Hemodynamics in pig-to-baboon heterotopic thoracic cardiac xenotransplantation: Recovery from perioperative cardiac xenograft dysfunction and impairment by cardiac overgrowth.

INTRODUCTION: Orthotopic cardiac xenotransplantation has seen notable improvement, leading to the first compassionate use in 2022. However, it remains challenging to define the clinical application of cardiac xenotransplantation, including the back-up strategy in case of xenograft failure. In this regard, the heterotopic thoracic technique could be an alternative to the orthotopic procedure. We present hemodynamic data of heterotopic thoracic pig-to-baboon transplantation experiments, focusing on perioperative xenograft dysfunction and xenograft overgrowth. METHODS: We used 17 genetically modified piglets as donors for heterotopic thoracic xenogeneic cardiac transplantation into captive-bred baboons. In all animals, pressure probes were implanted in the graft's left ventricle and the recipient's ascending aorta and hemodynamic data (graft pressure, aortic pressure and recipient's heart rate) were recorded continuously. RESULTS: Aortic pressures and heart rates of the recipients' hearts were postoperatively stable in all experiments. After reperfusion, three grafts presented with low left ventricular pressure indicating perioperative cardiac dysfunction (PCXD). These animals recovered from PCXD within 48 h under support of the recipient's heart and there was no difference in survival compared to the other 14 ones. After 48 h, graft pressure increased up to 200 mmHg in all 17 animals with two different time-patterns. This led to a progressive gradient between graft and aortic pressure. With increasing gradient, the grafts stopped contributing to cardiac output. Grafts showed a marked weight increase from implantation to explantation. CONCLUSION: The heterotopic thoracic cardiac xenotransplantation technique is a possible method to overcome PCXD in early clinical trials and an experimental tool to get a better understanding of PCXD. The peculiar hemodynamic situation of increasing graft pressure but missing graft's output indicates outflow tract obstruction due to cardiac overgrowth. The heterotopic thoracic technique should be successful when using current strategies of immunosuppression, organ preservation and donor pigs with smaller body and organ size.

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats.

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor's cardiac function. This technique's key points are as follows: transecting the donor's abdominal aorta before harvesting to unload the donor's heart; perfusing the donor's coronary arteries with a cold cardioplegic solution; and topical cooling of the donor's heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate. Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one, which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor's heart is harvested, a stiff guidewire is inserted into the donor's brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient's circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

A Heterotopic Mouse Model for Studying Laryngeal Transplantation.

Laryngeal heterotopic transplantation, although a technically challenging procedure, offers more scientific analysis and cost benefits compared to other animal models. Although first described by Shipchandler et al. in 2009, this technique is not widely used, possibly due to the difficulties in learning the microsurgical technique and time required to master it. This paper describes the surgical steps in detail, as well as potential pitfalls to avoid, in order to encourage effective use of this technique. In this model, the bilateral carotid arteries of the donor larynx are anastomosed to the recipient carotid artery and external jugular vein, allowing for blood flow through the graft. Blood flow can be confirmed intraoperatively by the visualization of blood filling in the graft bilateral carotid arteries, reddening of the thyroid glands of the graft, and bleeding from micro vessels in the graft. The crucial elements for success include delicate preservation of the graft vessels, making the correct size arteriotomy and venotomy, and using the appropriate number of sutures on the arterial-arterial and arterial-venous anastomoses to secure vessels without leakage and prevent occlusion. Anyone can become proficient in this model with sufficient training and perform the procedure in approximately 3 h. If performed successfully, this model allows for immunologic studies to be performed with ease and at low cost.

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia.

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs.

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Evaluation of an alternative heterotopic transplantation model for ovarian tissue to test pharmaceuticals improvements for fertility restoration.

BACKGROUND: Ovarian tissue cryopreservation and transplantation (OTCTP) is currently the main option available to preserve fertility in prepubertal patients undergoing aggressive cancer therapy treatments. However, a major limitation of OTCTP is follicle loss after transplantation. The mouse is a model of choice for studying ovarian function and follicle development after ovarian tissue grafting in vivo. In these mouse models, ovarian tissue or ovaries can be transplanted to different sites. Our aim was to evaluate a new alternative to heterotopic transplantation models that could be useful to test pharmaceutical improvement for ovarian grafts after OTCTP. METHODS: Slow frozen murine whole ovaries were transplanted into the mouse ears (between the external ear skin layer and the cartilage). Ovarian transplants were recovered after 3, 14 or 21 days. Grafts were analyzed by immunohistochemistry and follicle density analyses were performed. RESULTS: An increase of ovarian vascularization (CD31 and Dextran-FITC positive staining), as well as cellular proliferation (Ki67 staining) were observed 3 weeks after transplantation in comparison to 3 days. Fibrosis density, evaluated after Van Gieson staining, decreased 3 weeks after transplantation. Furthermore, transplantation of cryopreserved ovaries into ovariectomized mice favored follicle activation compared to transplantation into non-ovariectomized mice. CONCLUSION: The present study indicates that surgical tissue insertion in the highly vascularized murine ear is an effective model for ovarian grafting. This model could be helpful in research to test pharmaceutical strategies to improve the function and survival of cryopreserved and transplanted ovarian tissue.

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model.

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients' long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-side anastomosis to the recipient's aorta and inferior vena cava using stitches. In this model, the donor's heart is implanted by end-to-end anastomosis to the recipient's carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

Establishment of Heterotopic Hind Limb Transplantation Model in the Mouse.

BACKGROUND: The mouse is the most widely used animal for establishing in vivo models in transplant research. However, because of the advanced microsurgical skills required for these operations, the vascularized composite transplantation model in mouse has proven to be technically challenging. The purpose of this report is to describe novel modifications in surgical techniques to establish a consistent and reliable mouse model of hind limb transplantation. METHODS: Forty C57BL/6 male mice, half as donors and half as recipients, were used in this study. The donor hind limb was harvested and transplanted into the recipient's ipsilateral cervical region by anastomosing the donor femoral artery to the recipient common carotid artery with a modified sleeve technique. The donor femoral vein was mounted with a modified cuff and inserted into the recipient external jugular vein. The graft was evaluated at 2 weeks postoperatively. RESULTS: The modified cuff and modified sleeve technique facilitated anastomoses. The time spent on either of the donor operation and recipient operation was about 45 minutes. The graft survival rate was 80% (16 of 20) at 2 weeks after transplant. There was minimal blood loss and no infections were noted. CONCLUSIONS: Revised surgical techniques using a modified cuff proved to be a safe, reliable, and reproducible strategy in establishing a mouse model of hind limb heterotopic transplantation. The consistent graft survival in this syngeneic study demonstrates that this model can serve as a useful tool for further studies in vascularized composite transplantation.

Heterotopic Porcine Cardiac Xenotransplantation in the Intra-Abdominal Position in a Non-Human Primate Model.

Heterotopic cardiac transplantation in the intra-abdominal position in a large animal model has been essential in the progression of the field of cardiac transplantation. Our group has over 10 years of experience in cardiac xenotransplantation with pig to baboon models, the longest xenograft of which survived over 900 days, with rejection only after reducing immunosuppression. This article aims to clarify our approach to this model in order to allow others to share success in long-term survival. Here, we demonstrate the approach to implantation of a cardiac graft into the intra-abdominal position in a baboon recipient for the study of transplantation and briefly highlight our model's ability to provide insight into not only xenotransplantation but across disciplines. We include details that have provided us with consistent success in this model; performance of the anastomoses, de-airing of the graft, implantation of a long-term telemetry device for invasive graft monitoring, and ideal geometric positioning of the heart and telemetry device in the limited space of the recipient abdomen. We additionally detail surveillance techniques to assess long-term graft function.

Membrana amniótica, aplicaciones clínicas e ingeniería tisular. Revisión de su uso oftalmológico / Amniotic membrane, clinical applications and tissue engineering. Review of its ophthalmic use

La utilización de la membrana amniótica en la oftalmología está en alza en los últimos años debido a: sus múltiples propiedades biológicas y tectónicas, la mejora en el proceso de su obtención, la facilidad de uso y el avance en la ingeniería tisular. La membrana amniótica se ha convertido en uno de los principales tratamientos coadyuvantes, tanto en la cirugía oftalmológica como en otras especialidades médico-quirúrgicas. El desarrollo de la ingeniería tisular ha permitido su utilización no solo en su forma clásica sino también mediante gotas y otras presentaciones. A lo largo del artículo hemos realizado un resumen de los distintos pasos previos a su uso (preparación y conservación), de las distintas técnicas quirúrgicas y de sus principales aplicaciones clínicas

The use of amniotic membrane in ophthalmology has been increasing in recent years due to its multiple biological and tectonic properties, improvement in the process of obtaining, ease of use, and advancement in tissue engineering. The amniotic membrane has become one of the main adjuvant treatments, in ophthalmic surgery as well as in other medical-surgical specialties. The development of tissue engineering has allowed it to be used, not only in its classic form, but also by the use of drops and other presentations. The different steps prior to its use (preparation and conservation), the different surgical techniques, and their main clinical applications are described throughout the article

Amniotic membrane, clinical applications and tissue engineering. Review of its ophthalmic use. / Membrana amniótica, aplicaciones clínicas e ingeniería tisular. Revisión de su uso oftalmológico.

The use of amniotic membrane in ophthalmology has been increasing in recent years due to its multiple biological and tectonic properties, improvement in the process of obtaining, ease of use, and advancement in tissue engineering. The amniotic membrane has become one of the main adjuvant treatments, in ophthalmic surgery as well as in other medical-surgical specialties. The development of tissue engineering has allowed it to be used, not only in its classic form, but also by the use of drops and other presentations. The different steps prior to its use (preparation and conservation), the different surgical techniques, and their main clinical applications are described throughout the article.

Clinical effect of thumb finger reconstruction using dorsal foot flap transplant for treating thumb defects.

It was found that conventional toe graft alone could not meet the patients' needs for wound repair, so we hypothesized that it would be more effective to treat thumb and finger defect by toe graft with dorsalis foot flap. This prospective study was conducted in 104 thumb defect patients to investigate the clinical effect of thumb reconstruction using toe graft with dorsal foot flap for the treatment of thumb defects. These patients were randomly divided into the dorsal foot group and the control group by randomized double-blind method, with 52 patients in each group. The second toe was used for thumb reconstruction transplant in both the groups. After thumb reconstruction, the abdominal pedicled flap was used to repair the surgical wound in the control group whereas the dorsal foot flap was used to repair the surgical wound in the dorsal foot group. Three months after surgery, the efficacy of surgical treatment, evaluation of two-point discrimination, postoperative complications, function of reconstructed thumb, operation time, and hospitalization time were recorded and compared between the two groups. MHOQ questionnaire was used to evaluate and compare the patients' satisfaction with finger reconstruction in both the groups. The surgical therapeutic effect, the function of the reconstructed thumb, and satisfaction with finger reconstruction were significantly higher in the dorsal foot group compared to the control group (all p < 0.05). The postoperative two-point discrimination, postoperative complication rate, operation time, and hospitalization time of patients in the dorsal foot group were significantly lower compared to the control group (all p < 0.05). Thumb finger reconstruction using the second toe transplant with dorsal foot flap had a beneficial effect on thumb defect patients. It can effectively improve finger function and sensory recovery of patients while reducing complications.

Neovaginoplasty Using Nile Tilapia Fish Skin as a New Biologic Graft in Patients with Mayer-Rokitansky-Küster-Hauser Syndrome.

Mayer-Rokitansky-Küster-Hauser syndrome is the second most common cause of primary amenorrhea, trailing only to gonadal dysgenesis. Neovaginoplasty is an appropriate treatment option for patients who have failed dilation therapy. Several biomaterials have been used in this procedure, including peritoneum, amnion, skin grafts, and myocutaneous flaps. Nile Tilapia Fish Skin has noninfectious microbiota, morphologic structure comparable to human skin, and high in vivo bioresorption. In addition, it showed good outcomes when used as a xenograft for burn treatment. Thus, we suggest it as a new biologic graft for vaginal agenesis management. In this descriptive study, neovaginoplasty using Nile Tilapia Fish Skin offered 3 patients an anatomic and functional neovagina via a simple method with potential long-term effectiveness. When postsurgical dilation was performed correctly, a vaginal length greater than 6 cm was maintained at 180 days follow-up. Histologic and immunohistochemical analyses revealed the presence of stratified squamous epithelium with high expression of cytokeratins and fibroblast growth factor, matching the characteristics of normal adult vaginal tissue. We believe that further studies will show Nile Tilapia Fish Skin to be a relevant option in the therapeutic arsenal of Mayer-Rokitansky-Küster-Hauser syndrome.

Considerations for an Alternative Site of Islet Cell Transplantation.

Islet cell transplantation has been limited most by poor graft survival. Optimizing the site of transplantation could improve clinical outcomes by minimizing required donor cells, increasing graft integration, and simplifying the transplantation and monitoring process. In this article, we review the history and significant human and animal data for clinically relevant sites, including the liver, spleen, and kidney subcapsule, and identify promising new sites for further research. While the liver was the first studied site and has been used the most in clinical practice, the majority of transplanted islets become necrotic. We review the potential causes for graft death, including the instant blood-mediated inflammatory reaction, exposure to immunosuppressive agents, and low oxygen tension. Significant research exists on alternative sites for islet cell transplantation, suggesting a promising future for patients undergoing pancreatectomy.

Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs.

Implanted bioengineered livers have not exceeded three days of continuous perfusion. Here we show that decellularized whole porcine livers revascularized with human umbilical vein endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleens had been removed can sustain perfusion for up to 15 days. We identified peak glucose consumption rate as a main predictor of the patency of the revascularized bioengineered livers (rBELs). Heterotopic implantation of rBELs into pigs in the absence of anticoagulation therapy led to sustained perfusion for three days, followed by a pronounced immune responses directed against the human endothelial cells. A 10 day steroid-based immunosuppression protocol and a splenectomy at the time of rBEL implantation reduced the immune responses and resulted in continuous perfusion of the rBELs for over two weeks. We also show that the human endothelial cells in the perfused rBELs colonize the liver sinusoids and express sinusoidal endothelial markers similar to those in normal liver tissue. Revascularized liver scaffolds that can maintain blood perfusion at physiological pressures might eventually help to overcome the chronic shortage of transplantable human livers.

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique.

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation. Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

A Systematic Study on Reproductive Endocrine Function Recovery From Subcutaneous Ovarian Autotransplantation in Mice After 2 Weeks.

BACKGROUND: Heterotopic ovarian transplantation can not only restore the reproductive and endocrine function of animals but can also be studied with a specialized animal model. The aim of the study was to determine whether the reproductive endocrine function of the subcutaneously transplanted ovary was restored after 2 weeks. METHODS: The ovaries of 7-week-old mice were autologously transplanted into the back muscle. Fourteen days later, the ovarian structure was examined by hematoxylin and eosin staining. We continuously observed vaginal smears for changes in the estrous cycle. Estrogen and androgen concentrations were detected on the 14th day. The oocytes were collected and then used for in vitro maturation (IVM) and in vitro fertilization (IVF). RESULTS: The cyclical estrous cycle changes were similar to those of the control group. There were no differences in the serum androgen and estrogen levels between the graft and control groups. The oocytes were able to develop into blastocysts after IVM and IVF. These results indicated that ovarian endocrine and reproductive function were restored within 2 weeks. CONCLUSION: Our studies have shown that this ovarian heterotopic autotransplantation technique is able to restore steroidogenic and gametogenic functions at day 14 after transplantation. So far, the 14th day after transplantation is a landmark during the recovery from autologous heterotopic ovarian transplantation in the back of the mouse. This time point is the appropriate window to study heterotopic ovarian transplantation in mice.

Intraventricular placement of a spring expander does not attenuate cardiac atrophy of the healthy heart induced by unloading via heterotopic heart transplantation.

An important complication of the prolonged left ventricle assist device support in patients with heart failure is unloading-induced cardiac atrophy which proved resistant to various treatments. Heterotopic heart transplantation (HTx) is the usual experimental model to study this process. We showed previously that implantation of the newly designed intraventricular spring expander can attenuate the atrophy when examined after HTx in the failing heart (derived from animals with established heart failure). The present study aimed to examine if enhanced isovolumic loading achieved by implantation of the expander would attenuate cardiac post-HTx atrophy also in the healthy heart. Cardiac atrophy was assessed as the ratio of the transplanted-to-native heart weight (HW) and its degree was determined on days 7, 14, 21 and 28 after HTx. The transplantation resulted in 32±3, 46±2, 48±3 and 46±3 % HW loss when measured at the four time points; implantation of the expander had no significant effect on these decreases. We conclude that enhanced isovolumic loading achieved by intraventricular implantation of the expander does not attenuate the development of cardiac atrophy after HTx in the healthy heart. This indicates that such an approach does not represent a useful therapeutic measure to attenuate the development of unloading-induced cardiac atrophy.

A case report and follow-up of the first live birth after heterotopic transplantation of cryopreserved ovarian tissue in Eastern Europe.

BACKGROUND: Ovarian insufficiency is a major concern for long-term cancer survivors. Although semen freezing is well established to preserve male fertility, the possibilities to secure post-cancer female fertility are mostly limited to oocyte or embryo freezing. These methods require time-consuming ovarian stimulation with or without in vitro fertilization (IVF) that evidently delays cancer therapy. Ovarian tissue cryopreservation and subsequent thawed tissue autotransplantation are considered the most promising alternative strategy for restoring the fertility of oncology patients, which has not yet received the full clinical acceptance. Therefore, all successful cases are needed to prove its reliability and safety. CASE PRESENTATION: Here we report a single case in Estonia, where a 28-year-old woman with malignant breast neoplasm had ovarian cortex cryopreserved before commencing gonadotoxic chemo- and radiotherapy. Two years after cancer therapy, the patient underwent heterotopic ovarian tissue transplantation into the lateral pelvic wall. The folliculogenesis was stimulated in the transplanted tissue by exogenous follicle-stimulating hormone and oocytes were collected under ultrasound guidance for IVF and embryo transfer. The healthy boy was born after full-term gestation in 2014, first in Eastern Europe. CONCLUSION: Despite many countries have reported the first implementation of the ovarian tissue freezing and transplantation protocols, the data is still limited on the effectiveness of heterotopic ovarian transplant techniques. Thus, all case reports of heterotopic ovarian tissue transplantation and long-term follow-ups to describe the children's health are valuable source of clinical experience.