Non-suture Cuff Technique Research Articles

Arsenic Trioxide Combining Leflunomide Activates Nrf2-ARE-HO-1 Signaling Pathway and Protects Heart Xenografts.

To investigate the molecular mechanisms underlying the effects of arsenic trioxide (As2O3) in combination with leflunomide on the hamster-to-rat heart xenotransplant. Transplantation of LVG hamster hearts to Lewis rats was performed by anastomosis of vessels in the neck using end-to-end anastomosis with a non-suture cuff technique. Four groups of recipient rats (n=6 in each) were treated with normal saline (control), As2O3 [5 mg/(kg·day) intraperitoneally], leflunomide [5 mg/(kg·d) orally], or leflunomide [5 mg/(kg·d)+As2O3 [5 mg/(kg·d)] in combination. Donor hearts and/or rat spleens were harvested and analyzed 4 days after transplantation. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis were performed to detect the expression of the nuclear factor erythroid-derived factor 2-related factor (Nrf2) and its target gene heme oxygenase-1 (HO-1), Treg cell marker fork-head Box P3 (FOXP3), apoptosis-associated proteins Bcl-2, Bax, and cleaved caspase-3. Immunohistochemical staining was used to detect the levels of inflammatory natural killer cell and macrophage infiltration, intercellular cell adhesion molecule-1 (ICAM-1) and complement C3. Expression of Nrf2-ARE-HO-1 signaling pathway was upregulated in heart xenografts in rats treated with As2O3 plus leflunomide compared with control rats or rats treated with either drug alone (P<0.01), and this was accompanied by an increased Treg cells in the recipient rat spleen (P<0.01). In contrast, the expressions of Bax, cleaved caspase-3, ICAM-1, and complement C3, and infiltration of inflammatory cells in the xenografts were inhibited by As2O3 plus leflunomide treatment (P<0.01). Combination treatment with As2O3 and leflunomide protected hamster heart-xenografts in recipient rats.

A novel rat microsurgical model to study the immunological characteristics of male genital tissue in the context of penile transplantation.

Penis transplantation represents an exciting new avenue for restoration of male genitalia and function after devastating tissue loss. This animal model is designed to fill a critical void to study immunologic aspects related to reconstructive transplantation of male genitalia. A rat penile graft dissection was designed based on the internal pudendal arteries and dorsal penile vein and includes the skin of the prepuce. A nonsuture cuff technique was used to anastomose the graft vessels to the recipient superficial epigastric and femoral vessels. Seventy-seven penile transplantations were performed. Graft design yields suitable caliber and length of vessels at the radix of the penis. Anastomosis of the dorsal penile vein and the internal pudendal arteries insures optimal graft perfusion. The nonsuture cuff technique allows for successful microvascular anastomosis by a single surgeon with an average overall operative time of 2.5h. Long-term graft survival (>30days) was observed in syngeneic transplants. We have established a robust murine model with ideal vascular perfusion of penile tissue to study the unique immunobiology of male genitourinary allotransplantation. Heterotopic inset further allows for visual monitoring of graft viability, while the native penis serves as an optimal control.

Combined treatment with vitamin D3 and antibody agents suppresses secondary heart transplant rejection in the early postoperative period

BackgroundAccelerated transplant rejection mediated by donor reactive memory T cells is another barrier to the induction of graft tolerance. The aim of this study was to investigate the immunosuppressing effects of vitamin D (1,25(OH)2D3), administered alone or in combination with a costimulatory blockade treatment, on rejection of secondary heart allografts in a mouse model. MethodsCircular full-thickness skin grafts from BALB/c mice were cut and grafted onto the lumbar regions of C57BL/6 mice as allo-primed recipients. Four weeks after skin grafting, the vascularized hearts from the BALB/c mice were transplanted heterotopically into the allo-primed recipients using a non-suture cuff technique. The recipients were then randomly divided into four groups and given either intraperitoneal injection of isotype, Ab, 1,25(OH)2D3, or a combination of Ab and 1,25(OH)2D3. Allograft incidence was determined by hematoxylin-eosin staining, and cytokine expression was assessed by the quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and cytometric bead arrays. Spleen cells from the recipient were used to assess mixed lymphocyte reactions. Memory T cells and regulatory T cells (Tregs) in spleen cells were measured by flow cytometry. ResultsThe median allograft survival time was longer with the combined treatment with Ab and 1,25(OH)2D3 than with no treatment or with treatment with Ab or 1,25(OH)2D3 alone. The grafts were protected from infiltration by inflammatory cells and by inhibition of interleukin 2 and interferon gamma expression. Rejection was initially suppressed in the early postoperative period by a reduction in the number of memory T cells and induction of Foxp3+ Tregs, but this effect disappeared by day 15 after transplantation upon withdrawal of the treatment. ConclusionVitamin D3 administered as an immunosuppressive agent, when combined with monoclonal antibody treatment, may protect heart grafts from memory T cell responses in a secondary heart transplant model.

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.

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

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

Improved Cuff Technique and Intraoperative Detection of Vascular Complications for Hind Limb Transplantation in Mice.

BackgroundVascularized composite tissue allotransplantation (VCA) from a cadaveric donor has now become a clinical reality and the treatment modality of choice for patients with devastating injuries, deformities, and complex tissue defects. However, many VCA patients experience severe toxicities due to the strong immunosuppression required secondary to high antigenicity of the grafts. To improve immunosuppressive protocols for VCA, feasible and reliable preclinical models are necessary. The purpose of this study was to introduce new techniques to an established preclinical VCA model to accelerate future investigations.MethodsC57BL/6 (H-2b) and BALB/c (H-2d) mice were used to perform VCA as recipients and donors, respectively. Surgery time, success rate, associated complications, and mortality were analyzed. Blood flow in grafts was interrogated with laser speckle image (LSI).ResultsA nonsuture cuff technique was used with the abdominal aorta for end-to-end anastomosis. The cuff technique demonstrated efficiency for donor surgery (52 ± 10 minutes for donor vs. 45 ± 8 minutes for recipient surgery). Successful revascularization was achieved in 27 (90%) of 30 transplants. The majority of surgical complications occurred within 48 hours including artery occlusion, venous occlusion, cerebral stroke, and minor bleeding without mortality. LSI was useful in detecting intraoperative vascular complications with display patterns predictive of complication type.ConclusionsThe described techniques may facilitate a more efficient heterotopic hind limb transplantation mouse model of VCA.

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of >90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates. In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of >90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

A Mouse Model of Vascularized Skin Transplantation.

Allogeneic skin tissues are highly antigenic and induce intensive immune rejection after composite tissue allotransplantation. Mouse models have advantages in mechanistic studies of immune rejection. However, due to technical challenge in vascular anastomosis with suture technique, mouse vascularized skin allotransplantation models are not widely used in studies of immune rejection. Therefore, the authors propose vascular anastomosis through cuff technique during allotransplantation of mouse donor free groin skin flaps to either recipient inguinal or cervical site. Free groin skin flaps from BALB/c or C57BL/6 donor mice were transplanted to the groin (inguinal vascularized skin transplantation [IVST]) or to the neck of recipient sites (cervical vascularized skin transplantation [CVST]) of C57BL/6 mice. A nonsuture cuff technique was utilized to anastomose the donor vessels with either femoral vessels in IVST recipients or common carotid arteries and external jugular veins in CVST mice. Immunosuppressant drugs were used in the allogeneic skin group. The overall success rate was higher in the CVST (88.5%) when compared with the IVST (78.9%). Total operation time in CVST mice lasted 96 minutes (95% confidence interval, 92-101 minutes) that was shorter than that for IVST mice (136 minutes, 95% confidence interval, 127-176 minutes; P < 0.01). Complications, such as hindlimb necrosis and self-mutilation, were observed in IVST mice. Rapamycin (3 mg/kg, daily) significantly prolonged vascularized skin allografts survival with median survival time of 80 days. All syngeneic grafts survived for more than 80 days. We developed a novel mouse vascularized skin transplantation model that is feasible for the study of clinically relevant skin rejection and tolerance.

Abstract P14: A Novel Forelimb Orthotopic Allotransplantation Model in Mouse

PURPOSE: In translational research related to Vascularized composite allotransplantation (VCA), mouse models provide a profoundly relevant means to successfully studying of crucial aspects related to the immunobiology of graft rejection and acceptance, neuronal reinnervation of sensory and motor function and associated cortical reintegration of lost extremity function. The model developed here is designed to address this critical void to allow for mouse orthotopic forelimb transplantation, a tool to studying both aspects of functional recovery and immunobiology related to VCA in a genetically versatile mouse model system. METHODS: Male Balb/c donors and C57BL/6 recipients (8–10 weeks) mice were used to perform syngeneic and allogeneic orthotopic forelimb transplantation. Co-stimulation blockaded (CTLA4-Ig & MR-1) was used for immunosuppression. A circumferential skin incision was made at the shoulder. The pectoralis major muscle insertion was released at the humerus and reflected. The vascular pedicle was dissected from the axilla to the cubital fossa and transected at the level of the proximal axillary artery. Median and ulnar nerves were identified and transected proximal to the cubital fossa. The radial nerve was identified and transected dorsally at the intersection of medial and distal third of the humerus. Both flexor and extensor muscle groups were cauterized and transected at mid humerus level. In the recipient animal shoulder mobility was preserved and vessels and nerves were transected proximal to the cubital fossa. The graft was secured by muscle approximation with 6-0 Polysorb and by using a 26G spinal needle as an intramedullary rod for osteosynthesis. A non-suture cuff technique was employed to anastomose donor axillary vessels with recipient brachial (mid-arm) vessels. Tension free primary end-to-end neurorrhaphy was performed in median, ulnar and radial nerves using 11-0 microsuture. RESULTS: Long term animal and graft survival in syngeneic transplant combinations demonstrated the feasibility of orthotopic mouse forelimb transplantation with only limited behavioral disabilities (>60 days; N=3).The non-suture cuff technique allows for successful supermicrovascular anastomosis of the donor axillary vessel with the recipient brachial vessel. The feasibility of radial, median and ulnar nerve coaptation provides the basis for studying functional recovery as well as cortical reintegration of upper extremity function after VCA. Long term allograft survival (>4 months; N=3) underpins the applicability of orthotopic forelimb transplantation for studying the immunobiology of functional extremity reconstruction using VCA. CONCLUSION: The described orthotopic forelimb transplantation model in the mouse is designed to add an additional research model for VCA by providing the option of using the entire range of genetically engineered mice for research related to transplant immunology, neuroregeneration, and functional recovery after upper extremity transplantation.

Orthotopic Hind Limb Transplantation in the Mouse.

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models. Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

The role of soluble and insoluble gastric fluid components in the pathogenesis of obliterative bronchiolitis in rat lung allografts.

Repetitive gastric fluid aspirations have been shown to lead to obliterans bronchiolitis (OB), but the component or components of gastric fluid that are responsible are unknown. This study investigates the role of particulates and, separately, soluble material in gastric fluid during the development of OB. Whole gastric fluid (WGF) was collected from male Fischer 344 (F344) rats and separated by centrifugation into particle reduced gastric fluid (PRGF) and particulate components resuspended in normal saline (PNS). Orthotopic left lung transplants from male Wistar-Kyoto rats into F344 rats were performed using a modification of the nonsuture external cuff technique with prolonged cold ischemia. Rats were subjected to weekly aspiration of 0.5 ml/kg of WGF (n = 9), PRGF (n = 10), PNS (n = 9), or normal saline (control, NS; n = 9) for 8 weeks following transplantation. Lung allografts treated with WGF, PRGF, or PNS developed a significantly greater percentage of OB-like lesions compared with the control. No statistical difference was observed when comparing the fibrosis grades or the percentage of OB lesions of WGF, PRGF, and PNS groups, suggesting that both soluble and insoluble components of gastric fluid can promote the development of aspiration-induced OB and fibrosis in lung allografts.

The protective role of interleukin-18 binding protein in a murine model of cardiac ischemia/reperfusion injury.

IL-18, a proinflammatory cytokine, is produced by macrophages, epithelial cells, T cells, neutrophils, NK-T cells, and B cells, and has been implicated in the pathophysiology of a variety of inflammatory diseases including ischemia/reperfusion (IR) injury, transplant rejection, and autoimmune disease. Recent study indicated that neutralization of IL-18 with anti-IL-18 antibody or IL-18-binding protein (IL-18BP) ameliorates IR-induced myocardial injury. However, the mechanism needs to be further investigated. In our current study, syngeneic heterotopic heart transplantation was performed by a modified non-suture cuff technique. We found that IL-18BP treatment ameliorated cardiomyocyte necrosis and infiltration of CD4(+) T cells, neutrophils, and macrophages. IL-18BP-treated mice exhibited decreased expression of inflammatory cytokines including IL-1β, IL-23, IL-18, and IL-17. IL-18BP treatment suppressed Th17 differentiation in vivo and in vitro. Adoptive transfer of T cells from IL-18BP-treated mice showed alleviated cardiac IR injury when compared with that transferred from control mice. Furthermore, the decreased infiltration of mononuclear cells and production of troponin T (TnT) induced by IL-18BP treatment were both abrogated by additional administration of recombinant mouse IL-17 (rmIL-17). These data revealed a protective role of IL-18BP in cardiac IR injury. Above all, IL-18BP ameliorates cardiac IR injury in part through suppression of Th17 differentiation.

MEMS-Based Handheld Fourier Domain Doppler Optical Coherence Tomography for Intraoperative Microvascular Anastomosis Imaging

PurposeTo demonstrate the feasibility of a miniature handheld optical coherence tomography (OCT) imager for real time intraoperative vascular patency evaluation in the setting of super-microsurgical vessel anastomosis.MethodsA novel handheld imager Fourier domain Doppler optical coherence tomography based on a 1.3-µm central wavelength swept source for extravascular imaging was developed. The imager was minimized through the adoption of a 2.4-mm diameter microelectromechanical systems (MEMS) scanning mirror, additionally a 12.7-mm diameter lens system was designed and combined with the MEMS mirror to achieve a small form factor that optimize functionality as a handheld extravascular OCT imager. To evaluate in-vivo applicability, super-microsurgical vessel anastomosis was performed in a mouse femoral vessel cut and repair model employing conventional interrupted suture technique as well as a novel non-suture cuff technique. Vascular anastomosis patency after clinically successful repair was evaluated using the novel handheld OCT imager.ResultsWith an adjustable lateral image field of view up to 1.5 mm by 1.5 mm, high-resolution simultaneous structural and flow imaging of the blood vessels were successfully acquired for BALB/C mouse after orthotopic hind limb transplantation using a non-suture cuff technique and BALB/C mouse after femoral artery anastomosis using a suture technique. We experimentally quantify the axial and lateral resolution of the OCT to be 12.6 µm in air and 17.5 µm respectively. The OCT has a sensitivity of 84 dB and sensitivity roll-off of 5.7 dB/mm over an imaging range of 5 mm. Imaging with a frame rate of 36 Hz for an image size of 1000(lateral)×512(axial) pixels using a 50,000 A-lines per second swept source was achieved. Quantitative vessel lumen patency, lumen narrowing and thrombosis analysis were performed based on acquired structure and Doppler images.ConclusionsA miniature handheld OCT imager that can be used for intraoperative evaluation of microvascular anastomosis was successfully demonstrated.

Graft Function After Heterotopic Rat Heart Transplant With an Isolated Reperfused Working Heart: A Methodic Consideration

Assessment of graft function in experimental cardiac transplant has been underused by insufficient methods (echo-cardiography, magnetic resonance imaging). The isolated reperfused working-heart model is an excellent tool for hemodynamic evaluation of rodent hearts. So far, it has never been used in a cardiac transplant setting. Our study tries to combine the in vivo technique of a rat heart transplant model with the ex vivo method of an isolated working heart. Heterotopic heart transplants have been performed in rats with a nonsuture cuff technique. After 8 hours of cold ischemia and 24 hours of reperfusion, grafts were mounted on the working heart. To assess graft function, cardiac output was measured with increasing levels of afterload pressure. Nontransplanted hearts were mounted directly to the working heart apparatus to serve as a control group. Each heart was assessed subjectively by the Stanford score before being mounted on the working-heart apparatus. The working-heart assessment detected significantly impaired graft function in the transplant group compared with control hearts. In contrast, functional assessment with the score-system could not detect any difference between transplanted and native hearts. The isolated working-heart model is an excellent tool for assessing graft function after experimental heart transplant in rodents.

Mouse Hind Limb Transplantation: A New Composite Tissue Allotransplantation Model Using Nonsuture Supermicrosurgery

The development of microsurgical techniques has facilitated the establishment of vascularized composite tissue transplant models in small mammals. Because the mouse would be the ideal model to study various composite tissue allotransplantation (CTA)-related problems, we designed two new surgical techniques for orthotopic (ORT) and heterotopic (HET) hind limb transplantation. BALB/c hind limbs were transplanted to BALB/c or C57BL6 recipients using a nonsuture cuff technique. ORT: donor femoral vessels were anastomosed to recipient femoral vessels, the sciatic nerve approximated end-to-end and osteosynthesis was performed using an intramedullary rod. HET/cervical: Donor femoral vessels of a reduced size osteomyocutaneous hind limb CTA were anastomosed to recipient common carotid artery and external jugular vein without nerve approximation. Both procedures could be performed with a high success rate (ORT: 62%; HET: 90%). Donor operation lasted for 100±12 min and recipient operation 114±27 min (ORT) and 54±16 min (HET). Complication rates in terms of bleeding, and thrombosis at the cuff side was slightly higher in the ORT group. All syngeneic grafts survived long term (>100 days). FK506 (2 mg/kg) significantly prolonged graft survival (87±22 days) when compared with untreated controls (6±1 day). Functional evaluation of ORT grafts by means of video gait kinematics and CatWalk analysis revealed specific differences of gait parameters when compared with nontransplanted controls (P<0.05). The ORT hind limb transplant model seems to be best suited to study functional outcome and nerve regeneration in CTA. The technically less demanding HET/cervical model may be used to investigate basic immunology and clinically relevant questions related to acute and chronic rejection, and ischemia reperfusion injury in reconstructive transplantation.

Effect of L-N6-(1-tminoethyl) Lysine on ischemia-reperfusion injury in a rat model of lung transplantation

Objective To investigate the effect of L-N6-(1-iminoethyl) Lysine(L-NIL) on ischemia-reperfusion (I/R) -induced lung injury in a rat model of lung transplantation. Methods Pathogen free male SD rats weighing 250-350g were used as donor and recipient rats in this study. The animals were randomly divided into 3groups (n = 6 each): sham operation group (group S); lung tratsplantation group (group L) and lung transplantation + L-NIL (selective iNOS inhibitor) group (group L-NIL). In group L and L-NIL orthotopic left lung allograft transplantation was performed. In group L-NIL 3 mg/kg was injected iv at the beginning of reperfusion. The donor lungs were removed from live donor rats and placed in Euro-collins solution at 4 ℃. The lung transplantation was performed under microscope and non-suture cuff technique was used. The implanted donor lungs were ventilated and reperfused. 0.5% Evans blue 0.2 ml was injected iv during reperfusion. The donor lungs were removed after being implanted, ventilated and reperfused for 2 h for microscopic examination and determination of iNOS, endothelial NOS (eNOS) and myeloperoxidase (MPO) activity and malondialdehyde (MDA) and Evans blue content in the lung tissue and W/D lung weight ratio. Results Lung transplantation significantly inceased W/D ratio, iNOS and MPO activity, and Evans blue and MDA content in the lung tissue and decreased eNOS activity in group L as compared with group S. L-NIL iv significantly attenuated the increase in the variables mentioned above and ameliorated capillary congestion and inflammatory cell infiltration in the lung. Conclusion Intravenous L-NIL administered at the beginning of reperfusion can reduce I/R injury to the transplanted donor lungs. Key words: Lung transplantation; Reperfusion injury; L-N6-(1-Iminoethyl)Lysine

Comparison of surgical procedures for vascular and airway anastomoses that utilize a “modified” non-suture external cuff technique for experimental lung transplantation in rats

Background Lung transplantation in rats is technically difficult and results may vary. The non-suture cuff technique (non-suture [NS]), which involves using a cuff for anastomoses of vessels and airways, is commonly utilized to perform transplant procedures. However, the standard bronchial cuff may occlude the lumen, resulting in diminished graft survival. In contrast, the non-suture cuff technique for vascular anastomoses with bronchial suturing (bronchial suture [BS]), although more technically difficult, is known to have prolonged bronchial patency. We developed a “modified” NS technique that uses a larger and longer cuff for anastomoses and compared the efficacy of this procedure to the BS technique for lung transplantation. Methods The BS procedure was performed in 146 transplant procedures. The modified NS procedure, which uses a larger cuff than the standard NS procedure, was performed in 113 procedures. Results Although total ischemic times were comparable, the total operation time in the NS group was significantly shorter than in the BS group ( p < 0.0001). Bronchial leakage occurred in 9.6% of BS group patients and 0% of NS group patients. Survival rates were comparable between groups. Although the BS technique is widely acknowledged to provide prolonged patency of bronchial anastomosis, graft survival in the NS group was observed up 17 months post-transplant. Conclusions These data show that the NS technique, which is less technically difficult, results in shorter operative times compared with the BS procedure, and results in durable anastomoses of lung grafts.