Rat Model Of Intestinal Transplantation Research Articles

Increased NPC1L1 and serum cholesterol in a chronic rejection rat

To measure serum cholesterol and triglyceride levels and NPC1L1 mRNA and protein as an index of cholesterol absorption during the development of chronic rejection (CR) in a rat model of intestinal transplantation. Rats were randomly divided into two groups: Group 1 (n=20) underwent syngenic Lewis-to-Lewis transplantation and Group 2 (n=20) underwent allogenic F344-to-Lewis transplantation as well as treatment with FK506. Blood samples and intestinal tissue were procured on the 190th day after operation. Histological changes were analyzed and the semiquantitative scores of histological parameters were compared. The serum levels of cholesterol and triglyceride were determined. The expression of Niemann-Pick C1 Like 1(NPC1L1) mRNA and protein were analyzed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and immunohistochemistry, respectively. All the animals survived for the 190 days. The appearance and histology of isografts were almost normal whereas the allografts displayed thickened bowel wall and mesenteric fibrosis, concentric intimal thickening and interstitial fibrosis and inflammatory infiltration. The histology scores displayed a significant difference between the allografts and isografts (P < 0.001). No differences were observed for triglycerides for the two groups. The serum cholesterol levels increased significantly in the allogenic group in comparison with the syngenic group (P=0.034), while no difference was observed for triglyceride levels between groups. RT-PCR showed that the expression of NPC1L1 of allografts increased significantly (P=0.004). Immunohistochemistry confirmed RT-PCR findings. Neointima formation and mesenteric fibrosis were the dominant pathological features. The increased expression of NPC1L1 might contribute to hypercholesterolemia, which may be involved in the pathogenesis of transplant arteriosclerosis.

Short-Term Anti-CD4 Plus Anti-TNF-α Receptor Treatment in Allogeneic Small Bowel Transplantation Results in Long-Term Survival

Despite improved immunosuppression, intestinal transplantation is still complicated by severe rejection episodes. To further improve immunosuppressive concepts, we evaluated an anti-CD4 antibody and an anti-tumor necrosis factor (TNF)-alpha monoclonal antibody for their immunosuppressive efficacy in the standard rat model of intestinal transplantation. Intestinal transplantation was performed in the DA to Lewis combination, and recipients were treated perioperatively with either the anti-CD4 antibody RIB5/2 (day -1, 0, postoperative days 1, 2, 4, 7, 10, 14, 17, and 21), the anti-TNF antibody etanercept (60 min before reperfusion, postoperative days 3, 6, and 9) or a combination of both. Survival, histology and expression of immunologic mediator genes on days 3 and 4 after transplantation were investigated. Treatment with anti-CD4 antibody alone (19.71+/-5.94) and the antibody combination (171.58+/-122.76) prolonged survival. The chemokine MIP-1alpha was significantly decreased in both anti-CD4 antibody treatment groups, possibly indicating an additional effect of the TNF-alpha blockade on the immune modulation by RIB5/2. Our study demonstrated long-term graft survival in short-term treatment with a combination of an anti-CD4 antibody and a TNF-alpha antibody in more than 50% of the recipients of intestinal grafts. Such a combined approach could also be useful in clinical small bowel transplantation.

Refined Techniques for Intestinal Transplantation in Rat

Intestinal transplantation (IT), unlike other solid-organ transplantations, such as liver, kidney, and heart, has relatively disappointing results in humans. Significant advances have been made during the past 40 years, but rejection, graft-versus-host disease (GVHD), and infection remain the major obstacles to successful IT. These aspects may be studied using a rat model of IT. Without a microscope and traditional suture for vascular reconstruction, we simplified the procedure using a “three cuffs” technique for orthotopic intestinal transplantation (OIT). Technical modifications of our OIT model that induced good results included (1) adopting a “double cuffs” technique on the graft aorta, (2) using a portal cuff anastomosis to reconstitute the natural and physiologic portal graft drainage with the cuff fixed to the recipient first, and (3) administering a large volume of crystalloid or whole blood to maintain blood pressure and reduce ischemic injury to the graft during operation. In our group, the survival rate of recipients was 87.5% (21 of 24 rats), the average volume of bleeding in the recipient operation was less than 1 mL, and the cold ischemic time, 50 ± 11 minutes.

Simplified Rat Model of Intestinal Transplantation

Rat intestinal transplantation is an optimal model for research; nevertheless, it requires a complex and difficult microsurgical technique (1–5). We have recently developed a simplified rat model for intestinal transplantation using portal cuff anastomosis to overcome these technical difficulties (Fig. 1A).FIGURE 1.: (A) Figure illustrating the present model of intestinal transplantation. G, intestinal graft in shady color; SMA, superior mesenteric artery of the graft; VC, recipient vena cava; AO, recipient aorta. (B) Micropicture (16×) showing details of the portal cuff.Preoperative procedures, anesthesia and laparotomy are performed as previously described (1–4). In the donor operation, middle and descending colons are isolated from small bowel and ascending colon. Superior mesenteric vein is dissected and encircled. Splenic and pyloric veins are divided and portal vein is dissected up to hepatic hilum. Abdominal aorta is dissected and celiac trunk, right renal artery and lumbar branches are divided. Aorta is clamped at the diaphragm, tied below the mesenteric artery and this arterial segment is resected allowing a long aortomesenteric conduit. Entire small bowel, cecum and ascending colon are harvested on a vascular pedicle consisting of right colonic vessels, aortomesenteric conduit and portal vein. The graft is flushed and stored in cold preservation solution. Polyethylene catheter is used to create the cuff as described elsewhere (5). In this modified technique, the vein is introduced into the cuff and everted, covering the outer wall and fixed with three equidistant stitches (Fig. 1B). In the recipient, the superior mesenteric vein is exposed, and a segment between the first and second jejunal branch is dissected and prepared for cuff anastomosis. Eventually, the second jejunal branches and the right colonic vein may be divided in order to increase this segment. The graft is implanted in the recipient with an end-to-side aorto-aorta anastomosis as previously described (1–5). The dissected segment of mesenteric vein is isolated between clamps to insert the portal cuff and a 6-0 suture is used to fix the cuff into recipient’s mesenteric vein. The clamps are released and immediate graft reperfusion is observed. The recipient jejunum, ileum, cecum, and ascending colon are removed en bloc. Finally, anastomosis between the graft and the remaining intestine restores the digestive tract (Fig. 1A). The present model offers many possibilities for intestinal transplantation research with the following advantages: reconstitutes the natural and physiologic portal graft drainage; the portal cuff anastomosis is performed within few minutes without bleeding or venous thrombosis. This alternative technique avoids recipient visceral ischemia and hemodynamic instability because there is no obstruction on inferior vena cava and portal vein. Furthermore, it maintains grafts ileocecal valve and preserves the extrinsic enervation along the mesenteric-aortic segment that may improve bowel movement. This modified technique can be easily performed within two hours after a short period of training (8±3 week) and may reach excellent success rates. This rat model may surmount technical obstacles precluding microsurgical techniques and may ultimately spread this model in centers with low technical resources. Flávio H.F. Galvão Rafael M.N. Santos Marcel A.C. Machado Telesforo Bacchella Marcel C.C. Machado Transplantation and Liver Surgery Discipline University of Sao Paulo School of Medicine Sao Paulo, Brazil

Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation: effect of donor-specific cell augmentation

Introduction Immunomodulation by portal vein delivery of donor antigen reduces intestinal graft rejection. We investigated the impact of portal venous donor-specific cell augmentation (blood versus bone marrow) on cytokine expression in intestinal grafts versus native livers. Methods Ten groups of intestinal transplants (brown Norway male to Lewis female rats) varied by (1) the type of donor-specific cell augmentation and (2) the use and dose of tacrolimus-based immunosuppression. Tissue samples for histologic analysis and cytokine mRNA analysis were obtained at designated time points. Results Without immunosuppression, no type of cell augmentation reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood transfusion (versus bone marrow infusion). Irrespective of the type of cell augmentation, severe rejection caused strong intragraft expression of IL-1α, IL-1β, IFN-γ, and TNF-α; liver expression mainly involved TNF-α. Of note, nonimmunosuppressed, cell-augmented rats showed hardly any differences in cytokine expression in their grafts versus significant increases in their native livers. With immunosuppression, bone marrow infusion (versus blood transfusion) increased intragraft cytokine expression of IL-1α, IL-1β, IFN-γ, as well as TNF-α, and liver expression of IL-1β. Conclusions (1) Rejection and donor-specific cell augmentation independently caused differences in intragraft versus native liver cytokine expression after intestinal transplants. (2) Portal donor-specific blood transfusion (versus bone marrow infusion) lowered the incidence of rejection and diminished intragraft cytokine up-regulation. (3) In our study, TNF-α appeared to be the cytokine most strongly associated with rejection.

Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation with and without donor-specific cell augmentation

Background Immunomodulatory strategies such as donor-specific bone marrow or blood transfusions have been used to promote engraftment after intestinal transplants. We previously showed that delivery of donor antigen via the portal vein can effectively reduce the rate of intestinal graft rejection. The purpose of our current study was to investigate the impact of donor-specific cell augmentation (blood versus bone marrow) via the portal vein on cytokine expression in intestinal grafts versus native livers. Materials and methods We performed heterotopic small intestinal transplants between male Brown-Norway (donor) and female Lewis (recipient) rats. We studied 10 groups according to [1] the type of donor-specific cell augmentation and [2] the use and dose of immunosuppressive therapy. For cell augmentation, donor-specific blood or bone marrow was transfused via the donor portal vein immediately before graft implantation. For immunosuppression, tacrolimus was used post-transplant at a high or low dose. Control rats received neither immunosuppression nor cell augmentation. Tissue samples for histological assessment were obtained at designated time points. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements (IL-1 α, IL-1 β, IL-2, IL-4, IL-5, IL-6, IFN-γ, TNF-α, and TNF-β). Chimerism levels were determined using Q-PCR analysis. Results Without concurrent immunosuppression, neither portal donor-specific blood nor bone marrow transfusion reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood ( versus bone marrow) transfusion. Irrespective of the type of donor-specific cell augmentation, severe rejection caused strong cytokine expression in the grafts of IL-1 α, IL-1 β, IFN-γ, and TNF-α; in the native livers, mainly of TNF-α (with IFN-γ showing hardly any increase). In general, rejection caused stronger cytokine expression in the grafts than in the native livers. Mild rejection correlated well with strong intragraft expression of IL-6, TNF-α, and TNF-β (early rejection markers); severe rejection with IL-1 α, IL-1 β, IFN-γ, and TNF-α (late rejection markers). In addition to cell augmentation per se, the type of cell augmentation also had an impact on cytokine expression in both grafts and native livers. Cell-augmented ( versus tacrolimus-treated) rats showed hardly any differences in intragraft cytokine expression, but the expression of almost all cytokines was significantly stronger in the native livers. With immunosuppression, bone marrow infusion increased intragraft cytokine expression of IL-1 α, IL-1 β, IFN-γ, and TNF α, as well as liver cytokine expression of IL-1 β, compared to blood transfusion. This finding reflected the more advanced rejection stages in the bone marrow infused group; different types of donor-specific cell augmentation had similar effects on liver cytokine expression. In the absence of myoablative therapy, chimerism levels were low, in both cell-augmented and non-cell-augmented groups. Conclusions Rejection and donor-specific cell augmentation independently causes differences in intragraft versus native liver cytokine expression after intestinal transplants. Portal donor-specific blood transfusion, as compared with donor-specific bone marrow infusion, lowered the incidence of rejection and diminished intragraft cytokine up-regulation.

Diffuse mesenterial sclerosis: a characteristic feature of chronic small-bowel allograft rejection.

Chronic rejection is the major cause of late intestinal allograft dysfunction. The aim of this study was to analyze in detail the histopathological features of chronic rejection in the ACI-to-Lewis rat model of intestinal transplantation. Chronic rejection was achieved in orthotopic small-bowel allografts (ACI-Lewis) by limited immunosuppression with cyclosporin A (CyA). Isogeneic transplants (ACI-ACI) as well as native bowels (ACI) with and without immunosuppression served as controls. Bowels were removed together with the mesenteries 90 days postoperatively and analyzed using sections stained with hematoxylin and eosin as well as Masson's trichrome. The slides were coded, randomized and analyzed by grading of histological abnormalities. The most striking alterations of the allografts were noticed in the mesenteries exhibiting an extensive infiltration by mononuclear cells accompanied by a progressive diffuse fibrosis with shrinking of the mesenteries. These changes were most pronounced in the perivascular areas of the mesenteric arteriae and venae rectae. Three of five allografts showed vasculitis with myointimal proliferation of the arteriae rectae. Focally, there was spill-over of the inflammatory cells onto the intestinal muscularis propria. The mucosa of the allografts showed mild blunting, lymphocytic infiltration of the crypt epithelium and increased crypt cell apoptoses. The submucosa was unaffected, and there were no detectable abnormalities of the enteric ganglion cells. The present data support the view that chronic rejection of intestinal allografts is characterized by a diffuse sclerosing mesenteritis which may significantly contribute to late graft dysfunction. The present model may be useful to study the pathomechanisms of this inflammatory fibrosing process.

Apoptosis and rejection in rat intestinal transplantation: correlation with FK506 doses and donor specific bone marrow infusions.

Our purpose was to investigate the occurrence and the evolution of apoptosis of enterocytes during acute and chronic rejection in an experimental model of allogeneic heterotopic small bowel transplantation (SBTx). Forty-five rats were divided in 10 experimental groups according to the dose of FK506 administration and donor bone marrow infusions (DBMI). Groups 1 and 2 did not received BMI. Groups 3 and 4 received 150x106 cells at day 0, groups 5 and 6 received 75x106 cells at days 0-4, groups 7 and 8 received 75x106 cells at days 4 and 10, and groups 9 and 10 received 30x106 cells at days 4, 10, 15, 20, and 25. Animals of groups 1, 3, 5, 7, and 9 were immunosuppressed with 0.5 mg/kg FK 506, although the remaining groups with 1 mg/kg FK 506, from day 0 to 4 after transplant. Fragment end labeling of DNA was used to detect apoptosis. The number of apoptotic cells detected was highest at day 15 (184+/-154) and then progressively decreased thereafter (day 30=159+/-197; day 45=80+/-167; day 60=0). The number of apoptotic enterocytes was found increased during mild (151+/-108) and moderate (281+/-161) allograft rejection, although a low apoptotic rate was observed in cases without rejection (59+/-13) and during severe (53+/-131) and chronic rejection (46+/-136). Furthermore the number of labeled cells was found inversely correlated with fibrosis (P<0.0001). There was no correlation between apoptosis and the presence or absence of DBMI; however, at day 15 rats receiving 1 mg/day of FK 506 had a significantly lower number of apoptotic cells detected (127+/-103 vs. 233+/-174; P<0.02). In this study the number of apoptotic cells correlated positively with mild and moderate rejection episodes. In case of severe and chronic rejection a low apoptotic rate was found due probably to extensive necrosis and fibrosis of the mucosa. These data suggest an important role of apoptosis in acute and chronic intestinal rejection in a rat model of intestinal transplantation. Determination of apoptosis in allografts might represent an early sign of small bowel rejection and a useful marker in defining the degree of rejection and its outcome/prognosis.

Fat absorption after small intestinal transplantation in the rat.

Intestinal transplantation is now used for patients with severe malabsorption, however, little data exists quantifying the ability of the graft to absorb fat. This study tested the hypothesis that intestinal transplantation would not affect the lymphatic or venous uptake of fatty acids. A syngeneic rat model of intestinal transplantation (SIT) with caval drainage of the graft was used. Control animals underwent intestinal division and reanastomosis (n=15 in each group). The animals were followed for 6 weeks, and fat absorption in vivo was quantified. The animals were anesthetized, sampling catheters were placed in the jugular and superior mesenteric veins and in the mesenteric lymphatic duct, and a feeding tube was passed into the duodenum. Animals were allowed to recover, and a steady-state duodenal infusion of lauric (C12:0) and palmitic (C16:0) fatty acid emulsion was begun. A radiolabeled pulse of lauric (C12:0) and palmitic (C16:0) fatty acid was then given, and the subsequent appearance in the lymphatic and venous systems was quantified. In vivo absorption of dietary fat was preserved, but after transplantation the mesenteric lymphatic flow and cumulative lymphatic appearance of both labels was significantly reduced (flow reduced from 4.8+/-1.1 in controls to 1.0+/-0.29 ml/hr in transplant animals, whereas lauric acid absorption was 33+/-11.4% in controls vs. 7.5+/-2.5% in transplant animals). There was a modest increase in the jugular venous appearance of the fatty acids (2.0+/-1.1% in transplant animals vs. 0.75+/-0.55% in controls for lauric acid; P<0.05 for all comparisons). Absorption of lauric and palmitic acids was very similar, and there was no preferential absorption detected in the portal venous system. Dye studies demonstrated lymphatic recannulization around the vascular anastomosis, into the retroperitoneum. These results suggest that in this model of SIT, fat absorption via the mesenteric duct is reduced, but that compensatory collaterals form into the retroperitoneal lymphatics. There was no evidence of any significant increase in portal venous uptake of fatty acids after SIT, nor of preferential absorption of medium-chain fatty acids. These results may have implications for patients after SIT.

VIP receptors and content after bowel transplantation

Advances in immunosuppressive therapy have renewed interest in small bowel transplantation. Little is known, however, about the functional capacity of transplanted intestine. To clarify the potential for normal function, we investigated whether elements of the enteric nervous system are preserved after denervation in our rat model of intestinal transplantation. We investigated whether VIP, a major peptide neurotransmitter of the enteric nervous system, and its receptors are preserved in the bowel after transplantation. In our model of transplantation, avascular fetal jejunum from term Fisher rats is transplanted to the subcutaneous tissues of host syngeneic rats. This “neogut” becomes vascularized and develops characteristics of native small bowel. VIP content was measured by RIA and the in situ distribution of VIP receptors was determined by the technique of receptor autoradiography. Neogut was studied 1 and 3 weeks after transplantation and compared with age-matched rat pup jejunum. Autoradiographs showed high silver grain density, representing VIP binding sites, in the mucosal layers of all tissues studied. VIP content in the transplanted bowel was comparable to that of native gut and showed a rise with developmental age similar to that of native gut. VIP levels (pmole/mg protein, x ± SEM) were neogut 1 week, 0.26 ± 0.14; jejunum 1 week, 0.25 ± 0.07; neogut 3 weeks, 0.60 ± 0.21; and jejunum 3 weeks, 0.69 ± 0.16. These results show that VIP receptors and content are preserved in this model of transplantation. This suggests that the enteric nervous system and receptors for peptide neurotransmitters remain intact after transplantation and may retain the potential for regulatory function.