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.     

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37 °C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.     

食蟹猴肾移植模型的建立

目的建立食蟹猴的肾移植模型。方法通过显微外科手术对食蟹猴进行同种异体移植,将供体肾动脉、肾静脉分别端侧吻合到受体腹主动脉、下腔静脉,供体输尿管端端吻合到受体输尿管。结果154例肾移植食蟹猴无一只在手术过程中因意外死亡;无一只因手术感染死亡;无一只因手术并发症死亡;肾热缺血时间（30±4.5）min。结论应用食蟹猴建立肾移植模型,技术成熟,方法可靠。应用此模型评价新型免疫抑制药物,进行免疫耐受、异种移植研究可为临床试验提供更为准确可靠的依据。     

Heterotopic vascularized murine cardiac transplantation to study graft arteriopathy

The development of microsurgical techniques has facilitated the establishment of fully vascularized cardiac transplantation models in small mammals. A particularly useful model that has evolved for the study of cardiac allograft vasculopathy (CAV) is a heterotopic (abdominal) vascularized murine cardiac transplantation model. Using this model has permitted the elucidation of genetic, immune and non-immune factors contributing to the development of this inexorable pathological condition, which compromises half of all human cardiac transplants. This protocol details methods for performing the transplant, histomorphometric assessment of the graft vasculature and functional evaluation of the transplanted heart. In experienced hands, the surgical procedure requires approximately 75 min to complete, and vasculopathy results are obtained at 2 months. This model entails a fully vascularized implantation technique in which the donor ascending aorta and pulmonary artery are sutured end-to-side to the recipient abdominal aorta and inferior vena cava, respectively. As this model reliably reproduces immunological and non-immunological features of CAV, investigators can thoroughly explore contributory mechanisms, diagnostic modalities and therapeutic approaches to its mitigation.     

Tracer mixing: sites of tracer infusion and sampling

Controversy exists in the literature concerning the correct infusion and sampling sites in studies measuring substrate turnover rates. To investigate this problem, we examined the results obtained with various infusion and sampling sites in 7 anesthetized dogs. [1-14C]lactate was infused by a primed continuous infusion method in three different sites (the left ventricle, ascending aorta, and the aortic arch) in a sequential fashion; samples were obtained simultaneously from five sites (femoral artery, carotid artery, pulmonary artery, superior vena cava and inferior vena cava) for each of the three different infusion sites. [U-13C]lactate was also infused in a femoral vein and simultaneous samples were obtained in the carotid artery and femoral artery for analysis of the stable isotope. [14C]lactate analysis demonstrated that infusion of the tracer into the left ventricular chamber resulted in a uniform distribution in the systemic circulation. Infusion into the ascending aorta near the aortic valve resulted in uniform distribution of tracer in four out of five experiments. Tracer infusion into the aortic arch resulted in nonuniform systemic distribution of tracer. The [U-13C]lactate results showed that infusion into the femoral vein gives uniform systemic distribution, similar to that observed with left ventricular infusion. The pulmonary artery lactate specific activities varied from those in the superior vena cava. Thus, this study shows that the tracer must be infused in the left ventricle or upstream from this chamber to obtain optimal systemic distribution. Vena caval sampling, especially superior vena caval sampling, will not give a consistent mixed venous concentration of the lactate tracer. Therefore, aortic tracer infusion with vena caval sampling may lead to errors in determining substrate turnover values.     

Orthotopic Kidney Transplantation in Mice: Technique Using Cuff for Renal Vein Anastomosis

Mouse renal transplantation is a technically challenging procedure. Although the first kidney transplants in mice were performed over 34 years ago and refined some years later, the classical techniques of mouse renal transplantation required clamping both vena cava and aorta simultaneously and carry out suture anastomoses of the renal artery and vein in a heterotopic position. In our laboratory, we have successfully developed mouse orthotopic kidney transplantation for the first time, using a rapid “cuffed” renal vein technique for vessel anastomosis, wherein the donor’s renal vein was inserted through an intravenous catheter, folded back and tied. During grafting, the cuffed renal vein was directly inserted into the recipient’s renal vein without the need for the clamping vena cava and suturing of renal vein. This technique allowed for the exact transplantation of the kidney into the original position, compared to the classical technique, and has significantly shortened the clamping time due to a quicker and precise anastomosis of renal vein as described. This also allowed for a quicker recovery of the lower extremity activity, reduction in myoglobinuria with resultant kidney graft survival of 88.9%. Thus we believe that the cuffed renal vein technique simplifies microvascular anastomoses and affords important additional benefits.     

Correlation of endothelial vimentin content with hemodynamic parameters

 In mammalian species, vimentin is the sole intermediate filament protein of endothelial cells lining the chambers of the heart and the inner surface of large blood vessels. Obvious quantitative differences in the vimentin-like immunoreactivity of endothelial cells observed in different vascular segments led us to undertake a systematic survey on the endothelial content of vimentin throughout the heart chambers, the vena cava, the pulmonary trunk, and the aorta of the pig. Immunostaining and immunoblotting showed that vimentin in endothelial cells of cardiovascular segments exposed to high shear stress and blood pressure (pulmonary trunk, aorta, left ventricle) is approximately 2- to -3-fold higher than in endothelial cells exposed to lower levels of hemodynamic stress (vena cava, left and right atria, right ventricle). Throughout the aorta, an approximately 1.5-fold increase in the vimentin contents was observed in a proximal to distal direction. The total endothelial amount of vimentin was determined to be 1.2% (inferior vena cava) and 2–3.5% (aorta) of total cellular protein. These data support the notion that the endothelial vimentin cytoskeleton can adapt to different hemodynamic loads, indicating that vimentin might help endothelial cells to withstand the mechanical forces exerted by blood flow and blood pressure. Accepted: 2 February 1998     

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient’s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.     

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient’s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.     

Death-related gastric necrosis after Laparoscopic Adjustable Gastric Banding in the early post-operative period

Background

Although mediastinal tumors compressing or invading the superior vena cava represent the major causes of the superior vena cava syndrome, benign processes may also be involved in the pathogenesis of this medical emergency. One of the rarest benign causes is a pseudoaneurysm developing in patients previously having heart surgery.

Case report

We present the case of a large pseudoaneurysm of the ascending aorta, five years after primary surgery, with a significant compression of the right mediastinal venous system causing superior vena cava syndrome, detected at chest CT angiography. Perioperative findings showed two rush out points both coming from the distal aortic suture line which was performed five years ago. The patient underwent reoperation under circulatory arrest facilitating safe exploration and repair of the distal anastomotic leaks

Conclusion

Enhanced chest CT should be always undertaken in all patients with superior vena cava syndrome, especially in those previously having cardiac or aortic surgery to correctly evaluate the presence of a pseudoaneurysm. Mass effect to the superior vena cava makes necessary an open surgical treatment of the pseudoaneurysm so as to concurrently resolve the right mediastinal venous system's compression. Surgery should be performed in terms of safe approach to avoid exsanguination and cerebral malperfusion.     

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.     

Prenatal effects of maternal consumption of polyphenol‐rich foods in late pregnancy upon fetal ductus arteriosus

Fetal circulation has characteristic features, being morphologically and functionally different from extrauterine circulation. The ductus arteriosus plays a fundamental role in directing the blood flow to fetal inferior body parts. Basically, the ductus arteriosus directs 80–85% of the right ventricular output arising from the superior vena cava, coronary sinus, and a small part from the inferior vena cava to descending aorta. Its histological structure is made up predominantly by a thick muscular layer, differently from the aorta and the pulmonary artery, which increases with gestational age. The fibers have a circumferential orientation, especially at the external layers, facilitating and making effective ductal constriction. These factors may generate lumen alterations which may cause fetal and neonatal complications, such as heart failure, hydrops, neonatal pulmonary hypertension, and even death. Classically, maternal administration of indomethacin and/or other antiinflammatory drugs interfere in prostaglandins metabolism, causing ductal constriction. However, many cases of fetal ductal constriction, as well as of persistent neonatal pulmonary artery hypertension, remain without an established etiology, being referred as “idiopathic.” In recent years, a growing body of evidence has shown that herbs, fruits, nuts, and a wide diversity of substances commonly used in daily diets have definitive effects upon the metabolic pathway of inflammation, with consequent inhibition of prostaglandins synthesis. This antiinflammatory action, especially of polyphenols, when ingested during the third trimester of pregnancy, may influence the dynamics of fetal ductus arteriosus flow. The goal of this review is to present these new observations and findings, which may influence dietary orientation during pregnancy. Birth Defects Research (Part C) 99:256–274, 2013. © 2013 Wiley Periodicals, Inc.     

Surgical anatomy of the abdominal part of the thoracic duct

With the aim to determine local orienting points, that facilitate in searching the abdominal part of the thoracic duct, 43 corpses of mature persons have been investigated by means of a complex of anatomical techniques. The distance between the levels, where the superior mesenteric artery and renal arteries get off, can be used for preventing possible complications at certain surgical manipulations. In particular, when the distance from the beginning of the superior mesenteric artery is 37-64 mm, the thoracic duct is mainly situated behind the inferior vena cava; when this distance is 16-34 mm, the duct is situated between the inferior vena cava and the aorta, and when the distance between the superior mesenteric and renal arteries is 6-15 mm, the abdominal part of the thoracic duct should be searched for behind the inferior vena cava.     

Temporary assisting suspension suture technique for successful microvascular anastomosis of extremely small and thin walled vessels for mice transplantation surgery

Dissection and microsurgical anastomosis in small and thin-walled vessels is challenging. Temporary assisting suspension suture technique was developed to overcome those difficulties in establishing successful composite tissue allotransplantation in mice. The operations were performed in 12- to 16-week-old Balb/c mice weighing 25 to 30 grams as both donor and recipient animals. Extended vascularized groin cutaneous flaps based on the superficial epigastric vessels were used. A total of 10 groin cutaneous flaps were transplanted. Three temporary assisting suspension sutures of 11-0 nylon were placed at the 12-, 4-, and 8-o'clock positions to donor and recipient artery and vein before the anastomosis. This technique allowed atraumatic dissection of delicate and thin vessels, prevented vessel wall collapse, and facilitated adequate exposure of the lumen during placement of the permanent microvascular sutures. Thus, the microvascular anastomosis was performed in an unusual manner. The temporary assisting suspension sutures were removed just before the permanent suture was tied down. The mean operation time was 1 hour and 45 minutes with an ischemia time of 1 hour. Ninety-percent success in immediate and late-term patency rates was achieved, which was confirmed by transplant survival. This technique was proven to be useful for microvascular anastomosis in thin-walled vessels and is recommended.     

L'Anastomose cavo-pulmonaire dans les malformations tricuspidiennes: Evolution de 10 cas opérés

Certain congenital malformations affecting the rightsided cavities of the heart and accompanied by a diminished pulmonary blood flow can be improved by an end-to-side anastomosis between the right pulmonary artery and the superior vena cava. Nine out of the 10 patients who underwent the operation survived. The results have been very impressive in six patients with tricuspid atresia who were critically ill. The anoxemic episodes ceased completely and cyanosis has disappeared. Three cases of Ebstein''s anomaly have also been improved. This clinical study has given us the opportunity to review the physiological consequences of the anastomosis, its complications and its surgical indications. We believe that this type of shunt is especially indicated in cases of tricuspid atresia, in which only a palliative form of surgery can be performed.     

大鼠肾移植慢性排斥反应模型的建立

目的建立稳定而可靠的大鼠肾移植慢性排斥反应模型。方法选用30只Wistar大鼠为供体,30只SD大鼠为受体。取供体左肾,采用HC-A离体肾保存液原位灌注,将供肾动、静脉分别与受体腹主动脉、下腔静脉行端侧吻合,以输尿管膀胱植入法行尿路重建,建立大鼠同种异体肾移植模型。分别于术后3、6、9周取移植肾观察大体和组织形态学变化,观察术后并发症及排斥反应情况。结果移植肾脏大体和组织形态学呈渐进性变化,至术后9周可出现明显的慢性排斥反应病理改变。移植肾脏可顺利存活,部分出现肾积水并发症,但不影响排斥反应病理变化。结论本方法可建立稳定、可靠的肾移植慢性排斥反应模型,是研究慢性排斥反应的理想模型。     

大鼠腹腔异位心脏移植模型建立及改进

目的:建立一种快速有效的大鼠腹腔异位心脏移植模型。方法:采用SD大鼠作为受体,Wistar大鼠作为供体,行同种异位腹腔心脏移植,术后给以CsA5 mg/kg/d灌服,心脏移植手术方法采用改良的Ono术式,观察改良的腹腔异位心脏移植各步骤所需时间、术后成功率及主要并发症发生率。结果:共建立40只大鼠异位心脏移植模型,手术成功率92.5%。动脉吻合时间12.5±2.3min,静脉吻合时间12.3±1.5 min,供心缺血时间37±3.5 min,受体血管阻断时间34.2±2.6 min,总手术时间90.2±4.8 min,出现的主要并发症为出血和供心复跳失败(各占5%、2.5%)。结论:改进的大鼠腹部异位心脏移植技术是一种简便、快速、有效、成功率高的模型制作方法。     

Lipid composition of the vascular system during infancy, childhood, and young adulthood

The object of this study was to determine the changes in lipid composition that occur in blood vessels from infancy to young adulthood. Analyses included levels of total cholesterol, total triglyceride, phospholipid, and cholesteryl ester fatty acids, and the distribution of saturated and unsaturated fatty acids. Triglyceride, total and monoenoic fatty acids, and linoleic acid were lower in the ascending, thoracic, and abdominal aorta than in the pulmonary artery and inferior vena cava. Phospholipids and arachidonic acid were higher in aortic segments than in the other two vessels. Aortic lipids showed significant changes with increasing age: total cholesterol and total fatty acids decreased from <1 wk to 5 yr, then increased to 22 yr of age. Triglycerides decreased whereas cholesteryl esters increased from 10 to 22 yr of age. Saturated fatty acids decreased from 1 wk to 10 yr, then remained relatively constant. Linoleic acid (3.7-9.8% of total fatty acids) and arachidonic acid (15.8-21.7%) both increased with age; the increase in cholesteryl linoleate was highly significant. After 10 yr of age, total cholesterol and total fatty acids were significantly higher in abdominal than in ascending and thoracic segments of aorta.     

Distribution of hepatic venous blood in the total cavo pulmonary connection: an in vitro study into the effects of connection geometry.

The total cavo pulmonary connection, or TCPC, is a surgical correction to congenital heart defects. The geometry of this connection has been shown to determine the fluid power loss as well as the distribution of hepatic fluid that enters through the inferior vena cava. In vitro studies were performed to measure the power loss and hepatic fluid distribution in models of the TCPC with four different geometries. It was found that a zero offset straight geometry provided good hepatic fluid distribution but large power loss. A zero offset flared geometry provided low power loss but poor hepatic fluid distribution. The optimal geometry from those tested was found to be the zero offset cowl geometry whereby an enlargement was made on one side of the inferior and superior vena cava. So long as the cowl was directed toward the pulmonary artery of lowest flow rate, low power loss and relatively good distribution of hepatic flow could be obtained.     

大鼠异位工作型心脏移植模型的建立

目的 建立大鼠腹腔异位工作型心脏移植动物模型.方法 Wistar大鼠30只,将供心主肺动脉与左房吻合,再将供心移植到受体腹部,将升主动脉与供心腹主动脉端侧吻合,使得左心房获得前负荷,成为工作型心脏.结果 成功建立大鼠腹腔异位工作型心脏移植模型,15例成功11例,手术成功率为73%.手术平均时间为(38.8±5.7)min.移植心脏获得长期存活,在28 d后通过超声观察可见供体心脏工作正常.结论 手术的关键是提高供心的摘取技术及受体的血管吻合技术,同时术后给与供心适当的按摩辅助也非常必要.此模型可以更好的用于移植免疫学的研究.