High-volume hydrodissection of intramuscular perforators of deep inferior epigastric perforator (DIEP) flaps in Sprague-Dawley rats has previously demonstrated a significant reduction in dissection time while simultaneously increasing the safety of dissection. However, increasing volumes of fluid injected into the closed rectus compartment may have adverse consequences on perfusion to the overlying flap. A prospective experimental animal study was performed to define a safe upper limit of volume injection for high-volume hydrodissection. Eight Sprague-Dawley rats underwent bilateral "DIEP" flap dissections with varying volumes of fluid (1, 3, 6, and 9 mL) injected into the study side. The primary outcome measurement was overlying tissue perfusion, measured using laser Doppler imaging, at 6 separate time points during the flap harvest. Although no significant difference in perfusion was noted between study and control sides despite increasing volumes of injection, a trend toward significant altered perfusion was noted immediately after injection in the 9-mL study group. Six milliliters is defined as the safe upper limit volume of injection into the closed rectus compartment without significantly altering overlying flap perfusion in our Sprague-Dawley rats. Using volumetric analysis, these data translate to 425 mL as the safe upper limit for high-volume hydrodissection for a single average sized human rectus sheath during DIEP flap harvesting. The mechanical and potentially pharmacologic implications of these data in humans remain to be seen.
Objective. To describe the role of imaging in vascular composite allotransplantation based on one institution's experience with upper extremity allotransplant patients. Methods. The institutional review board approved this review of HIPAA-compliant patient data without the need for individual consent. A retrospective review was performed of imaging from 2008 to 2011 on individuals undergoing upper extremity transplantation. This demonstrated that, of the 19 patients initially considered, 5 patients with a mean age of 37 underwent transplantation. Reports were correlated clinically to delineate which preoperative factors lead to patient selection versus disqualification and what concerns dictated postoperative imaging. Findings were subdivided into musculoskeletal and vascular imaging criterion. Results. Within the screening phase, musculoskeletal exclusion criterion included severe shoulder arthropathy, poor native bone integrity, and marked muscular atrophy. Vascular exclusion criterion included loss of sufficient arterial or venous supply and significant distortion of the native vascular architecture. Postoperative imaging was used to document healing and hardware integrity. Postsurgical angiography and ultrasound were used to monitor for endothelial proliferation or thrombosis as signs of rejection and vascular complication. Conclusion. Multimodality imaging is an integral component of vascular composite allotransplantation surgical planning and surveillance to maximize returning form and functionality while minimizing possible complications.
Alveolar type II (ATII) epithelial cells play a crucial role in the repair and remodeling of the lung following injury. ATII cells have the capability to proliferate and differentiate into alveolar type I (ATI) cells in vivo and into an ATI-like phenotype in vitro. While previous reports indicate that the differentiation of ATII cells into ATI cells is a complex biological process, the underlying mechanism responsible for differentiation is not fully understood. To investigate factors involved in this differentiation in culture, we used a PCR array and identified several genes that were either up- or downregulated in ATI-like cells (day 6 in culture) compared with day 2 ATII cells. Insulin-like growth factor-I (IGF-I) mRNA was increased nearly eightfold. We found that IGF-I was increased in the culture media of ATI-like cells and demonstrated a significant role in the differentiation process. Treatment of ATII cells with recombinant IGF-I accelerated the differentiation process, and this effect was abrogated by the IGF-I receptor blocker PQ401. We found that Wnt5a, a member of the Wnt-Frizzled pathway, was activated during IGF-I-mediated differentiation. Both protein kinase C and ?-catenin were transiently activated during transdifferentiation. Knocking down Wnt5a using small-interfering RNA abrogated the differentiation process as indicated by changes in the expression of an ATII cell marker (prosurfactant protein-C). Treatment of wounded cells with either IGF-I or Wnt5a stimulated wound closure. These results suggest that IGF-I promotes differentiation of ATII to ATI cells through the activation of a noncanonical Wnt pathway.
The emerging field of vascular composite allotransplantation (VCA) has become a clinical reality. Building upon cutting edge understandings of transplant surgery and immunology, complex grafts such as hands and faces can now be transplanted with success. Many of the challenges that have historically been limiting factors in transplantation, such as rejection and the morbidity of immunosuppression, remain challenges in VCA. Because of the accessibility of most VCA grafts, and the highly immunogenic nature of the skin in particular, VCA has become the focal point for cross-disciplinary approaches to developing novel approaches for some of the most challenging immunological problems in transplantation, particularly the early diagnoses and assessment of rejection. This paper provides a historically oriented introduction to the field of organ transplantation and the evolution of VCA.
Organ/tissue transplantation has become an effective therapy for end-stage diseases. However, immunosuppression after transplantation may cause severe side effects. Donor-specific transplant tolerance was proposed to solve this problem. In this study, we report a novel method for inducing and maintaining heart allograft tolerance rats. First, we induced indefinite vascularized hind-limb allograft survival with a short-term antilymphocyte serum?+?Cyclosporine A treatment. Peripheral blood chimerism disappeared 6-7 weeks after immunosuppression was withdrawn. Then the recipients accepted secondary donor-strain skin and heart transplantation 200 days following vascularized hind-limb transplantation without any immunosuppression, but rejected third party skin allografts, a status of donor-specific tolerance. The ELISPOT results suggested a mechanism of clone deletion. These findings open new perspectives for the role of vascularized hind-limb transplant in the induction and maintenance of organ transplantation tolerance.
Skin is the most immunogenic component of a vascularized composite allograft (VCA) and is the primary trigger and target of rejection. The skin is directly accessible for visual monitoring of acute rejection (AR) and for directed biopsy, timely therapeutic intervention, and management of AR. Logically, antirejection drugs, biologics, or other agents delivered locally to the VCA may reduce the need for systemic immunosuppression with its adverse effects. Topical FK 506 (tacrolimus) and steroids have been used in clinical VCA as an adjunct to systemic therapy with unclear beneficial effects. However, there are no commercially available topical formulations for other widely used systemic immunosuppressive drugs such as mycophenolic acid, sirolimus, and everolimus. Investigating the site-specific therapeutic effects and efficacy of systemically active agents may enable optimizing the dosing, frequency, and duration of overall immunosuppression in VCA with minimization or elimination of long-term drug-related toxicity.
Reconstructive transplantation has emerged as clinical reality over the past decade. Long-term graft acceptance has been feasible in extremity and facial vascularized composite allotransplantation (VCA) under standard immunosuppression. Minimizing overall burden of lifelong immunosuppression is key to wider application of these non-life saving grafts. Allograft tolerance is the holy grail of many cell-based immunomodulatory strategies. Recent protocols using mesenchymal stem cells from bone marrow and adipose tissue offer promise and potential in VCA. This article provides an overview of the experimental basis, the scientific background and clinical applications of stem cell-based therapies in the field of reconstructive allotransplantation.
Caring for upper extremity transplant recipients can offer challenges and opportunities to nursing staff in combining new patient procedures, new technologies, and complex patient care needs including unique physical care, monitoring and observation, rehabilitation expectations, and psychiatric/psychosocial support. Medical professionals continue to be apprehensive about the risks of immunosuppressive therapy and the possibility of acute and chronic rejection. The sustained development and research into reliable, reduced-dose immunosuppression or immunomodulatory strategies could expand the life-enhancing benefits of reconstructive transplantation.
Upper extremity transplantation is an innovative reconstructive strategy with potential of immediate clinical application and the most near-term pay-off for select amputees, allowing reintegration into employment and society. Routine applicability and widespread impact of such strategies for the upper extremity amputees with devastating limb loss could be enabled by implementation of cellular therapies that integrate and unify the concepts of transplant tolerance induction with those of reconstructive transplantation. Such therapies offer the promise of minimizing the risks, maximizing the benefits and optimizing outcomes of these innovative procedures.
To date, 78 upper extremity transplants have been performed in 55 recipients around the world. The purpose of this article is to provide an overview of acute and chronic rejection (CR) and to summarize collective insights in upper extremity transplantation. To date, almost all patients experienced AR that is pathophysiologically similar to that in solid organs. The spectre of chronic rejection is just emerging. Upper extremity transplantation has significant potential as a reconstructive option only if efforts are invested in strategies to reduce risks of prolonged immunosuppression and in approaches to better diagnose, monitor and treat AR and CR.
Vertebral bone marrow is a rich and easily accessible source of hematopoietic and mesenchymal stem cells that has been used to promote chimerism and transplantation tolerance in connection with cadaveric organ transplantation. The purpose of this study is to provide a detailed account of the procedure used to prepare the first five vertebral bone marrow products for infusion in conjunction with the first hand/hand-forelimb transplants performed at the University of Pittsburgh (PA, USA).
Both hyperoxia and mechanical ventilation can independently cause lung injury. In combination, these insults produce accelerated and severe lung injury. We recently reported that pre-exposure to hyperoxia for 12 hours, followed by ventilation with large tidal volumes, induced significant lung injury and epithelial cell apoptosis compared with either stimulus alone. We also reported that such injury and apoptosis are inhibited by antioxidant treatment. In this study, we hypothesized that apoptosis signal-regulating kinase-1 (ASK-1), a redox-sensitive, mitogen-activated protein kinase kinase kinase, plays a role in lung injury and apoptosis in this model. To determine the role of ASK-1 in lung injury, the release of inflammatory mediators and apoptosis, attributable to 12 hours of hyperoxia, were followed by large tidal volume mechanical ventilation with hyperoxia. Wild-type and ASK-1 knockout mice were subjected to hyperoxia (Fi(O(2)) = 0.9) for 12 hours before 4 hours of large tidal mechanical ventilation (tidal volume = 25 ?l/g) with hyperoxia, and were compared with nonventilated control mice. Lung injury, apoptosis, and cytokine release were measured. The deletion of ASK-1 significantly inhibited lung injury and apoptosis, but did not affect the release of inflammatory mediators, compared with the wild-type mice. ASK-1 is an important regulator of lung injury and apoptosis in this model. Further study is needed to determine the mechanism of lung injury and apoptosis by ASK-1 and its downstream mediators in the lung.
Starting a hand transplant program poses tremendous challenges. Solid organ transplantation and hand replantation are time-tested procedures and are now standard of care. Hand transplantation is the amalgamation of the scientific principles of reconstructive surgery and the concepts of organ transplantation. Thus, for any hand transplant program to be successful, there must be collaboration within a multidisciplinary team comprising a core group of hand and transplant surgeons. Such a joint effort can overcome the challenges that are inherent in a complex therapeutic option that integrates different disciplines and organizations during the planning, procedural, and posttransplant phases.
Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K(+) channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this study we hypothesized that hyperoxia affects expression of Trek-1 in alveolar epithelial cells and that Trek-1 is involved in regulation of cell proliferation and cytokine secretion. We found gene expression of several K2P channels in mouse alveolar epithelial cells (MLE-12), and expression of Trek-1 was significantly downregulated in cultured cells and lungs of mice exposed to hyperoxia. Similarly, proliferation cell nuclear antigen (PCNA) and Cyclin D1 expression were downregulated by exposure to hyperoxia. We developed an MLE-12 cell line deficient in Trek-1 expression using shRNA and found that Trek-1 deficiency resulted in increased cell proliferation and upregulation of PCNA but not Cyclin D1. Furthermore, IL-6 and regulated on activation normal T-expressed and presumably secreted (RANTES) secretion was decreased in Trek-1-deficient cells, whereas release of monocyte chemoattractant protein-1 was increased. Release of KC/IL-8 was not affected by Trek-1 deficiency. Overall, deficiency of Trek-1 had a more pronounced effect on mediator secretion than exposure to hyperoxia. This is the first report suggesting that the K(+) channel Trek-1 could be involved in regulation of alveolar epithelial cell proliferation and cytokine secretion, but a direct association with hyperoxia-induced changes in Trek-1 levels remains elusive.
The ultimate goal of hand allotransplantation is to achieve graft survival and useful long-term function. To achieve these goals, selection of the appropriate patient, detailed preoperative planning, and precise surgical technique are of paramount importance. Transplantation should be reserved for motivated consenting adults in good general heath, who are psychologically stable and have failed a trial of prosthetic use. While the key surgical steps of transplantation are similar to those of replantation, there are major differences. This article describes the steps in hand allotransplantation, and the importance of patient selection as well as preoperative and postoperative care.
Donor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs.
The goal of hand allotransplantation is to achieve graft survival and useful long-term function. To achieve these goals, precise surgical technique is of critical importance. The key surgical steps and sequence of events in hand allotransplantation are similar to major upper extremity replantations, but are modified to accommodate major conceptual differences that exist between the two procedures.
Both prolonged exposure to hyperoxia and large tidal volume mechanical ventilation can each independently cause lung injury. However, the combined impact of these insults is poorly understood. We recently reported that preexposure to hyperoxia for 12 h, followed by ventilation with large tidal volumes, induced significant lung injury and epithelial cell apoptosis compared with either stimulus alone (Makena et al. Am J Physiol Lung Cell Mol Physiol 299: L711-L719, 2010). The upstream mechanisms of this lung injury and apoptosis have not been clearly elucidated. We hypothesized that lung injury in this model was dependent on oxidative signaling via the c-Jun NH(2)-terminal kinases (JNK). We, therefore, evaluated lung injury and apoptosis in the presence of N-acetyl-cysteine (NAC) in both mouse and cell culture models, and we provide evidence that NAC significantly inhibited lung injury and apoptosis by reducing the production of ROS, activation of JNK, and apoptosis. To confirm JNK involvement in apoptosis, cells treated with a specific JNK inhibitor, SP600125, and subjected to preexposure to hyperoxia, followed by mechanical stretch, exhibited significantly reduced evidence of apoptosis. In conclusion, lung injury and apoptosis caused by preexposure to hyperoxia, followed by high tidal volume mechanical ventilation, induces ROS-mediated activation of JNK and mitochondrial-mediated apoptosis. NAC protects lung injury and apoptosis by inhibiting ROS-mediated activation of JNK and downstream proapoptotic signaling.
To improve the degree of functional return and sensibility provided by composite tissue allotransplantation, enhanced nerve regeneration is essential. Chondroitin sulfate proteoglycans are found in the extracellular matrix of nerves and inhibit regenerating axons after injury. Treatment with chondroitinase to remove chondroitin sulfate proteoglycans has been shown to improve nerve regeneration in isolated nerve graft and transection-and-repair models. This study assesses the efficacy of chondroitinase as a neurotherapeutic agent in the setting of composite tissue allotransplantation.
Recent advances in transplant immunology are shifting the focus from immunosuppression to immunoregulation, making composite tissue allotransplantation with novel and less potent immunosuppressive regimens a possibility. Hand transplantation has been the most frequently performed human composite tissue allotransplantation, with more than 50 upper extremity-based transplants done worldwide. Further research is needed regarding immunomodulating protocols, and careful oversight and individualized screening procedures will be required as patients seeking improved quality of life through human composite tissue allotransplantation come to accept a certain level of risk in these experimental procedures. Still, composite tissue allotransplantation offers to advance transplant medicine and reconstructive surgery.
In the past decade, more than 100 different composite tissue allotransplantation (CTA) procedures have been performed around the world including more than 50 hand and 8 facial transplants with encouraging graft survival and excellent functional outcomes. Broader clinical application of CTA, however, continues to be hampered by requirement for long-term, high-dose, multidrug maintenance immunosuppression to prevent graft rejection mediated particularly by composite tissue allografts highly immunogenic skin component. Medication toxicity could result in severe adverse events including metabolic and infectious complications or malignancy. Notably, unlike in solid organs, clinical success is dictated not only by graft acceptance and survival but also by nerve regeneration, which determines ultimate functional outcomes. Novel strategies such as cellular and biologic therapies that integrate the concepts of immune regulation with those of nerve regeneration have shown promising results in small and large animal models. Clinical translation of these insights to reconstructive transplantation and CTA could further minimize the need of immunosuppression and optimize functional outcomes. This will enable wider application of such treatment options for patients in need of complex reconstructive surgery for congenital deformities or devastating injuries that are not amenable to standard methods of repair.
Despite the development of successful immunosuppression protocols and tremendous improvement in short-term graft survival rates, the problem of chronic graft loss remains the bane of clinical transplantation. The induction and maintenance of transplantation tolerance is the "Holy Grail" of transplantation. The recent identification and characterization of regulatory T cells has opened up exciting opportunities for tolerance induction, immunotherapy, and immunomodulation in transplantation. This review focuses on current understanding of regulatory T cells and their role in transplantation tolerance.
Hand/forearm/arm transplants are vascularized composite allografts, which, unlike solid organs, are composed of multiple tissues including skin, muscle, tendons, vessels, nerves, lymph nodes, bone, and bone marrow. Over the past decade, 26 upper extremity transplantations were performed in the United States. The University of Pittsburgh Medical Center has the largest single center experience with 8 hand/forearm transplantations performed in 5 recipients between January 2008 and September 2010. Anesthetic management in the emerging field of upper extremity transplants must address protocol and procedure-specific considerations related to the role of regional blocks, effects of immunosuppressive drugs during transplant surgery, fluid and hemodynamic management in the microsurgical setting, and rigorous intraoperative monitoring during these often protracted procedures.
Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability.
Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1?, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CXCR4 signaling is known to be important in immune cell migration, the role of this chemokine-receptor interaction has not been studied in alveolar epithelial repair mechanisms. In this study, we demonstrated that secretion of CXCL12 was increased in the bronchoalveolar lavage of rats ventilated with an injurious tidal volume (25 ml/kg). We also found that CXCL12 secretion was increased by primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell line following scratch wounding and that both types of cells express CXCR4. CXCL12 significantly increased ATII cell migration in a scratch-wound assay. When we treated cells with a specific antagonist for CXCR4, AMD-3100, cell migration was significantly inhibited. Knockdown of CXCR4 by short hairpin RNA (shRNA) caused decreased cell migration compared with cells expressing a nonspecific shRNA. Treatment with AMD-3100 decreased matrix metalloproteinase-14 expression, increased tissue inhibitor of metalloproteinase-3 expression, decreased matrix metalloproteinase-2 activity, and prevented CXCL12-induced Rac1 activation. Similar results were obtained with shRNA knockdown of CXCR4. These findings may help identify a therapeutic target for augmenting epithelial repair following acute lung injury.
Devastating facial deformities can cause significant functional and psychosocial injury. Significant facial disfigurement can preclude meaningful human interaction. Allotransplantation of facial tissues for reconstruction of devastating deformities has become a clinical reality, with 15 transplants performed at various centers around the world. Restoration of aesthetics and functionality has been superior to that achieved by conventional reconstruction, without the morbidity of multiple surgeries. Unlike solid organ transplantation which can be life saving, facial transplantation is considered by many to be life enhancing, highlighting the ethical argument against justification of these procedures given the risks of lifelong immunosuppression. Meticulous patient selection is mandatory, and a multidisciplinary team approach is key for the programs success. The overriding goal of screening for candidacy is to identify and select subjects who have the best chance for a positive immunologic, functional, and quality-of-life outcome. This article reviews the pertinent considerations and screening approach for appropriate patient selection in facial tissue transplantation.
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