantation (OLT) in the rat model remains the animal model of choice for liver transplantation due to the availability of inbred strains, technically simple procedure and the lack of need for immunosuppression. A major limiting factor in the rat liver transplantation is the short anhepatic phase which is poorly tolerated due to ischemia reperfusion injury. Portosystemic shunt induced by subcutaneous transposition of spleen (STS) may help in preventing reperfusion injury. To study this hypothesis 8 rats were subjected to STS which was followed 3 weeks later by portal pedicle clamping for 60 min in 4 rats (STS-60) and 90 min in 4 rats (STS-90). The control group consisted of 8 rats undergoing sham laparotomy by left subcostal incision followed by portal pedicle clamping 3 weeks later for 60 min- 4 rats (SHAM-60) and for 90 min in 4 rats (SHAM-90). These groups were compared on the basis of survival, SGOT levels, intestinal congestion during clamping, collateral formation in the subcutaneous pouch and the histopathological examination of the liver. The ability of the shunt to prevent ischemia reperfusion injury was shown by the survival rate of 100% in STS-60 and STS-90 groups while sham operated groups SHAM-60 and SHAM-90 had survival rates of 25% and 0%, respectively. Intestinal congestion was noted during clamping in the sham operated group but was absent in the STS group. SGOT levels which rose in the first post portal clamping day returned to baseline levels by day 7 in the STS group. Histopathological examination of the livers in STS rats showed maintenance of hepatocellular architecture, absence of sinusoidal dilatation and absence of polymorphonuclear infiltrate. Subcutaneous transposition of spleen can therefore provide an effective portosystemic shunt which is easy to perform. It prolongs the anhepatic phase of rat liver transplantation and can be used to study various factors and mediators involved in ischemia reperfusion injury.

Key words: Liver transplantation, Rat, Subcutaneous transposition of spleen, Ischemia reperfusion injury.

INTRODUCTION

Orthotopic liver transplantation (OLT) in the rat was first described by Lee et al¹ and the modification proposed by Kamada and Calne² greatly simplified the procedure. Rat model has become the standard investigative tool in experimental liver transplantation. Despite several innovations, it remains perhaps the most difficult experimental microsurgical transplant to perform due the short an hepatic phase of 26 min². This phase is characterized by a combination of intestinal congestion and hypovolemic shock during clamping, and acidosis and endotoxemia on removal of the clamp. This is ischemia reperfusion injury and this causes liver damage which is poorly tolerated by rats. A portosystemic shunt may prevent these ill effects of portal clamp release by diverting splanchnic blood from liver. Subcutaneous transposition of the spleen STS was first performed by Bengmark et al³ as an experimental study to induce portosystemic collateral formation for decompressing the portal system in portal hypertension. The purpose of the study was to test the ability of STS to provide effective decompression of the portal system during prolonged portal pedicle clamping as occurs in OLT. This can increase the anhepatic phase of OLT in rats which can prove useful for beginners in the surgical learning phase of transplantation. The biochemical and physiological changes occurring during prolonged portal vein clamping have also been studied as these factors are responsible for ischemia reperfusion injury.

MATERIAL AND METHODS

This prospective animal study was conducted in the Department of Surgery, UCMS & GTB Hospital on 16 male Wistar rats weighing between 150-400 gms. Rats were cared for in controlled conditions of temperature and light/dark cycles with rat feed and water freely available in their cages pre and post operatively. 8 rats underwent a sham laparotomy by a left subcostal incision while subcutaneous transposition of spleen (STS) was carried out in 8 rats. Three weeks later, four of the sham operated rats underwent portal vein clamping for 60 minutes (SHAM-60 group) while four rats underwent portal vein clamping for 90 minutes (SHAM-90 group). Similarly, rats subjected to STS underwent portal vein clamping of 60 minutes in four rats (STS-60 group) and clamping 90 minutes in four rats (STS-90 group).

All procedures were carried out under inhalational ether anaesthesia. Ether was delivered using open drop method through a face mask. The plane of anaesthesia was controlled by varying the distance of face mask from the nostrils. Sham laparotomy comprised giving a left subcostal incision of about 2 cm length and subsequent closure in layer. STS was carried out by making a similar left subcostal incision and creating a subcutaneous pouch by blunt and sharp dissection. Spleen was delivered into this subcutaneous pouch and muscle borders were approximated by taking a vicryl 4-0 stitch around the splenic vessel to prevent retraction of the spleen. Three weeks later, portal pedicle clamping was performed through a midline laparotomy incision in the SHAM and STS group. The hepatic artery and portal vein were defined and clamped separately. Degree of intestinal discoloration during clamping was noted at this stage. Blood samples for estimation of SGOT levels were drawn after excising the tail under anesthesia and collecting blood from the tail vein at Day 0, Day1 and Day 7. All samples were centrifuged immediately and sera preserved at 4^o C. The standard kinetic method of enzyme assay was utilised for SGOT estimation in all cases.

All the rats dying in the first seven days of the post operative period were subjected to necropsy and liver preserved in formalin for biopsy. Half the number of rats in each group surviving at Day 7 was sacrificed by exsanguination of the abdominal aorta under ether anaesthesia and liver preserved. Haemotoxylin and eosin (H&E) stained sections fixed in paraffin were used for histopathological examination.

The statistical analysis of the SGOT levels in the STS groups was carried out using the unpaired Student's T test. The level of significance was taken as P<0.005.

RESULTS

The ability of the portosystemic shunt induced by STS was tested by portal pedicle clamping for extended periods over the one normally tolerated. Sham operated rats subjected to 60 minutes clamping time had survival rate of 25% 1 out of 4 while none of the rats subjected to 90 minutes clamping survived beyond 8 hours in the post operative period. In contrast, STS rats submitted to 60 and 90 minutes clamping rapidly recovered from the procedure and 100% survival at Day 7 was noted.

SGOT levels in the single surviving rat in the Sham group returned to normal levels at Day 7 after an initial rise. In the STS 60 group SGOT levels rose to 664 +/- 228 IU/litre Day 1 post clamping and returned to baseline levels, 80 +/- 22 IU/litre at Day 7. SGOT levels in STS 90 group rose to 867 +/- 259 IU/litre at Day 1 and returned to normal at Day 7 106 +/- IU/litre. The changes in SGOT levels were statistically significant in the STS 60 group at Day 1 and Day 7, and in the STS 90 group at Day 7 (Table 1)

Congestion of the bowel noted during clamping as noted by the bluish discoloration of gut in the Sham group was absent in the STS group. Well formed collaterals around spleen were observed in the subcutaneous pouch in the STS group. The livers subjected to histopathological examination were reviewed by the pathologist unaware of the group involved. The H&E slides belonging to the Sham group showed areas of necrosis (Figure 1), areas of sinusoidal dilation (Figure 2) prominent polymorphonuclear infiltration and prominent Kupffer cells (Figure 3). These features were suggestive of an endotoxin induced injury with activation of the reticuloendothelial system. On the other hand, livers of both STS 60 and STS 90 groups had better preserved hepatocellular architecture, minimal polymorphonuclear infiltrate, absent venous congestion and Kupffer cells were unremarkable (Figure 4).

DISCUSSION

The subject of ischemia reperfusion injury and its decisive role in graft survival and organ dysfunction has been extensively researched in the past decade. The basis of reperfusion injury is the venous stasis in the gut and subsequent translocation of bacteria. The various factors involved in the cell injury which have been studied are endotoxins, superoxide radicals, interleukins, potassium and acidic pH.^4-6 In an attempt to increase the anhepatic phase, researchers have focused on modifying the local environment of the gut like using preoperative antibiotics for bowel decontamination, intravenous infusions of prostaglandins PGE₁ for maintaining cell integrity and metabolism under hypoxia and using nitrogen saturated buffers to decrease superoxide radical formation. Research incorporating models with and without portosystemic shunt can be used for a quantitative analysis of reperfusion injury.⁷ These shunts can be created by using an extracorporeal shunt: portocaval, jugulocaval or subcutaneous transposition of spleen (STS).

Of the various methods of creating portosystemic shunt, STS is performed with ease, has minimal morbidity and alteration of the physiological system. STS provides effective portosystemic decompression as can be seen by the results of our study. The mortality in the STS-90 group is 0% compared to 100% in the SHAM-90 group. The liver biopsies of SHAM group rats showed disruption of the hepatocellular architecture, polymorphonuclear infiltration and sinusoidal dilatation while these changes were absent in the STS group. The liver functions as quantified by SGOT levels returned to baseline levels by the 7^th post-clamping day in the STS group.

While studying the effect of a portosystemic shunt in preventing warm ischemia perfusion injury it is also important to consider the effect of cold preservation ischemia and the possible `steal' phenomenon through the shunt after vascular anastomoses. In addition, experimental data showing benefits of portosystemic shunt on preserved grafts should be considered cautiously when applied to venovenous bypass in the clinical setting as a variety of physiological manipulations are used during liver transplantation to minimise adverse ischemia reperfusion squealae of acidosis and hypertension.

STS can play an important role in studies focused on ischemia reperfusion injury. The effective portosystemic shunt and the ease of performing this procedure are the two important factors which may make it the experimental model of choice amongst researchers.

REFERENCES

1. Lee S, Charter AC, Orloff MJ. Simplified technique for Orthotopic liver transplantation in the rat. Am J Surg 1975;130:38-40.

2. Kamada N, Calne RY. Orthotopic liver transplantation in the rat - Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 1979;28:47-50.

3. Bengmark S, Borjesson B, Olin T. Development of Portosystemic shunt after subcutaneous transposition of the spleen in the rat. Am J Surg 1973;125:757-62.

4. Delriviere, L, Kamada, N, Kobayashi, E, Enosawa S, Goto S. Portosystemic Shunt for Orthotopic Liver Transplantation in the Rat. J Surg Res 1994;56;457-60.

5. Omokawa S, Arai Y, Saito H, Faruya T, Sato T, Sato T, et al. A simple experimental model of Total hepatectomy, Hepatic ischemic and extrahepatic portal Obstruction in rats using splenic transposition. Jpn J Surg 1991;21:50-6.

6. Miyata T, Todo S, Imventarza O, Ueda Y, Furukawa H, Starzl TE. Endogenous Endotoxemia during Orthotopic Liver Transplantation in Dogs. Transplant Proc 1989;21:3861-2.

7. Sankary HN, Yin DP, Chong AS, Ma LL, Shen J, Foster P et al. The portosystemic shunt protects liver against ischemic repefusion injury. Transplantation 1999;68:958-63.

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