Orthotopic Rat Forelimb Transplantation

Vascularized composite allotransplantation (VCA) involves the transplantation of multiple tissue types -- including skin, muscle, bone, and nerves -- ...

Johns Hopkins University School of Medicine

Northwestern University Feinberg School of Medicine

Since the beginning of transplant medicine in the 1950s, advances in surgical technique and immunosuppressive therapy have created the success story of modern organ transplantation. However, today more than ever, we are facing a huge discrepancy between organ supply and demand, limiting the potential for transplantation to save and improve the lives of millions. To address the current limitations and shortcomings, a variety of emerging new technologies focusing on either maximizing the availability of organs or on generating new organs and organ sources hold great potential to eventully overcoming these hurdles. These advances are mainly in the field of regenerative medicine and tissue engineering. This review gives an overview of this emerging field and its multiple sub-disciplines and highlights recent advances and existing limitations for widespread clinical application and potential impact on the future of transplantation.

Emerging technologies in organ preservation, tissue engineering and regenerative medicine: a blessing or curse for transplantation?

Vascularized composite allotransplantation (VCA) has become a valid therapeutic option to restore form and function after devastating tissue loss. However, the need for high-dose multidrug immunosuppression to maintain allograft survival is still hampering more widespread application of VCA. In this study, we investigated the immunoregulatory potential of costimulation blockade (CoB; CTLA4-Ig and anti-CD154 mAb) combined with nonmyeoablative total body irradiation (TBI) to promote allograft survival of VCA in a fully MHC-mismatched mouse model of orthotopic hind limb transplantation. Compared with untreated controls (median survival time [MST] 8 days) and CTLA4-Ig treatment alone (MST 17 days), CoB treatment increased graft survival (MST 82 days), and the addition of nonmyeloablative TBI led to indefinite graft survival (MST > 210 days). Our analysis suggests that VCA-derived BM induced mixed chimerism in animals treated with CoB and TBI + CoB, promoting gradual deletion of alloreactive T cells as the underlying mechanism of long-term allograft survival. Acceptance of donor-matched secondary skin grafts, decreased ex vivo T cell responsiveness, and increased graft-infiltrating Tregs further indicated donor-specific tolerance induced by TBI + CoB. In summary, our data suggest that vascularized BM-containing VCAs are immunologically favorable grafts promoting chimerism induction and long-term allograft survival in the context of CoB.

Vascularized composite allotransplantation combined with costimulation blockade induces mixed chimerism and reveals intrinsic tolerogenic potential.

We herein investigate the safety and efficacy of single-agent anti-rejection regimens in a mouse vascularized composite allotransplantation (VCA) model. Orthotopic hind-limb transplantations (Balb/c → C57BL/6) were performed using 6- to 8-week-old mice. A thirty-day regimen of either rapamycin, tacrolimus (both 1, 3, 5 mg/kg/day) or cyclosporine (25, 35, 50 mg/kg/day) was used. Primary endpoints were animal and graft survival, and secondary chimerism and regulatory T-cell levels. For rapamycin and tacrolimus given at 1, 3, and 5 mg/kg/day, median graft survival time (MST) was 23 days (18-28 days), 30 days (23-30 days), and 30 d (30-30 days) and 14 days (13-18 days), 30 days (16-30 days), and 30 days (30-30 days), respectively. For cyclosporine dosed at 25 and 35 mg/kg/day, MST was 15 days (12-18 days) and 21 days (14-27 days). Toxicity from CsA 50 mg/kg led to 100% mortality. Mixed chimerism levels were higher in rapamycin-treated animals than in tacrolimus-treated recipients (P = 0.029). Tacrolimus was superior in preventing leukocyte recruitment to the allograft. In murine VCA, no standardized immunosuppressive regimen exists, limiting comparability of outcomes and survival. Our data demonstrate that rapamycin and tacrolimus maintenance treatment at 5 mg/kg/day both yielded allograft survival (<grade 3 rejection) in all animals. Rapamycin displayed less toxicity and maintained mixed chimerism but was not as potent in controlling leukocyte recruitment compared with tacrolimus.

Efficacy of single-agent immunosuppressive regimens in a murine model of vascularized composite allotransplantation.

Functional recovery following peripheral nerve injury worsens with increasing durations of delay prior to repair. From the time of injury until re-innervation occurs, denervated muscle undergoes progressive atrophy that limits the extent to which motor function can be restored. Similarly, Schwann cells (SC) in the distal nerve lacking axonal interaction progressively lose their capacity to proliferate and support regenerating axons. The relative contributions of these processes to diminished functional recovery is unclear. We developed a novel rat model to isolate the effects of SC vs. muscle denervation on functional recovery. Four different groups underwent the following interventions for 12 weeks prior to nerve transfer: 1) muscle denervation; 2) SC denervation; 3) muscle + SC denervation (negative control); 4) no denervation (positive control). Functional recovery was measured weekly using the stimulated grip strength testing (SGST). Animals were sacrificed 13 weeks post nerve transfer. Retrograde labeling was used to assess the number of motor neurons that regenerated their axons. Immunofluorescence was performed to evaluate target muscle re-innervation and atrophy, and to assess the phenotype of the SC within the distal nerve segment. Functional recovery in the muscle denervation and SC denervation groups mirrored that of the negative and positive control groups, respectively. The SC denervation group achieved better functional recovery, with a greater number of reinnervated motor endplates and less muscle atrophy, than the muscle denervation group. Retrograde labeling suggested a higher number of neurons contributing to muscle reinnervation in the muscle denervation group as compared to SC denervation (p > 0.05). The distal nerve segment in the muscle denervation group had a greater proportion of SCs expressing the proliferation marker Ki67 as compared to the SC denervation group (p < 0.05). Conversely, the SC denervation group had a higher percentage of senescent SCs expressing p16 as compared to the muscle denervation group (p < 0.05). The deleterious effects of muscle denervation are more consequential than the effects of SC denervation on functional recovery. The effects of 12 weeks of SC denervation on functional outcome were negligible. Future studies are needed to determine whether longer periods of SC denervation negatively impact functional recovery.

Defining the relative impact of muscle versus Schwann cell denervation on functional recovery after delayed nerve repair.

In vascularized composite allotransplantation (VCA), invasive tissue biopsies remain the gold standard in diagnosing rejection carrying significant morbidity. We aimed to show feasibility of tape-stripping for noninvasive immune monitoring in VCA. Tape-stripping was performed on allografts and native skin of upper extremity transplant recipients. Healthy nontransplanted individuals served as controls. The technique was also used in swine on naïve skin in nontransplanted animals, native skin of treated, transplanted swine, nonrejecting VCAs, and rejecting VCAs. Extracted protein was analyzed for differences in cytokine expression using Luminex technology. Significantly decreased levels of INFγ and IL-1Ra were seen between human allograft samples and native skin. In swine, rejecting grafts had increased IL-1Ra compared to naïve and native skin, decreased levels of GM-CSF compared to native skin, and decreased IL-10 compared to nonrejecting grafts. Unsupervised hierarchical clustering revealed rejecting grafts separated from the nonrejecting (P = 0.021). Variable importance in projection scores identified GM-CSF, IL-1Ra, and IL-2 as the most important profiles for group discrimination. Differences in cytokine expression are detectable in human VCA patient native skin and VCA graft skin using a noninvasive tape-stripping method. Swine studies suggest that differences in cytokines between rejecting and nonrejecting grafts are discernable.

Noninvasive evaluation of intragraft immune responses in upper extremity transplantation.

Static cold storage is the cheapest and easiest method and current gold standard to store and preserve donor organs. This study aimed to compare the preservative capacity of gluconate-lactobionate-dextran (Unisol) solutions to histidine-tryptophan-ketoglutarate (HTK) solution. Murine syngeneic heterotopic heart transplantations (Balb/c-Balb/c) were carried out after 18 h of static cold storage. Cardiac grafts were either flushed and stored with Unisol-based solutions with high-(UHK) and low-potassium (ULK) ± glutathione, or HTK. Cardiac grafts were assessed for rebeating and functionality, histomorphologic alterations, and cytokine expression. Unisol-based solutions demonstrated a faster rebeating time (UHK 56 s, UHK + Glut 44 s, ULK 45 s, ULK + Glut 47 s) compared to HTK (119.5 s) along with a better contractility early after reperfusion and at the endpoint on POD 3. Ischemic injury led to a significantly increased leukocyte recruitment, with similar degrees of tissue damage and inflammatory infiltrate in all groups, yet the number of apoptotic cells tended to be lower in ULK compared to HTK. In UHK- and ULK-treated animals, a trend toward decreased expression of proinflammatory markers was seen when compared to HTK. Unisol-based solutions showed an improved preservative capacity compared with the gold standard HTK early after cardiac transplantation. Supplemented glutathione did not further improve tissue-protective properties.

Gluconate-Lactobionate-Dextran Perfusion Solutions Attenuate Ischemic Injury and Improve Function in a Murine Cardiac Transplant Model.

Static cold storage (SCS), the current clinical gold standard for organ preservation, provides surgeons with a limited window of time between procurement and transplantation. In vascularized composite allotransplantation (VCA), this time limitation prevents many viable allografts from being designated to the best-matched recipients. Machine perfusion (MP) systems hold significant promise for extending and improving organ preservation. Most of the prior MP systems for VCA have been built and tested for large animal models. However, small animal models are beneficial for high-throughput biomolecular investigations. This study describes the design and development of a multiparametric bioreactor with a circuit customized to perfuse rat abdominal wall VCAs. To demonstrate its concept and functionality, this bioreactor system was employed in a small-scale demonstrative study in which biomolecular metrics pertaining to graft viability were evaluated non-invasively and in real time. We additionally report a low incidence of cell death from ischemic necrosis as well as minimal interstitial edema in machine perfused grafts. After up to 12 h of continuous perfusion, grafts were shown to survive transplantation and reperfusion, successfully integrating with recipient tissues and vasculature. Our multiparametric bioreactor system for rat abdominal wall VCA provides an advanced framework to test novel techniques to enhance normothermic and sub-normothermic VCA preservations in small animal models.

Design of a Multiparametric Perfusion Bioreactor System for Evaluating Sub-Normothermic Preservation of Rat Abdominal Wall Vascularized Composite Allografts.

Ex vivo machine perfusion (EVMP) is a versatile platform utilized in vascularized composite allotransplantation (VCA) to prolong preservation, salvage tissue, and evaluate graft viability. However, there is no consensus on best practices for VCA. This article discusses the common components, modifications, and considerations necessary for a successful VCA perfusion.

A Guide to the Implementation and Design of Ex Vivo Perfusion Machines for Vascularized Composite Allotransplantation.

Vascularized composite allotransplantation (VCA) has revolutionized restorative surgery of devastating injuries. Unfortunately, these grafts undergo significant injury during prolonged cold ischemia and subsequent reperfusion.  machine perfusion (EVMP) is a technique that has shown significant promise in solid organ transplant, but study of its utility in VCA has been limited. A systematic review was conducted to identify preclinical publications investigating perfusion in limb VCAs. Articles published through June 2023 were screened. 29 articles met inclusion criteria, comprising 370 VCA limbs from swine, rats, canines, and humans. EVMP was conducted under normothermic (n = 6), near-normothermic (n = 11), sub-normothermic (n = 3), or hypothermic (n = 13) conditions. While each study used a unique perfusate recipe, most were based on a premade medium. Many incorporated additives, including antibiotics and red blood cells. The duration varied from 3 to over 24 h. Multiple studies showed improved or equivalent biomarkers, histology, and outcomes for normothermic or near-normothermic EVMP (n = 4) and hypothermic EVMP (n = 8) compared to static cold storage, suggesting that EVMP may be a superior storage method to SCS. While there is no definitive evidence regarding the optimal temperature, perfusate composition, or perfusion time for VCAs, each perfusion factor should be chosen and adapted based on the individual goals of the study. This review offers a summary of the current literature to serve as an accessible reference for the design of future protocols in this field.

 Perfusion of Limb Vascularized Composite Allotransplants: A Systematic Review of Published Protocols.

Antibiotic resistance is a global public health challenge. The gut microbiota serves as a reservoir for antibiotic resistance genes (ARGs), facilitating their transfer between bacteria. With the rising incidence of major depressive disorders (MDD), antidepressant prescriptions have surged. Previous pure-culture studies suggest that antidepressants exhibit antibiotic-like side effects, but their impact on gene transfer in microbial communities remains unclear. Here, we report that clinically relevant doses of antidepressants duloxetine and sertraline enhance the transfer of a broad-host range conjugative plasmid across bacterial phyla from mice gut microbiota. Tests in human gut simulators confirmed that duloxetine facilitates plasmid transfer in human gut microbiota. Mechanistic analyses revealed that antidepressants increase reactive oxygen species levels and alter bacterial cell membrane permeability. Using fluorescence-activated cell sorting and 16S rRNA gene sequencing, we discovered that antidepressants alter plasmid transfer patterns at both phylum and genus levels, driving ARG exchange among opportunistic pathogens. Our findings suggest that antidepressant use may promote the spread of antibiotic resistance between commensal and pathogenic bacteria, raising important public health concerns.

Article	Year
Emerging technologies in organ preservation, tissue engineering and regenerative medicine: a blessing or curse for transplantation? Transplant international : official journal of the European Society for Organ Transplantation\| PubMed ID: 30920056	2019
Vascularized composite allotransplantation combined with costimulation blockade induces mixed chimerism and reveals intrinsic tolerogenic potential. JCI insight\| PubMed ID: 32271163	2020
Efficacy of single-agent immunosuppressive regimens in a murine model of vascularized composite allotransplantation. Transplant international : official journal of the European Society for Organ Transplantation\| PubMed ID: 32299127	2020
Defining the relative impact of muscle versus Schwann cell denervation on functional recovery after delayed nerve repair. Experimental neurology\| PubMed ID: 33607079	2021
Noninvasive evaluation of intragraft immune responses in upper extremity transplantation. Transplant international : official journal of the European Society for Organ Transplantation\| PubMed ID: 33626223	2021
Gluconate-Lactobionate-Dextran Perfusion Solutions Attenuate Ischemic Injury and Improve Function in a Murine Cardiac Transplant Model. Cells\| PubMed ID: 35626690	2022
Design of a Multiparametric Perfusion Bioreactor System for Evaluating Sub-Normothermic Preservation of Rat Abdominal Wall Vascularized Composite Allografts. Bioengineering (Basel, Switzerland)\| PubMed ID: 38671729	2024
A Guide to the Implementation and Design of Ex Vivo Perfusion Machines for Vascularized Composite Allotransplantation. Plastic and reconstructive surgery. Global open\| PubMed ID: 39534373	2024
Perfusion of Limb Vascularized Composite Allotransplants: A Systematic Review of Published Protocols. Transplant international : official journal of the European Society for Organ Transplantation\| PubMed ID: 40458543	2025
Antidepressant drugs promote the spread of broad-host-range plasmid in mouse and human gut microbiota. Gut microbes\| PubMed ID: 40462285	2025

Yinan Guo

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