Heterotopic Transplantation Research Articles

Inhibiting S100A8/A9 Attenuates Airway Obstruction in a Mouse Heterotopic Tracheal Transplantation Model

<h3>Purpose</h3> Chronic lung allograft dysfunction (CLAD) is a major cause of mortality after lung transplantation (LTx). The main pathogenesis of CLAD is fibrosis of the small airways or lung parenchyma. Neutralizing antibodies against S100A8/A9, an inflammatory protein, have been shown to inhibit lung fibrosis in mouse models. In the present study, we tested the hypothesis that S100A8/A9 antibodies suppress airway fibrosis in a mouse model. <h3>Methods</h3> A mouse heterotopic tracheal transplantation model was used. The anti-S100A8/A9 monoclonal antibody or control IgG were intraperitoneally administered twice a week. The rate of graft lumen occlusion, the positivity of the fibroblast marker α-smooth muscle actin (SMA), and mRNA expression of cytokines and chemokines were evaluated. <h3>Results</h3> The luminal obstruction ratio at day 21 was significantly higher in the treatment group than in the control group (p = 0.0023) (Fig.1A, 1B and 2A). The αSMA-positive area in submucosal layer was significantly smaller in the treatment group than in the control group (p = 0.0376) (Fig 1C, 1D and 2B). The expression levels of TGFβ (p = 0.0026) and Collagen 3a1 (p = 0.0238) at day 21 by qPCR were significantly lower in the treatment group than in the control group. Conversely, expression levels of inflammatory cytokines in the treatment group was equivalent to those in the control group. <h3>Conclusion</h3> S100A8/A9 antibody ameliorated airway obstruction by reducing collagen production of αSMA positive myofibroblast without suppression of inflammatory responses in a mouse model. The heterodimer S100A8/A9 can be a novel therapeutic target for CLAD after LTx.

A new approach to use of oral mucosa in reconstructive urethral surgery: micrografts

Introduction. Treatment of patients with complex urethral strictures is an actual problem of reconstructive urology. After multi-staged urethral surgery with multiple revisions the new reconstruction is limited by paucity of plastic material (for grafts and flaps). In-thing, new materials for urethral reconstruction (various auto, allo and xenografts) are still being developed in reconstructive urethral surgery. Purpose of the study . To study the possibility of using oral mucosa micrografts to form the urethral plate in the multi-stage surgery for patients with extended urethral strictures. Materials and methods. In the experimental study, male Wistar rats (22 individuals) weighing 300 – 400 g underwent a full-thickness skin wound after intramuscular sedation. We used the wound chamber (12 mm diameter) to exclude the wound contraction. The oral mucosa graft was harvested (6 mm in diameter). After pre-fabrication, the graft was minced to fragments &lt; 1 mm 2 . Micrografts with fibrin-thrombin glue were applied to the wound. By day 45, the epithelial plate was excised for histological examination. In the clinical study, 4 patients with recurrent penile urethral strictures were treated with staged urethroplasty with urethral plate formation using oral mucosa micrografts. The average length of the stricture was 7.5 ± 1.2 cm (with extremely narrow and obliteration sites). The urethral plate was formed as the first stage. The preparation of the graft bed and oral mucosa grafts harvesting was carried out according to the standard procedure. Micrografts preparation and implantation was carried out as in experimental part of this study. After 6 months, neourethra tubularization was performed. The patients were evaluated every 3 months after the final stage of urethroplasty (uroflowmetry, ultrasound, X-ray, PROM-USS). The median follow-up was 9 months (3 – 18 months). Results. On day 15, in the experimental study, in 16 of 22 (72.7%) rats, the wound chambers had focal growth of the oral mucosa epithelium. On day 45, the wounds healed completely healed with oral mucosa. The final area of the plate was 78 ± 12 mm 2 . In the clinical study, 6 months after the first stage, all patients (n = 4) had a urethral plate covered with an oral mucosa epithelium without scar formation and sufficient for neurethra tubularization. All patients underwent urethral tubularization. After catheter removal, all men urinated. After 9 months (median follow-up, n = 3), the Q max was 22.7 ± 4.2 ml/s, the post-void residual urine was 34.8 ± 2.2 ml, the total PROM-USS score was 7.4 ± 1.2, urethral lumen is preserved. All patients showed high satisfaction with the treatment. Conclusion. The oral mucosa micrografts showed good take in heterotopic transplantation (72.7%) with the formation of an epithelial layer on the wound surface. The final mucosal plate area 3 times exceeded the initial micrografts area. This initial clinical experience of using oral mucosa micrografts shows the new possibility of this technology in reconstructive urethral surgery, especially in patients with complex urethral strictures.

Microsurgical Transplantation of Pedicled Muscles in an Isolation Chamber—A Novel Approach to Engineering Muscle Constructs via Perfusion-Decellularization

Decellularized whole muscle constructs represent an ideal scaffold for muscle tissue engineering means as they retain the network and proteins of the extracellular matrix of skeletal muscle tissue. The presence of a vascular pedicle enables a more efficient perfusion-based decellularization protocol and allows for subsequent recellularization and transplantation of the muscle construct in vivo. The goal of this study was to create a baseline for transplantation of decellularized whole muscle constructs by establishing an animal model for investigating a complete native muscle isolated on its pedicle in terms of vascularization and functionality. The left medial gastrocnemius muscles of 5 male Lewis rats were prepared and raised from their beds for in situ muscle stimulation. The stimulation protocol included twitches, tetanic stimulation, fatigue testing, and stretching of the muscles. Peak force, maximum rate of contraction and relaxation, time to maximum contraction and relaxation, and maximum contraction and relaxation rate were determined. Afterwards, muscles were explanted and transplanted heterotopically in syngeneic rats in an isolation chamber by microvascular anastomosis. After 2 weeks, transplanted gastrocnemius muscles were exposed and stimulated again followed by intravascular perfusion with a contrast agent for µCT analysis. Muscle constructs were then paraffin embedded for immunohistological staining. Peak twitch and tetanic force values all decreased significantly after muscle transplantation while fatigue index and passive stretch properties did not differ between the two groups. Vascular analysis revealed retained perfused vessels most of which were in a smaller radius range of up to 20 µm and 45 µm. In this study, a novel rat model of heterotopic microvascular muscle transplantation in an isolation chamber was established. With the assessment of in situ muscle contraction properties as well as vessel distribution after 2 weeks of transplantation, this model serves as a base for future studies including the transplantation of perfusion-decellularized muscle constructs.

Re-Endothelialization of Decellularized Liver Scaffolds: A Step for Bioengineered Liver Transplantation.

Bioengineered livers (BELs) are an attractive therapeutic alternative to address the donor organ shortage for liver transplantation. The goal of BELs technology aims at replacement or regeneration of the native human liver. A variety of approaches have been proposed for tissue engineering of transplantable livers; the current review will highlight the decellularization-recellularization approach to BELs. For example, vascular patency and appropriate cell distribution and expansion are critical components in the production of successful BELs. Proper solutions to these components of BELs have challenged its development. Several strategies, such as heparin immobilization, heparin-gelatin, REDV peptide, and anti-CD31 aptamer have been developed to extend the vascular patency of revascularized bioengineered livers (rBELs). Other novel methods have been developed to enhance cell seeding of parenchymal cells and to increase graft functionality during both bench and in vivo perfusion. These enhanced methods have been associated with up to 15 days of survival in large animal (porcine) models of heterotopic transplantation but have not yet permitted extended survival after implantation of BELs in the orthotopic position. This review will highlight both the remaining challenges and the potential for clinical application of functional bioengineered grafts.

Offspring from oocytes grown in frozen-thawed ovarian tissues transplanted to male and female bodies

Abstract Objective: There are few reports of live births from heterotopic transplantation of frozen-thawed ovarian tissue. The purpose of this study is to assess the follicular development in the frozen-thawed ovarian tissues following heterotopic transplantation in both female and male bodies. Methods: Cluster of differentiation 1 (CD1) mice (6-8 weeks) were used in this study as ovarian tissue donors and foster mothers for embryo transfer. Sperm from CD1 male mice were used for insemination by intracytoplasmic sperm injection (ICSI). Nude severe combined immunodeficiency mice (8 weeks) were employed as recipients of ovarian tissue transplantation. The frozen-thawed ovarian tissues were transplanted to 4 sites on each mouse, female and male, subcutaneously. After 3 months, both female and male mice were injected with 5.0 IU gonadotropins intraperitoneally. Post 48 hours of injection, the mouse was killed for ovarian transplant collection. Only fully grown oocytes with contacted cumulus cells (cumulus-oocyte complexes) were then selected for maturation in vitro. In vitro matured oocytes were inseminated with fresh sperm by ICSI, and the developed blastocysts were frozen using the vitrification method and stored until embryo transfer. After thawing, the thawed blastocysts were incubated for at least 2 hours before the transfer. The foster mice mothers mated with vasectomised male 3 days previously. Live birth was monitored at 19 days after transfer, and the resulted offspring was raised for fertility test. Results: The relatively high recovery rates of the transplanted ovarian tissues were collected in both frozen-thawed and fresh ovarian tissue transplants from both female and male bodies. The fully grown immature oocytes became mature in vitro and the fertilized zygotes developed to blastocyst stage. There are no differences between frozen-thawed and fresh ovarian transplants in term of oocyte quality and embryo development to blastocyst rates. Nineteen-day post-transfer, 3 foster mothers from the frozen-thawed ovarian tissue transplant group delivered 13 pups and the 4 foster mothers of the fresh ovarian tissue transplant group delivered 12 live pups. The produced offspring were normal in appearance and grew healthy and fertile. Conclusions: Our results attest that the follicles can survive and develop in the frozen-thawed ovarian tissues following the subcutaneous transplant to adult male mouse's body regardless of basal endocrinal environment. Those fully grown oocytes can produce healthy and fertile offspring which will provide the possibility for further mechanistic understanding of endocrinology of folliculogenesis.

Erector Spinae Plane Block For Postoperative Analgesia After Kidney Transplant.

In this report, we assessed the effectiveness of erector spinae plane block for postoperative analgesia after kidney transplant. Our study included 28 total patients who underwent heterotopic kidney transplant from living related donors. In the retrospective control group, patients (n = 14) received conventional postoperative analgesia. In the study group, patients (n = 14) received continuous erector spinae plane block in addition to conventional analgesics. Study endpoints were pain intensity evaluated using numerical rating scale at rest and during movement, opioid consumption, and postoperative nausea and vomiting incidences during the first postoperative day. Patients in the study group demonstrated significantly less pain (2.1 ± 1.09 at rest and 3.8 ± 1.18 during movement per the numerical rating scale) compared with the control group (3.3 ± 1.17 and 5.2 ± 2.15, respectively) (P = .009 and P = .042) and less opioid requirements (15.9 ± 7.1 vs 4.7 ± 6.2 mg of morphine; P < .001). In addition, incidences of nausea and vomiting were less frequent in the study group. Continuous erector spinae plane block in combination with nonopioid analgesics provided adequate pain control and significantly reduced postoperative opioid requirements after kidney transplant.

Effect of a C5a receptor antagonist on macrophage function in an intestinal transplant rat model

BackgroundC5a promotes alloreactivity via the C5a receptor 1 (C5aR1) on immune cells, but this has not been confirmed in the case of small intestine transplantation immunity. In the present study, we examined the effect of C5aR1 antagonist (PMX53) on macrophage function in small intestinal transplantation. MethodsThe model was created by heterotopic intestinal transplantation using donor Dark Agouti and recipient Lewis rats. PMX53 was administered starting on the day of operation until postoperative day 7. The graft survivals were compared, and HE staining of grafts, lymphocyte mixed reaction test (MLR, mixed culture of T cells from lymph nodes and spleen cells from donors), and changes in macrophage and T cell accumulation in grafts on day 6 after transplantation were evaluated. In addition, the effect of PMX53 on macrophage differentiation and activation was assessed using macrophages derived from bone marrow (BMDM). ResultsGraft survival was significantly prolonged in the therapeutic group compared to the untreated group. Histological evaluation showed that PMX53 inhibited the shortening of the graft villus, and the stimulation index of MLR was significantly lower in the therapeutic group compared to the untreated group. In the therapeutic group, the accumulation of macrophages in intestinal graft and monocyte in blood were reduced, compared with the untreated group. PMX53 decreased the differentiation in BMDM and the mRNA expression of IL-1β and TNF-α in activated BMDM. ConclusionInhibition of C5a/C5aR1 signaling appears to regulate macrophage differentiation and suppress rejection in small intestine transplantation immunity.

Comparative and mechanistic study of pharmacodynamic difference in anti-breast cancer activity between water and liquor extracts of Xiaojin Pills

Ethnopharmacological relevanceXiaojin Pills was first recorded during the Qing Dynasty and have a history of nearly 300 years. It is the first choice among Chinese patent medicines for the clinical treatment of diseases of the mammary glands in contemporary traditional Chinese medicine. It was also widely used in the treatment of breast cancer, lung cancer, thyroid cancer, and other malignant tumors. Its initial administration method was “taken orally after soaking with Chinese baijiu”; however, the method was changed to “taken orally with water” within the last 40 years. There is no scientific evidence for the difference in efficacy against breast cancer between the two methods of administration. Aim of the studyIn vitro and in vivo experiments were carried out to confirm the therapeutic advantages of the liquor extract of Xiaojin Pills to improve the efficacy against breast cancer, and the mechanism was explained in terms of metabolomics and molecular biology. Materials and methodsIn vitro, a cell counting kit-8 cell activity assay and flow cytometry were used to detect the activity and apoptosis of MCF-7 breast cancer cells. In vivo, pharmacodynamic evaluation was performed by constructing a heterotopic transplantation model of breast cancer in BALB/c-nu mice. TUNEL staining was used to observe the apoptosis of cells in tumor tissues. The expression of proteins associated with the phosphoinositide 3-kinase (PI3K)/Akt pathway in BALB/c-nu mice tissue was investigated by metabolomics analysis, immunohistochemistry and western blotting. ResultsCCK-8 assay showed that the IC50 of XJP-L for the inhibition of the activity of MCF-7 cells was less than that of XJP-W at different times. Flow cytometry assay suggested that the apoptosis rate in the XJP-L group was higher than that in the normal control group (p < 0.01). Animal experiment results indicated that both XJP-W group and XJP-L group reduced the volume and quality of the tumor after administration, and the reduction was more significant in the XJP-L group (p < 0.01). Metabolomics analysis results demonstrated that there are about 26 different metabolites have been screened in the serum metabolites between the liquor and water extract, mainly involved in glycerophospholipid, glutamic acid, aspartic acid, nitrogen and pyrimidine metabolism. In addition, immunohistochemistry and WB results showed that compared with the model group, the protein expression of PTEN, AKT, BAX and in tumor tissues of XJP-L and XJP-W groups both exhibited an upward trend, while the expression of BCL-2, p-PI3K and p-AKT exhibited a downward trend, which was much more obvious in XJP-L group. ConclusionsThis study demonstrates that the liquor extract of Xiaojin Pills had a stronger anti-breast cancer effect than that of the water extract. The PI3K/Akt signaling pathway might play an important role in the mechanism of the liquor extract of Xiaojin Pills and thus improve the efficacy against breast cancer.

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

Donor-Transmitted Cancer in Orthotopic Solid Organ Transplant Recipients: A Systematic Review.

Donor-transmitted cancer (DTC) has major implications for the affected patient as well as other recipients of organs from the same donor. Unlike heterotopic transplant recipients, there may be limited treatment options for orthotopic transplant recipients with DTC. We systematically reviewed the evidence on DTC in orthotopic solid organ transplant recipients (SOTRs). We searched MEDLINE, EMBASE, PubMed, Scopus, and Web of Science in January 2020. We included cases where the outcome was reported and excluded donor-derived cancers. We assessed study quality using published checklists. Our domains of interest were presentation, time to diagnosis, cancer extent, management, and survival. There were 73 DTC cases in liver (n = 51), heart (n = 10), lung (n = 10) and multi-organ (n = 2) recipients from 58 publications. Study quality was variable. Median time to diagnosis was 8 months; 42% were widespread at diagnosis. Of 13 cases that underwent re-transplantation, three tumours recurred. Mortality was 75%; median survival 7 months. Survival was worst in transmitted melanoma and central nervous system tumours. The prognosis of DTC in orthotopic SOTRs is poor. Although re-transplantation offers the best chance of cure, some tumours still recur. Publication bias and clinical heterogeneity limit the available evidence. From our findings, we suggest refinements to clinical practice. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020165001, Prospero Registration Number: CRD42020165001.

The embryonic node behaves as an instructive stem cell niche for axial elongation

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

Arterial Embolization of Polycystic Kidneys for Heterotopic Transplantation

Introduction: The purpose of this study was to evaluate the efficacy of polycystic kidney embolization, performed to reduce kidney volume before heterotopic kidney transplantation, as this technique could be an alternative to pretransplant nephrectomy. Materials and Methods: All patients who underwent pretransplant embolization of polycystic kidneys were included in a prospective register from June 2014 to February 2020. All patients underwent computed tomography (CT) scan with volumetric reconstruction (OsiriX, Bernex, Switzerland) before embolization and were then followed up at 3 and 6 months after embolization. Primary outcome was percentage of kidney volume reduction. Secondary outcomes were 30 day mortality and morbidity. Results: Thirty-one embolizations performed on 29 patients (medium age = 55.6; 62.1% male) were included between June 2014 and February 2020. All patients were under dialysis before embolization (9 peritoneal dialysis and 20 hemodialysis). Technical success was observed in 96.8% of cases. Mean procedural time was 65 minutes (range = 35–106 minutes) and mean length of in-hospital stay was 3.8 days (range = 3–6 days). A volume reduction allowing a kidney transplant was obtained for 28 patients (96.5%). The mean volume reduction was 39.9% (range = 6.01–68.2). The main observed complication was postembolization pain in 10 cases (32.2%). One patient needed complementary nephrectomy due to insufficient volume reduction. Twenty-three patients (79.3%) received renal transplant during follow-up with a mean delay of 19.5 month (range = 4–54). Conclusion: Polycystic kidney embolization is an effective and safe minimally invasive technique. It can be proposed as the first-choice technique for kidney transplant recipients as an alternative to pretransplantation nephrectomy.

Cytosporone B (Csn-B), an NR4A1 agonist, attenuates acute cardiac allograft rejection by inducing differential apoptosis of CD4+T cells

CD4+T cell-mediated acute rejection remains a major factor that affects the early survival of transplanted organs post-transplantation. Here, we reveal that nuclear receptor subfamily 4 Group A member 1 (Nr4A1) was upregulated during cardiac allograft rejection and that the increased Nr4A1 was primarily localized in intragraft-infiltrating CD4+T cells. Nr4A1 acts as a transcription factor with an important role in CD4+T cell apoptosis, differentiation and T cell dysfunction, which indicates that Nr4A1 may play a critical role in transplant rejection. Cytosporone B (Csn-B) is a naturally occurring agonist of Nr4A1, and the role of Csn-B in the physiological process of cardiac rejection is poorly defined. This study constructed an acute rejection model of abdominal heterotopic cardiac transplantation in mice and investigated whether Csn-B could attenuate acute transplant rejection by modulating the CD4+T lymphocyte response. The results showed that Csn-B prolonged murine cardiac allograft survival and reduced inflammation in allografts. Subsequently, it was confirmed that Csn-B functions by inducing non-Treg apoptosis and promoting Treg cell differentiation. Finally, we also confirmed that Csn-B attenuates acute rejection by directly targeting Nr4A1 in CD4+T cells. Our data suggest that Csn-B is a promising novel therapeutic approach for acute cardiac allograft rejection.

Integrin αvβ6 contributes to the development of intestinal fibrosis via the FAK/AKT signaling pathway

Intestinal fibrosis is one of the most severe complications of inflammatory bowel disease (IBD) and frequently requires surgery due to intestinal obstruction. Integrin αvβ6, which is mainly regulated by the integrin β6 subunit gene (ITGB6), is a special integrin subtype expressed only in epithelial cells. In our previous study, we found integrin αvβ6 can promote the development of IBD, but the role of integrin αvβ6 in intestinal fibrosis remains unclear. In this study, we observed a gradual increase of ITGB6 mRNA expression from normal region to stenotic region of IBD patients’ intestinal specimens. Next, we established a dextran sulfate sodium (DSS)-induced intestinal fibrosis model and a heterotopic intestinal transplant model, and found intestinal fibrosis was decreased in ITGB6-deficient mice compared to wild-type (WT) mice. Furthermore, we performed RNA-sequencing and KEGG pathway analysis on intestinal tissues from ITGB6-overexpressing transgenic mice and WT mice, and found multiple pathways containing ITGB6, are related to the activation of focal adhesion kinase (FAK); finding was confirmed by Western blot. At last, we generated a heterotopic intestinal transplant model found the FAK/AKT pathway was inhibited in ITGB6-deficient mice. In conclusion, our data demonstrate that integrin αvβ6 promotes the pathogenesis of intestinal fibrosis by FAK/AKT pathway, making integrin αvβ6 a potential therapeutic target to prevent this condition.

Endocrine Function of Ovarian Tissue Grafts After Normo- and Hypothermic Storage

Morphological characteristics and function of ovarian tissue after 2–120 hrs of storage at 4 and 37°C in incubation media with different composition (mannitol-containing solution (MСS) and phosphate buffered saline (PBS)) were investigated. The function of ovarian tissue after normo- and hypothermic storage was studied by heterotopic transplantation to recipient animals simultaneously with bilateral ovariectomy. After 2-hour storage at 37ºC and 24-hour one at 4ºC in all the studied media, the estradiol and progesterone levels in plasma of recipient animals were experimentally proved to be 18.2–27.5 and 13.5–18.5 ng/ml, respectively, (after fresh tissue transplantation these values for estradiol and progesterone made (28.1 ± 3.6) pg/ml and (19.1 ± 2.8) ng/ml, respectively). Prolongation of tissue storage time at 37ºC increased the number of degenerative follicles, augmented TBARS concentration and reduced sex hormone levels after transplantation using both MCS and PBS. A protective effect of MCS only, manifested in endocrine function restoration in recipient animals after transplantation ((13.5 ± 2.5) pg/ml of estradiol) was found after 48 hrs of ovarian tissue hypothermic storage. If PBS was used, the estradiol level was within the indices for ovariectomized animals ((7.1 ± 1.5 (pg/ml)).

Abstract 14368: Valproic Acid Activates Irg1-Itaconate and Improve Donor Heart Function After Extended Storage Time

Background: Cold ischemic storage and subsequent reperfusion injury contributes to the primary graft dysfunction after heart transplantation. Valproic acid (VPA) has been shown to reduce myocardial infarction injury. However, the role of VPA in donor heart preservation is unknown. Methods: We investigate the role of VPA on donor heart preservation using both ex-vivo perfusion and heterotopic transplantation in murine models. Results: Our results demonstrate the administration of VPA to histidine-tryptophan-ketoglutarate (HTK) cardioplegia can significantly improve donor heart function after perfusion with extend storage time (16 hours, n=8). Specifically, VPA reduced the accumulation of succinate, a metabolite that involved in reperfusion injury, during cold storage. We further show that VPA increases the Irg1 expression. Irg1 is known to produce itaconate and suppress the succinate accumulation during ischemia. We found that the effect of VPA in donor heart function is significantly reduced in Irg1 knockout mice compared to wild type mice after 16 hours of cold storage. Furthermore, VPA reduced oxidative stress of donor heart via Nrf2 antioxidant pathway activation. However, Irg1 deletion decreased the induction of antioxidant genes expression by VPA. Administration of 4-octyl-itaconate rescued donor heart function after extended storage. Conclusion: Our results demonstrate that VPA could improve donor heart function and ischemic tolerance. This is in part through Irg1/itaconate mediated anti-oxidative activities. Importantly, VPA preserved cardiac function after perfusion with extended storage times. This strategy can extend the tolerable ischemic time for donor heart for heart transplant and may reduce the incidence of primary graft dysfunction.

Abstract 14312: Inhibition of the Electron Transport Chain and Inflammasome Pathway Preserve Function in Donor Hearts After Circulatory Death

Introduction: Hearts by donation after circulatory death (DCD) have the potential to increase the number of transplantable hearts. However, the DCD process promotes ischemia and reperfusion injury (IRI). Mitochondrial damage and NLRP3 inflammasome activation contribute to the DCD heart damage. Blockade of electron transport chain (ETC) with the reversible inhibitor amobarbital (AMO) or the NLRP3 inhibitor 16773340 (NLRP3-I) can mitigate IRI. Hypothesis: AMO and/or NLRP3-I given to the DCD hearts at the moment of heterotopic heart transplantation afford lasting protection to the DCD hearts. Methods: We induced the DCD process in anesthetized rats that were intubated and ventilated by paralyzing the animal with vecuronium bromide and removing the ventilatory support. Treatment groups were AMO (1 mM), NLRP3-I, AMO+NLRP3-I, and vehicle (N=8-10/group). The DCD hearts were procured after 25 min of in-body ischemia then flushed with ice-cold cardioplegia solution supplemented or not with the AMO. NLRP3-I was administered orally (100 mg/kg) to the recipient before HTx and once a day. Control beating-heart donors (CBD) were procured without ischemia by giving the cardioplegia. The donor hearts underwent heterotopic transplantation in the abdomen of recipients. After 14 days, the function of the DCD heart was measured using an intraventricular balloon. Results: The developed pressure (DP), the peak -dP/dt, and the rate pressure product (RPP) of DCD hearts were significantly reduced compared to the CBD controls (63±7% vs 99±8%; -851±113 vs -1221±123; 20,729±2,268 vs 11,874±1,987, respectively; all p&lt;0.05). AMO, NLRP3-I, and AMO+NLRP3-I preserved the DCD heart function (all parameters, p&lt;0.03). Double treatment AMO+NLRP3-I trended toward a better improvement without reaching statistical significance over single treatments. Conclusions: Inhibition of ETC with AMO alone or combined with NLRP3 inhibition preserved the function of DCD hearts.

Optimization of Ex Vivo Machine Perfusion and Transplantation of Vascularized Composite Allografts

Machine perfusion is gaining interest as an efficient method of tissue preservation of Vascularized Composite Allografts (VCA). The aim of this study was to develop a protocol for ex vivo subnormothermic oxygenated machine perfusion (SNMP) on rodent hindlimbs and to validate our protocol in a heterotopic hindlimb transplant model. In this optimization study we compared three different solutions during 6 h of SNMP (n = 4 per group). Ten control limbs were stored in a preservation solution on Static Cold Storage [SCS]). During SNMP we monitored arterial flowrate, lactate levels, and edema. After SNMP, muscle biopsies were taken for histology examination, and energy charge analysis. We validated the best perfusion protocol in a heterotopic limb transplantation model with 30-d follow up (n = 13). As controls, we transplanted untreated limbs (n = 5) and hindlimbs preserved with either 6 or 24 h of SCS (n = 4 and n = 5). During SNMP, arterial outflow increased, and lactate clearance decreased in all groups. Total edema was significantly lower in the HBOC-201 group compared to the BSA group (P = 0.005), 4.9 (4.3-6.1) versus 48.8 (39.1-53.2) percentage, but not to the BSA+PEG group (P = 0.19). Energy charge levels of SCS controls decreased 4-fold compared to limbs perfused with acellular oxygen carrier HBOC-201, 0.10 (0.07-0.17) versus 0.46 (0.42-0.49) respectively (P = 0.002). Six hours ex vivo SNMP of rodent hindlimbs using an acellular oxygen carrier HBOC-201 results in superior tissue preservation compared to conventional SCS.

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents.

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

Experimental models in the study of pathogenesis and the development of treatments for ovarian cancer (systematic review)

A systematic review of modern methods of experimental study of ovarian cancer using traditional (immunocompetent, genetically engineered and immunodeficient) and non-traditional (that don’t belong to the mammals) animal models,established and primary cultures of human ovarian cancer, including three-dimensional organotypic spheroids (3D- models ex vivo) is presented. The prospects of the considered models for studying of the pathogenesis of various molecular-genetic and histological variants of ovarian cancer, as well as for developing methods of personalized treatment, are discussed. The limitations of modern animal models are indicated. The greatest attention is paid to studies on immunodeficient animals using xenografts based on established cultures of human ovarian cancer cells and on tumor tissue obtained directly from the patients (patient derived xenografts, PDX). The questions of various variants of xenograft transplantation with an emphasis on the problems of orthotopic transplantation of human ovarian cancer into immunodeficient mice and the relevance of methods for local humanization in heterotopic transplantation are considered. The most promising, from the point of the author’s view, approaches to studying the effectiveness of drug therapy for ovarian cancer in immunodeficient animal models are outlined. To prepare a systematic review, a literature search was carried out on the Scopus, Web of Science, Med Line, PubMed, Cyber Leninka, RSCI databases. The analysis used literature sources indexed in the Scopus and Web of Science databases (97%) and the RSCI. More than 60% of the works amount has been published over the past 5 years.