Tolerance to limb tissue allografts between swine matched for major histocompatibility complex antigens

Transplantation of limb tissue allografts would greatly expand the realm of reconstructive surgery. However, the toxicity of chronic immunosuppression has adversely tilted the risk-benefit balance for clinical transplant. In this study, a procedure was sought to achieve host tolerance to limb tissue allografts through matching of the major histocompatibility complex (MHC) antigens between donor and host swine using only a 12-day course of cyclosporine. Massachusetts General Hospital (MGH) miniature swine were used as a large animal model with defined MHC, and musculoskeletal grafts from the donor hind limb were transplanted heterotopically to the recipient femoral vessels. Allografts from MHC-mismatched donors treated with cyclosporine (n = 4) were rejected in less than 6 weeks by gross inspection and histologic sections. Allografts from MHC-matched, minor antigen mismatched donors not treated with cyclosporine (n = 4) were rejected between 9 and 12 weeks. Allografts from similarly matched donors treated with 12 days of cyclosporine (n = 7) showed no evidence of rejection until sacrifice between 25 and 47 weeks. Thus allograft tolerance was achieved between MHC-matched swine using a limited course of cyclosporine. Demonstration of limb tissue allograft survival in a large animal model without long-term immunosuppression represents an important step toward clinical transplantation.     

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.^1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.⁴ In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.^5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.  The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor’s portal vein was carried out via the recipient’s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient’s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. ⁷In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3±0.8 g to 9.8±1 g. At this time, the graft’s weight decreased from 3.3±0.8 g to 2.3±0.8 g.We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.     

Development of a New Technique for Reconstruction of Hepatic Artery during Liver Transplantation in Sprague-Dawley Rat

Background

Sleeve anastomosis is the most common technique used to rearterialize orthotopic liver transplants (OLT). However, this technique has a number of disadvantages, including difficulty of performance of the technique visually unaided. We herein describe a novel rearterialized OLT model in the rat.

Materials and Methods

Forty-six male Sprague Dawley rats (300–400 g) were used as donors and recipients. Based on Kamada’s cuff technique, the new model involved performing a modified “sleeve” anastomosis between the celiac trunk of the donor and common hepatic artery of the recipient to reconstruct blood flow to the hepatic artery. An additional ten male Sprague Dawley rats underwent liver transplantation without artery reconstruction. Liver grafts were retrieved from the two groups and histological examination was performed following surgery.

Results

Total mean operating times were ~42 minutes for the donor liver extraction and 57 minutes for the recipient transplantation. Graft preparation took an additional 15 minutes and the time to fix the arterial bracket was ~3 minutes. During transplantation, the anhepatic phase lasted 18 ± 2.5 min and the artery reconstruction only required ~3 minutes. The patency rate was 94.44% and the 4-week survival rate was 90%. Histology indicated obvious fibrosis in the liver grafts without artery reconstruction, while normal histology was observed in the arterialized graft.

Conclusions

This new method allows for the surgical procedure to be performed visually unaided with good survival and patency rates and represents an alternative model investigating OLT in rats.     

Posttransplant administration of donor leukocytes induces long-term acceptance of kidney or liver transplants by an activation-associated immune mechanism

Donor leukocytes play a dual role in rejection and acceptance of transplanted organs. They provide the major stimulus for rejection, and their removal from the transplanted organ prolongs its survival. Paradoxically, administration of donor leukocytes also prolongs allograft survival provided that they are administered 1 wk or more before transplantation. Here we show that administration of donor leukocytes immediately after transplantation induced long-term acceptance of completely MHC-mismatched rat kidney or liver transplants. The majority of long-term recipients of kidney transplants were tolerant of donor-strain skin grafts. Acceptance was associated with early activation of recipient T cells in the spleen, demonstrated by a rapid increase in IL-2 and IFN-gamma at that site followed by an early diffuse infiltrate of activated T cells and apoptosis within the tolerant grafts. In contrast, IL-2 and IFN-gamma mRNA were not increased in the spleens of rejecting animals, and the diffuse infiltrate of activated T cells appeared later but resulted in rapid graft destruction. These results define a mechanism of allograft acceptance induced by donor leukocytes that is associated with activation-induced cell death of recipient T cells. They demonstrate for the first time that posttransplant administration of donor leukocytes leads to organ allograft tolerance across a complete MHC class I plus class II barrier, a finding with direct clinical application.     

Experiences of Microsurgical Reconstruction for Variant Hepatic Artery in Living Donor Liver Transplantation

There is an emergent need for improving the microsurgical technique of variant arterial anastomosis to reduce the often seen surgery-related complications. We describe in this article our experience in improving this technique, in 73 living donor liver grafts (64 right lobes, 9 left lobes) in patients with end-stage liver disease during living donor liver transplantation. The hepatic arteries were evaluated preoperatively with computed tomography and magnetic resonance angiography. In this series, 13 grafts (17.80 %) with variant hepatic artery were conducted arterioplasty on a back-table under a loupe or a high-power microscope, which included one recipient in situ interposition vessel graft of recipient proper hepatic artery for artery reconstruction. The back-table reconstruction time was 16 ± 5.6 min. No arterial thrombosis was found in these cases during the 6-month postoperative follow-up. On the basis of our experience, we suggest that back-table microsurgical plasty for graft with arterial variation should be applied to minimize operative difficulties and to avoid arterial complications in living donor liver transplantation.     

Model of occlusion-reperfusion arrhythmias of the transplanted heart in rats

Rats with heterotopically transplanted heart were used for modeling of occlusion-reperfusion arrhythmias. Heart transplantation was carried out by performing the anastomoses between the recipient carotid artery and jugular vein and innominate artery and pulmonary artery of the transplant respectively. The reversible ischemia of myocardium was achieved through temporal occlusion of recipient carotid artery supplying transplanted heart. The model is characterised by total denervation of the heart, the absence of pump function, total nature of myocardial ischemia during occlusion of supplying artery, high reproducibility of arrhythmias and the opportunity of repeated use of the animals.     

The isolated perfused rat liver: standardization of a time-honoured model

For many years, the isolated perfused rat liver (IPRL) model has been used to investigate the physiology and pathophysiology of the rat liver. This in vitro model provides the opportunity to assess cellular injury and liver function in an isolated setting. This review offers an update of recent developments regarding the IPRL set-up as well as the viability parameters that are used, with regards to liver preservation and ischaemia and reperfusion mechanisms.A review of the literature was performed into studies regarding liver preservation or liver ischaemia and reperfusion. An overview of the literature is given with particular emphasis on perfusate type and volume, reperfusion pressure, flow, temperature, duration of perfusion, oxygenation and on applicable viability parameters (liver damage and function).The choice of IPRL set-up depends on the question examined and on the parameters of interest. A standard technique is cannulation of the portal vein, bile duct and caval vein with pressure-controlled perfusion at 20 cm H2O (15 mmHg) to reach a perfusion flow of approximately 3 mL/min/g liver weight. The preferred perfusion solution is Krebs-Henseleit buffer, without albumin. The usual volume is 150-300 cm3, oxygenated to a pO2 of more than 500 mmHg. The temperature of the perfusate is maintained at 37 degrees C. Standardized markers should be used to allow comparison with other experiments.     

Protein synthesis in heterotopically transplanted rat hearts

This study examines protein synthesis in heterotopically transplanted rat hearts and several tissues of recipient rats. Donor hearts and recipient tissues synthesized many of the normally occurring proteins observed in tissues of unstressed rats. In addition, a stress-induced protein with a molecular mass of 71 kilodaltons was synthesized in donor heart, recipient heart and lung. Donor hearts incorporated more L-[35S]-methionine than did recipient hearts. Tissues of recipient rats also incorporated more label than the respective tissues of sham-recipient rats. These results suggest that ischemia, endured by the donor hearts during transplantation, induced these changes in protein synthesis.     

Germ-line chimera by lower-part blastoderm transplantation between diploid goldfish and triploid crucian carp

Germ-line chimerism was successfully induced by blastoderm transplantation from donor triploid crucian carp, which reproduces gynogenetically, to recipient diploid goldfish, which reproduces bisexually. Lower part of donor blastoderm including primordial germ cells (PGCs) was sandwiched between recipient blastoderm at the mid- to late-blastula stage. When donor grafts were prepared from intact embryos or ventralized ones by removing vegetal yolk hemisphere at the 1- to 2-cell stage, malformations including double axes were observed in the resultant chimeras transplanted with grafts from intact embryos at the hatching stage, while a few malformations in those from ventralized embryos. PGCs originated from donor grafts were observed around the gonadal anlage at 10 days post-fertilization in chimeras. When ploidy of erythrocytes and epidermal cells in chimeric fish was examined by flow-cytometry, no triploid cells were detected at 1- and 5-year-old chimeras. Three-year-old chimeric fish (n=5) laid eggs originated from the donor together with those from the recipient. The frequency of eggs from the donor crucian carp blastoderm varied from 3.1 to 89.3% between chimeras.     

Surgical technique in the rat model of kidney transplantation

Today successful kidney transplantation procedures, techniques and immunosuppression protocols are a consequence of extensive research on animal models. During every transplantation surgery there are two crucial points for the success of the entire procedure: vascular (arterial end venous) and ureteral or ureterovesical anastomosis. Renal artery and vein of the donor kidney can be anastomosed end-to-side to the abdominal aorta and vena cava of the recipient (heterotopic transplantation), or end-to-end to the remains of renal artery and vain of the recipient (orthotopic transplantation) after nephrectomy. The ureter can be anastomosed also end-to-end or we can connect it directly to the urinary bladder (ureterocystoneostomy). The aim of this study was to elucidate which technique has better results according to: animal survival, reperfusion and perfusion of the transplanted kidney, elimination of the urine from the transplanted kidney and procedure costs. The study included 240 (120 donors and 120 recipients) male Wistar rats (3 months old; weight 250-300 g Our results are clearly showing that the end-to-end vascular anastomosis, and Paquins ureterovesical anastomosis have better results in transplanted rat kidneys survival and urine drainage compared to end-to-side vascular anastomosis and end-to-end ureteral anastomosis. Based on our experience we can conclude that described methods of end-to-end vascular anastomosis and Paquins ureterovesical anastomosis are less technically demanding and have a shorter learning curve. Therefore, we can recommend the use of described methods in kidney transplantation related researches.     

Small bowel transplantation in mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.     

Effect of foreign innervation on contractile and histochemical properties of the transplanted levator ani muscle of the rat.

Heterotopic transplantation of the levator ani (LA) muscle into the bed of the fast tibialis anterior (TA) or slow soleus (SOL) muscle respectively results in transformation of contractile and histochemical properties of the muscle dependent on the new "foreign" innervation. This transformation is observed after transplantation of minced muscle tissue and of free grafts. The result of transformation is more pronounced in the case of free LA-TA grafts which show progressive shortening of contractile response, whereas the LA-SOL shows slight shortening. The heterotopically transplanted free LA-SOL and the LA-TA grafts become relatively faster than the respective original muscle, suggesting operation of myogenic factors related to the fast LA muscle. Maximal tetanic tension output of the free heterotopic grafts 60 days after transplantation recovers to only about a quarter of the correspondong control muscles. Recovery of speed of contraction in the transplanted LA muscle is similar to that observed after selfreinnervation after crushing the pudendal nerve close to its entry into the muscle. In the heterotopically transplanted muscles the reversal of the originally uniform histochemical fibre pattern to a mixed fibre pattern in respect to ATPase and SDH activity is dependent on the type of innervation. After selfreinnervation of the LA muscle by the pudendal nerve a uniform fibre pattern is maintained with regeneration of the nerve.     

Immunofluorescent study of the hemopoietic organs of xenogeneic mouse radiation chimeras]

An immunofluorescent study of hemopoietic organs in xenogenic (mouse-rat) radiation chimaeras has been carried out by means of specific antiserum against hemopoietic cells of the rat bone marrow. The presence of donor cells was tested at different times after the transplantation in the bone marrow, spleen, lymph nodes, thymus and liver of radiochimaeras. The transplanted cells were shown to populate all hemopoietic organs of the recipient, first of all tissues of the bone marrow type and, then, lymphoid organs. The donor (bone marrow) origin of the extramedullar foci of hemopoiesis in the liver was established.     

Association between IL-4 polymorphism and acute rejection of solid organ allograft: A meta-analysis

Background

Cytokines have been implicated in the acute rejection of solid organ transplantation. Many studies have investigated the association between recipient or donor IL-4 polymorphism and acute rejection, with different studies reporting inconclusive results.

Methods

We searched PUBMED and EMBASE until June 2012 to identify eligible studies investigating the association between IL-4 polymorphism with acute rejection after solid organ transplantation. Statistical analysis was performed using STATA10.0.

Results

A total of 12 studies were included. Pooled ORs suggested 1) no significant association was detected between recipient or donor IL-4 − 590C/T polymorphism and acute rejection of solid allograft; 2) no significant association was detected between recipient IL-4 − 33C/T polymorphism and acute rejection of solid allograft; 3) when stratified by transplantation type, IL-4 − 590C/T polymorphism was associated with acute rejection of liver transplantation (T/T + C/T vs. C/C: OR = 0.36, 95%CI = 0.14–0.90); 4) significantly decreased risk of acute rejection was detected in recipient IL-4 − 590*T-negative/donor T-positive genotype pairs than all other recipient–donor IL-4 − 590T/C pairs (OR = 0.14, 95%CI = 0.03–0.66).

Conclusions

Our meta-analysis suggested that recipient IL-4 − 590C/T polymorphism was associated with acute rejection of liver transplantation, but nor renal or heart transplantation. It was also suggested that combined recipient IL-4 − 590*T-negative/donor T-positive genotype may suffer decreased risk of acute rejection of solid allograft. Further well-designed studies with larger sample size were required to verify our findings, with focus on the association of IL-4 polymorphism with acute rejection in patients with liver transplantation and studies investigating combined recipient–donor genotype.     

Pretreatment of cisplatin in recipients attenuates post-transplantation pancreatitis in murine model

Pancreas transplantation is the definite treatment for type 1 diabetes that enables the achievement of long-term normoglycemia and insulin independence. However Post-Transplantation Pancreatitis (PTP) due to ischemia reperfusion (IR) injury and preservation is a major complication in pancreas transplantation. Owning the potential anti-inflammatory effect of Cisplatin (Cis) in liver IR injury, we have examined if Cis could attenuate PTP using a murine model. We found that Cis is able to prevent inflammatory response in PTP. Pretreatment of Cis in recipient mice reduce the impairments of the grafts and hyperamylasimea in the recipients. We documented that the protective mechanism of Cis in PTP involves improvement of microcirculation, reduction of the mononuclear cellular infiltration and apoptosis, suppression of inflammatory cytokine-cascade and inhibition of translocation of high-motility group box protein-1 (HMGB-1) from nucleus to cytoplasm. In short, our study demonstrated that pretreatment of Cis in recipients may reduce the onset of PTP in pancreas transplantation.     

带血管髂骨移植动物模型的建立与评估

目的:探索一种相对简单稳定的大鼠髂骨皮瓣移植手术方式,为髂骨移植诱导免疫嵌合状态的研究建立可靠的动物模型。方法:实验分为3组:空白对照组,不做任何处理;旧术式组,将供者髂骨肌皮瓣移植入受者腹股沟区,吻合供者髂腰动静脉和受者股动静脉;实验组,剥离供者髂骨臀大肌,取供者的髂骨连带肌肉及皮瓣移植于受者的腹股沟区域,将供者的髂总动脉和髂腰静脉分别与受者的股动静脉吻合,随后缝合皮肤。术后所有大鼠给予环孢素A 28天,始剂量16 mg/kg/d,第三周始每周剂量减半,第28天处死大鼠并选取髂骨做病理学检测,观测骨髓细胞量,骨陷窝是否有骨细胞等,以此判断髂骨是否成活。结果:实验组共进行手术15例,皮瓣成活11例,成功率73%;病理检测皮下髂骨成活7例,成功率47%;旧术式组共进行手术8例,皮瓣成活4例,成功率50%,病理检测皮下髂骨成活3例,成功率37.5%,其中实验组动脉吻合时间(24.7±2.3)min,明显短于旧术式组(36.7±1.5)min(均P0.05),而静脉吻合时间、缺血时间,供受体准备时间并无差异(均P0.05)。结论:通过剥离供者臀大肌,改变动脉吻合对象,可以建立相对简单、稳定的髂骨皮瓣移植模型,为探索带血管骨髓移植诱导嵌合状态提供了新的动物模型。     

Crucial Role for Neuronal Nitric Oxide Synthase in Early Microcirculatory Derangement and Recipient Survival following Murine Pancreas Transplantation

Objective

Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation.

Background

Tetrahydrobiopterin donor treatment has been shown to specifically attenuate these derangements following pancreas transplantation, and tetrahydrobiopterin-mediated protective effects to rely on its NOS-cofactor activity, rather than on its antioxidant capacity. However, the NOS-isoform mainly involved in this process has still to be defined.

Methods

Using a murine pancreas transplantation model, grafts lacking one of the three NOS-isoforms were compared to grafts from wild-type controls. Donors were treated with either tetrahydrobiopterin or remained untreated. All grafts were subjected to 16 h cold ischemia time and transplanted into wild-type recipients. Following 4 h graft reperfusion, microcirculation was analysed by confocal intravital fluorescence microscopy. Recipient survival was monitored for 50 days.

Results

Transplantation of the pancreas from untreated wild-type donor mice resulted in microcirculatory damage of the transplanted graft and no recipient survived more than 72 h. Transplanting grafts from untreated donor mice lacking either endothelial or inducible NOS led to similar outcomes. In contrast, donor treatment with tetrahydrobiopterin prevented microcirculatory breakdown enabling long-term survival. Sole exception was transplantation of grafts from untreated donor mice lacking neuronal NOS. It resulted in intact microvascular structure and long-term recipient survival, either if donor mice were untreated or treated with tetrahydrobiopterin.

Conclusion

We demonstrate for the first time the crucial involvement of neuronal NOS in early microcirculatory derangements following solid organ transplantation. In this model, protective effects of tetrahydrobiopterin are mediated by targeting this isoform.     

Anorectal Transplantation in Human Cadavers: Mock Anorectal Allotransplantation

Background

Anorectal transplantation is a method for patients who have lost their anorectal function or suffer from congenital anorectal dysfunction to recover this function, and this has been investigated in experimental animal models using pigs, dogs, and rats. In this study, we performed an examination of anorectal transplantation in human cadavers to investigate whether this procedure could be performed in patients.

Methods

A 77-year-old woman cadaver 1 was used as the donor and a 98-year-old woman cadaver 2 was used as the recipient. Initially, abdominoperineal excision of the anus and rectum (the Miles’ operation) was performed on the recipient. Next, an anorectal graft containing the pudendal nerve (PN), pudendal artery (PA), pudendal vein (PV), inferior mesenteric artery (IMA), and inferior mesenteric vein (IMV) was harvested from the donor. The donor graft was transplanted into the recipient by intestinal anastomosis and microneurovascular anastomoses orthotopically.

Results

The diameters of the PN (right/left), IMA, and IMV were 2.5 mm/2.5 mm, 2.0 mm, and 1.5 mm, respectively, in cadaver 1, and 2.0 mm/2.0 mm, 2.0 mm, and 2.0 mm, respectively, in cadaver 2. The length of the PN, PA, PV, IMA, and IMV in the graft was sufficient to allow proper anastomosis.

Conclusion

This preliminary study indicated that human anorectal transplantation was possible anatomically and technically. We anticipate our study will aid in the potential future application of this procedure to human patients.     

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.