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Background: Studies have shown that lymphocytes support hepatic oval cell (HOC)-dependent liver regeneration and FK506（Tacrolimus） is known as an immunosuppressor. Therefore, we studied the role of FK506 in HOC activation and/or proliferation to guide the clinical use of FK506., Methods: Thirty male Lewis rats were randomly divided into 4 groups: (A) intervene in activation (n = 8), (B) intervene in proliferation (n = 8), (C) control HOC model (n = 8), and (D) pure partial hepatectomy (PH) (n = 6). The HOC model was established by 2AAF(2-acetylaminofluorene)/PH in groups A to C. FK506 (at a dose of 1 mg/kg/d) was given subcutaneously in group A except on operation day, and not until day 8 post-operation (PO) in group B. Half of the animals were euthanized on days 10 and 14 PO, respectively. The remnant liver was weighed and stained by hematoxylin and eosin and immunohistochemical staining of proliferating cell nuclear antigen and epithelial cell adhesion molecule enabled HOC proliferation analysis., Results: FK506 intervention exacerbated liver damage and hindered the recovery of the HOC model rat. Weight gain was severely retarded or even negative. Liver weight and the liver body weight ratio were lower than control group. HE and immunohistochemistry showed pooer proliferation of hepatocytes and fewer HOC numbers in group A., Conclusion: FK506 inhibited HOC activation by affecting T and NK cells, ultimately blocking liver regeneration. Poor liver regeneration after auxiliary liver transplantation might be associated with the inhibition of HOC activation and proliferation caused by FK506 treatment., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Transplantation of the hand or face, known as vascularized composite allotransplantation (VCA), has revolutionized reconstructive surgery. Notwithstanding, there are still several areas of improvement to mitigate immune rejection while sparing systemic adverse effects. The goal of this study was to evaluate the engraftment and viability of a genetically modified cell population pre-engrafted into a VCA transplant, to potentially act as a local biosensor to report and modify the graft in vivo. A rat fibroblast cell line genetically modified to secrete Gaussia-Luciferase (gLuc), which served as a constitutive biomarker of cells, was incorporated into a VCA to study the viability of biosensor cells in a syngeneic rat heterotopic partial hindlimb transplantation model., Results: Five perfusions were first performed as engineering runs to have a stable limb perfusion protocol, followed by 3 perfusions to analyze the cell engraftment during machine perfusion, and finally 4 perfusions to study in vivo persistence of the cell biosensors. Blood samples were collected to monitor gLuc secretion during perfusion and postoperatively. A time-dependent increase in gLuc secretion in the limb perfusion outflow during machine perfusion indirectly verified the presence of biosensors within the graft. After the ex vivo perfusion, VCA hindlimbs were analyzed for near infrared fluorescence emission that showed a presence of dyed engineered cells in all areas of the limbs. Postoperatively, gLuc was detectable 4 to 5 days after transplantation (W = 16, P = .02857). This study demonstrated that engineered cells could be successfully preimplanted into VCAs-an important step toward development of an in vivo biosensor platform to use in modulating acute VCA outcomes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The main cause of chronic renal allograft dysfunction (CRAD) still remains unclear. Insulin resistance (IR) may be a potential inducement, but there is insufficient evidence about this association. We aimed to establish a rat model of CRAD complicated with IR and to explore the function and pathologic changes of the renal allograft induced by IR., Methods: F344-to-Lewis rats of CRAD were fed a high-fat diet to induce IR. They were divided into 3 groups: IR (CRAD+IR), CRAD, and control (CTL). Serum levels of blood urea nitrogen (BUN) and serum creatinine (Scr) were measured to evaluate the renal function. The Homeostasis Model Assessment (HOMA)-IR index was detected by comparing the values of fasting serum insulin levels (FINS) with fasting blood glucose levels (FBG). The pathologic analysis was conducted by the degree of renal lesions including glomerular lesions, renal tubular lesions, hemorrhage, inflammatory cell infiltration, fibrillation, and hyperplasia of the renal interstitium., Results: In the second, third, and fourth month after surgery, serum levels of Scr and BUN in the IR group were reduced more than those in the CRAD group, while they were both higher compared to the CTL group, suggesting that renal function in the CRAD group was declined. The HOMA-IR in the IR group was greater than that in the CRAD and CTL groups, showing that simple high-fat diet feeding significantly and steadily increased FINS and FBG in CRAD complicated with IR rats. Pathologic changes indicated that the CRAD rat model was successfully constructed and was still in the early-middle stages of renal lesions 4 months after surgery, yet IR presented a significant effect on CRAD., Conclusion: These results indicate that the stable CRAD complicated with IR rat model can be established through a high-fat diet in CRAD rats in 4 months, and IR could be an influencing factor., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: New strategies to inhibit acute rejection are needed for further applications of composite tissue allotransplantation. The nuclear receptor subfamily 4 group A member 1 (NR4A1) is considered a key controller of maintaining tolerance homeostasis. However, the effect of NR4A1 in suppressing rejection responses after allotransplantation remains unknown., Methods: Brown Norway rat groin flaps were transplanted into Lewis rat recipients. The recipients were administrated cytosporone B, an NR4A1 activator. NR4A1 expression and graft survival time were assessed. T helper type 1 and regulatory T cell populations in the second lymphoid organ were detected by flow cytometry. Furthermore, a retrovirus containing NR4A1 was constructed and transfected to T cells in vitro. After stimulation, interleukin 2 and interferon gamma secretions were detected in the T cells., Results: Administration of cytosporone B activated NR4A1 expression in allotransplant recipients and was associated with prolonged survival time of the vascularized free flap allograft. T helper type 1 cells in the recipient secondary lymphoid organs were decreased, whereas the population of regulatory T cells did not change. Interleukin 2 and interferon gamma were suppressed in vitro in the T cells overexpressing NR4A1., Conclusions: We demonstrated that the overexpressed NR4A1 is associated with suppressed graft rejection response. The suppression effect may attribute to induction of T-cell anergy and blockade of key immunologic cytokines., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Intravenous immunoglobulin (Ig) therapy is used based on empirical findings for treatment of antibody-mediated rejection (AMR) in cases of renal transplantation, although its therapeutic efficacy has not been proven and the functional mechanism of an administered Ig remains elusive. In this study, the therapeutic effects of an Ig were examined in a preclinical rat renal transplant model of AMR to investigate this mechanism., Methods: To establish an AMR renal graft model, skin graft specimens were obtained from Brown-Norway (BN) rats and transplanted to Lewis rats to produce donor-specific antibodies (DSAs), after which kidney transplantation from the Brown-Norway to Lewis rats was performed. AMR model rats were administered the Ig at a dose of 2 g/kg or saline as a control. Survival period, renal graft histopathology, complement factors, and DSA levels were assessed., Results: The survival period of the group administered the Ig was significantly prolonged. Histopathological examinations of renal grafts also showed significant suppression of glomerulitis and peritubular capillaritis in the Ig group, and real-time polymerase chain reaction analysis results demonstrated significantly lower levels of expression of the complement factors C1q and C3. When the Ig was given to rats that underwent skin grafting but not renal transplantation, DSA was decreased after 6 hours and remained lower than the baseline level for at least 7 days., Conclusion: Ig administration suppressed DSA production. The present results showed that suppression of the complement system contributed to effective Ig treatment for AMR., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Expression of genes with immunoregulatory capacity can potentially decrease rejection of allograft. According to recent studies, viral interleukin (IL)-10 can reduce immune response during allotransplantation and is one of the most promising methods for the prevention of rejection. Our study aimed to analyze the immunosuppressive potential of recombinant adenovirus-mediated rat IL-10 in rat skin allograft., Methods: We performed skin graft surgery 1 hour after infecting the donated skin with adenovirus-mediated rat IL-10. On day 7 postoperatively, the skin allografts were harvested, and acute rejection was graded histologically., Results: Viral IL-10 gene transfer into rat skin allografts improved graft survival and reduced acute rejections., Conclusion: The results of our study suggest that the therapeutic potential of graft viral IL-10 gene transfer is an effective immunosuppressive method for preventing skin allograft rejection., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Macrophages may be important in chronic rejection after organ transplantation. This study aimed to investigate the possibility of depleting macrophages for a certain amount of time to alleviate chronic rejection in a heart transplant model of Fischer to Lewis rats., Methods: Clodronate liposome was injected abdominally to deplete macrophages for 2 time frames. The expression levels of ectodysplasin 1, arginase 1 (Arg1), chitinase-like lectin (Ym1), interferon gamma, tumor necrosis factor α (TNF-α), smooth muscle α-actin (α-SMA), monocyte chemoattractant protein 1 (MCP-1), and interleukin 10 (IL-10) were detected., Results: 1. The expression levels of α-SMA, interferon gamma, TNF-α, and MCP-1 and the transformation of peripheral T cells were lower after macrophage depletion for 2 or 4 weeks. 2. The expression levels of α-SMA, TNF-α, and MCP-1 and the transformation of peripheral T cells were even lower after 4 weeks compared with 2 weeks, except for interferon gamma. 3. A higher level of expression of Arg1 and Ym1 after macrophage depletion for 2 weeks was observed. 4. A higher level of expression of IL-10 after macrophage depletion for 2 weeks, but not 4 weeks, was also observed., Conclusions: Macrophage clearance after heart transplantation alleviated chronic rejection probably via M2 polarization of regenerated macrophages, reduced T-lymphocyte proliferation, and changed the expression levels of interferon gamma, TNF-α, MCP-1, and IL-10., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background Context: Smoking is detrimental to obtaining a solid spinal fusion mass with previous studies demonstrating its association with pseudoarthrosis in patients undergoing spinal fusion. Varenicline is a pharmacologic adjunct used in smoking cessation which acts as a partial agonist of the same nicotinic receptors activated during tobacco use. However, no clinical or basic science studies to date have characterized if varenicline has negative effects on spinal fusion and bone healing by itself., Purpose: Our study's aim was to elucidate whether varenicline affects the frequency or quality of posterolateral spinal fusion in a rodent model at an endpoint of 12 weeks., Study Design: Randomized control trial., Patient Sample: Fourteen male Lewis rats randomly separated into two experimental groups., Outcome Measures: Manual palpation of fusion segment, radiography, μCT imaging, and four-point bend., Methods: Fourteen male Lewis rats were randomly separated into two experimental groups undergoing L4-L5 posterior spinal fusion procedure followed by daily subcutaneous injections of human dose varenicline or saline (control) for 12 weeks postsurgery. Spine samples were explanted, and fusion was determined via manual palpation of segments by two independent observers. High-resolution radiographs were obtained to evaluate bridging fusion mass. μCT imaging was performed to characterize fusion mass and consolidation. Lumbar spinal fusion units were tested in four-point bending to evaluate stiffness and peak load. Study funding sources include $5000 OREF Grant. There were no applicable financial relationships or conflicts of interest., Results: At 3 months postsurgery, 12 out of 14 rats demonstrated lumbar spine fusion (86% fused) with no difference in fusion frequency between the varenicline and control groups as detected by manual palpation. High-resolution radiography revealed six out of seven rats (86%) having complete fusion in both groups. μCT showed no significant difference in bone mineral density or bone fraction volume between groups in the region of interest. Biomechanical testing demonstrated no significant different in the average stiffness or peak loads at the fusion site of the varenicline and control groups., Conclusion: Based on the results of our rat study, there is no indication that varenicline itself has a detrimental effect on the frequency and quality of spinal fusion., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Brain-dead (BD) donor is the main source for liver transplantation (LT). We aim to investigate the effect of brain death on donor liver inflammatory activity and its association with ischemia-reperfusion (I/R) injury and biliary tract injury after LT., Material and Method: A brain death model using male Lewis rats was established, in both BD and non-BD groups; livers were harvested for transplantation using a 2-cuff technique. The rats were sacrificed 12 hours (n = 10) or 4 weeks (n = 10) after transplantation. I/R injury and long-term biliary tract injury were observed after transplantation., Results: All rats survived for 4 weeks after transplantation. At 12 hours after BD-donor LT (BDDLT), liver injury worsened; serum transaminases, bilirubin, oxidative stress, inflammatory responses and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining level substantially increased (P < .05). At 4 weeks after BDDLT, serum bilirubin and bile lactate dehydrogenase and γ-glutamyl transpeptidase levels were elevated (P < .05). Biliary fibrosis and epithelial-mesenchymal transition (EMT) were detectable and NDRG1 gene expression was decreased., Conclusions: These results suggested that brain death-induced inflammatory response in donor organs and resulted in a worse I/R injury and biliary tract injury after LT in rats. The brain death-related biliary tract injury may be associated with the regulation of EMT through NDRG1., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Subcutaneous pockets provide an extrahepatic transplant site for islet grafting to treat type 1 diabetes. However, a hypoxic environment may cause central necrosis to islets and lead to graft failure. Our previous studies focused on a pre-treated subcutaneous site with basic fibroblast growth factor (bFGF) for the formation of vascular bed. In addition to neovascularization, bFGF was also shown to protect islets against oxidative stress and chemical-induced damage in vitro. Accordingly, we propose that subcutaneous islet transplantation with a bFGF-slow releasing device simultaneously can improve islet survival in vivo., Methods: A bFGF-impregnated collagen sheet was implanted in the right back of a streptozotocin-induced diabetic mouse for neovascularization. After 10 days, the sheet was removed and the rat islet-embedding gel within the immune-isolation device was transplanted (2-time operation [OP]). In another group, the diabetic mice received bFGF-impregnated gel with rat islets within the immune-isolation device simultaneously (1-time OP)., Results: Diabetic mice in 2-time OP group experienced a decrease in their non-fasting blood glucose level for a period of 10 days, and the glucose levels were lower than those of untreated diabetic mice post-implantation. However, the mice in the 1-time OP group remained hyperglycemic post-operation and showed no improvements in body weight or the area under curve in intraperitoneal glucose tolerance test. Furthermore, mice in the 2-time OP had relatively higher serum insulin levels with improved renal and metabolic biomarkers., Conclusion: Our findings suggest that bFGF had no beneficial effect on a 1-time operation in subcutaneous islet transplantation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to evaluate the impact of static cold storage preservation on skeletal muscle metabolism using a rodent model., Methods: Sixteen male Lewis rats (250 ± 25 g) were distributed into 4 groups, including naive control, warm ischemia for 2 hours, static warm storage for 6 hours, and static cold storage for 6 hours. Energy status, metabolomics profiling, and histopathology of the muscle were analyzed., Results: In the warm ischemia and static warm storage groups, glycolytic pathway metabolites decreased, but the Krebs cycle metabolite of succinate and the purine degradation product of hypoxanthine accumulated. Increased succinate and hypoxanthine levels were associated with increased injury severity scores. During static cold storage, the glycolytic pathway activity and the energy status were preserved. Succinate and hypoxanthine levels showed no significant difference from the naive group., Conclusion: Warm ischemia results in reduced glycolysis and Krebs cycle metabolites. Static cold storage preserves the glycolytic pathway and represents a favorable contribution to cellular energy demand. Succinate and hypoxanthine might be used as novel potential biomarkers for the assessment of viability and injury severity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Human umbilical cord-derived mesenchymal stem cells (HuMSCs) have been shown to suppress cardiac fibrosis; however, the underlying mechanisms are not fully understood. Recent studies have shown that endothelial-mesenchymal transition (EndMT) plays a crucial part in myocardial fibrosis. In the present study, we investigated the suppressive role of HuMSCs in cardiac fibrosis and related mechanisms in a rat dilated cardiomyopathy (DCM) model., Methods: Male Lewis rats were randomly divided into 3 groups. Rats without any treatment served as a negative control group, while the DCM rats, which were generated by immunization with porcine myosin, were divided into 2 groups: a HuMSC group, in which HuMSCs (1 × 10 6 cells/rat) were injected intravenously, and a vehicle group, in which rats were injected with volume-matched solution containing no HuMSCs. Histologic and immunofluorescent measurements were used to evaluate the effects of HuMSCs on cardiac fibrosis and EndMT., Results: We observed a significant increase in myocardial fibrosis, and elevated EndMT in rats of the vehicle group were observed compared with those in the negative control group along with the increased activity of transforming growth factor (TGF)-β1/extracellular signal-regulated kinase (ERK) 1/2 signaling. Treatment with HuMSCs repressed the increase in myocardial fibrosis and EndMT observed in DCM rats, which correlated with decreased activity of TGF-β1/ERK1/2 signaling., Conclusion: The HuMSCs attenuated cardiac fibrosis at least partly through the inhibition of TGF-β/ERK-induced EndMT., (Copyright © 2019. Published by Elsevier Inc.)

Objective: A rat model of right whole ovary transplantation is difficult to establish because of an unfit location or absence of the right ovarian artery. The purpose of this study is to develop a rat model of left whole ovary transplantation., Methods: Two methods of whole ovary transplantation were performed. In Group A (n = 40), the rats underwent right whole ovary grafting via conventional orthotopic allotransplant, with donor abdominal aorta and inferior vena cava end-to-side anastomoses to the recipient abdominal aorta and inferior vena cava. In Group B (n = 40), the rats underwent left whole ovary graft via orthotopic allotransplant, with donor abdominal aorta and left renal vein end-to-side anastomoses to the recipient abdominal aorta and inferior vena cava. Bilateral native ovariectomy in the recipient was performed immediately in both groups. The operative times, recipient survival rates, and transplanted ovarian function were measured., Results: There was no significant difference in the success rates of transplant between the 2 groups, with 92.5% and 90% success, respectively. All the living recipients recovered ovarian function. However, the operative time for the donor, the vein anastomosis time, and the cold ischemia time decreased significantly in Group B (P < .05)., Conclusion: The modified method of left whole ovary transplantation is reliable and easy to perform, which could significantly shorten the times of graft harvest and cold ischemia. The method is a good supplement and improvement of the conventional model of rat whole ovary transplantation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background Context: Degenerative disc disease (DDD) is associated with longitudinal remodeling of paravertebral tissues. Although chronic vertebral changes in advanced stages of DDD are well-studied, very little data exists on acute vertebral bone remodeling at the onset and progression of DDD., Purpose: To longitudinally characterize bony remodeling in a rodent model of disc injury-induced DDD., Study Design: In vivo animal study involving a rat annulus fibrosus injury model of DDD., Methods: Eight female Lewis rats were assigned to intervertebral disc (IVD) injury (Puncture) or sham surgery (Sham). All rats underwent anterior, transperitoneal approach to the lumbar spine, and Puncture rats underwent annulus fibrosus injury at the L3-L4 and L5-L6 IVDs (n = 8 per group). Live micro computed tomography imaging (10-μm voxel size) was performed 1 week before surgery and postoperatively at 2-week intervals up to a 12-week endpoint. Bone morphology and densitometry of the cranial vertebral body and bony endplate were analyzed and reported with respect to the preoperative baseline scan. Sagittal Safranin-O/Fast-Green and Toluidine Blue histology evaluated using the Rutges IVD score and a custom vertebral endplate score., Results: Vertebral trabecular tissue mineral density (TMD), vertebral trabecular spacing, endplate TMD, and endplate apparent bone mineral density were all significantly greater in Puncture compared with Sham at 4 weeks and each subsequent timepoint. Puncture rats exhibited marginally lower endplate total volume. Anterior endplate osteophyte formation and central physeal ossification were observed in Puncture rats. Endpoint histological analysis demonstrated moderate evidence of IVD degeneration, indicating that vertebral bone adaptation occurs in the acute phases of DDD onset and progression., Conclusions: Annulus injury-induced DDD leads to acute and progressive changes to the morphology and densitometry of bone in the adjacent vertebral bodies and endplates., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The goal of this study was to analyze the protective role of ARA290 in early renal allograft injury by using a rat model of renal allograft., Methods: Lewis rats were divided into 3 groups: sham, University of Wisconsin solution (UW), and ARA290. A rat model of renal allograft was established by anastomosis using a titanium ring pin stapler. The kidneys were removed 24 hours after transplantation to accomplish the following: (1) examine the protective effect of ARA290 on renal morphology and function; (2) investigate the underlying mechanism by determining the binding affinity of nuclear factor-κB (NF-κB) to DNA by using an electrophoretic mobility shift assay; (3) observe the effect of ARA290 on macrophage infiltration using immunohistochemistry; and (4) detect messenger RNA (mRNA) expression of inflammatory mediators by using reverse transcriptase polymerase chain reaction., Results: Serum creatinine and blood urea nitrogen levels in the ARA290 group were significantly lower than those in the UW group. Kidney tissue samples from the UW group exhibited morphologic abnormalities and marked macrophage infiltration compared with those in the sham group. In the ARA290 group, renal morphology was greatly improved with decreased macrophage infiltration. The binding affinity of NF-κB to DNA in the ARA290 group was markedly lower than that in the UW and sham groups. The mRNA expression of NF-κB downstream effectors (monocyte chemotactic protein-1; regulated on activation, normal T cell expressed and secreted; intercellular adhesion molecule-1; and vascular cell adhesion molecular-1) was significantly downregulated in the ARA290 group compared with that in the UW group., Conclusions: ARA290 protects against early renal allograft injury in rats by reducing macrophage infiltration, improving renal morphology, inhibiting mRNA expression of inflammatory mediators, and weakening the binding affinity of NF-κB to DNA., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Hepatocyte transplantation is a promising alternate for the treatment of hepatic diseases. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in the quantity sufficient and the quality required for biotherapy. Isolated fresh hepatocytes include damaged cells that are also early apoptotic cells, which is not ideal for hepatocyte transplantation. However, this does not reflect cell viability, although it is considered that it adversely affects cell survival after transplantation. We aimed to harvest these hepatocytes and filter the apoptotic cells using a magnetic method to provide a transplantation source., Materials and Methods: Rat hepatocytes were isolated from caudate lobes using manual enzymatic perfusion. The hepatocyte yield was 5.3 ± 0.66 × 10 9 cells/g of liver tissue, with a viability of 82.3 ± 3.5%. Two samples of hepatocytes were freshly isolated, one using the magnetic method, and the other without. The magnetic method was performed using DynaMag-15 Magnet, and Annexin V Antibody was used on the early apoptotic cells. We evaluated the viability and plate efficiency of the cells after 24 hours at 37°C. Hepatocytes were isolated using cell separation method, and 30 × 10 6 cells were mixed with 1.0 mL of Dulbecco's Modified Eagle's Medium (DMEM) and directly injected into the spleen of Lewis rats (150-250 g) using 24-gauge needles. Blood samples were collected on days 0, 3, 7, and 14, and the blood albumin level was measured using enzyme-linked immunosorbent assay (ELISA):G1, control (medium injection); G2, fresh hepatocyte transplant using the magnetic method; and G3, fresh hepatocyte transplant without the magnetic method., Results: The viability was 84.9 ± 2% for fresh hepatocytes and 80.7 ± 1.2% for hepatocytes isolated using the magnetic method. The magnetic method does not damage the cells (73.5 ± 2% vs 35.2 ± 2% after 24 hours), preserving hepatocyte. The albumin level accepted significantly increased in the magnet-treated group compared with the nonmagnet group. Simultaneously, the spleen in which these hepatocytes were transplanted could be used to observe the hepatocytes; the cells were transplanted 14 days later, and the magnet-treated group had significantly higher levels of hepatocytes than the nonmagnet group., Conclusion: We developed an effective technique for hepatocyte isolation for short-term preservation. As a result, we believe that transplantation not only improves the cell transplantation effect but also allows the cells to be stored efficiently using the magnetic method. These results demonstrate the usefulness of hepatocyte hypothermic preservation for cell transplantation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Previously, high levels of CXCR3+ T-cell recruitment was demonstrated in the prolonged ischemia-accelerated acute allograft rejection in rat kidney transplant. In the present study, the effect of chemokine IP-10 was investigated and the expression of chemokine-related PRINS (Psoriasis susceptibility-related RNA gene induced by stress) lncRNA determined in the allografts subjected to ischemia., Methods: F344-to-Lewis rat kidney transplantation was performed, and renal grafts were stored for 2 hours or 16 hours. Samples were removed at 24 hours and 7 days after operation. Cellular infiltration was determined with the use of immunohistochemistry, and messenger RNA expression was assessed with the use of real-time polymerase chain reaction., Results: The 16-hour-ischemia kidney displayed acute tubule damage and up-regulation of PRINS lncRNA expression. On day 7, IP-10 expression and CD3-positive T cells were increased in allografts compared with control samples, which were inhibited by the IP-10 antibody treatment accompanied by reduced serum creatinine., Conclusions: These observations provide evidence for IP-10 in a regulatory role in cold ischemia-elicited acute allograft rejection and in PRINS lncRNA expression. Our data enhance the understanding of the mechanism underlying between prolonged ischemia and acute rejection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background Context: Bone morphogenetic protein (BMP)-2/7 heterodimer is a stronger inducer of bone regeneration than individual homodimers. However, clinical application of its potent bone induction ability may be hampered if its use is accompanied by excessive inflammatory reactions., Purpose: We sought to quantitatively evaluate bone induction and inflammatory reactions by BMP heterodimer and corresponding BMP homodimers using ultra-high resolution magnetic resonance imaging (MRI) and micro-computed tomography., Study Design: An experimental animal study was carried out., Methods: A total of 32 absorbable collagen sponge implantations into dorsal muscle were performed in rats of four different groups (control group, 0 µg BMP; recombinant human (rh)BMP-7 group, 3 µg rhBMP-7; rhBMP-2 group, 3 µg rhBMP-2; rhBMP-2/7 group, 3 µg rhBMP-2/7). Inflammatory reactions were evaluated by 11.7-T MRI (axial T2-weighted imaging using rapid acquisition with relaxation enhancement) at postoperative days 2 and 7. Bone volumes (BVs) of the induced ectopic bone were quantified at postoperative day 7. In addition, immunohistochemical staining for interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α was performed in samples obtained on postoperative day 2. Bone formation (BF)-to-inflammation (IM) ratios were calculated by dividing BVs by values of inflamed areas., Results: At postoperative day 2, the mean volume of T2 high area on MRI scans in BMP-2 group was significantly larger than that in control group. In contrast, the BMP-2/7 had no difference in the mean volume of T2 high area compared with the control group; however, there was no difference between the BMP-2/7 compared with BMP-2 group. At postoperative day 7, the volumes of T2 high area were not different between the groups. Mean BV of the newly formed bone on postoperative day 7 was significantly greater in BMP-2/7 group than in BMP-7 groups. No new bone formation was observed in control group. BF-to-IM ratio in BMP-2/7 group was significantly higher than those in BMP-2 and BMP-7 homodimer groups. Immunohistochemistry experiments did not reveal differences in expression levels of IL-1β, IL-6, or TNF-α in samples from BMP-2, BMP-7, and BMP-2/7 groups., Conclusions: This study demonstrated that BMP-2/7 heterodimer has stronger bone induction ability without accompanying increased inflammatory reactions (the increased BF-to-IM ratio) than those observed by BMP-2 or BMP-7 homodimers. These results suggest that BMP-2/7 heterodimer can be an alternative to BMP-2 and BMP-7 homodimers in clinical applications, although further translational studies, including whether lower doses of BMP heterodimer may produce similar bone formation compared with the BMP homodimers but produce a reduced inflammatory response, are required., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: This study aimed to investigate the acute rejection patterns of outbred mice and rats on a xenotransplantation model., Materials and Methods: Thirty male Balb/c mice weighing from 30 to 40 g were used as donors, and 30 male outbred Sprague-Dawley rats weighing from 150 to 200 g were used as recipients for heterotopic heart xenotransplantation models. Animals were allocated into 5 groups according to their killed days, and each group had 6 animals. Animals in group 1 were followed for 1 day and then killed; animals in groups 2, 3, 4, and 5 were followed up 2, 3, 4, and 5 days after transplantation, respectively. Operative findings, the appearance and heartbeat strength of the grafts, and histopathologic examinations of the grafts were evaluated to determine acute rejection., Results: There were no mortalities during the study period, and the animal survival rate was 100%. Heartbeat strengths were strong (4.5 ± 0.5) and graft appearances were normal in group 1 animals. The heartbeat scores of the xenografts dramatically declined thereafter. Histopathologically, there were inflammation patterns in all xenografts. The infiltration of neutrophils and the formation of platelet and fibrin thrombi were seen in the first postoperative day and gradually increased daily., Conclusions: This study concluded that outbred Balb/c mouse to Sprague-Dawley rat combination has reliable acute xenograft rejection patterns microscopically and macroscopically in the heterotopic heart xenotransplantation model., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: We describe a simple and reliable orthotopic kidney transplantation method in rats with the use of sleeve arterial anastomosis and a modified stenting technique for anastomosis of the renal vein (RV)., Methods: Male Fischer and Lewis rats were used as kidney donors and recipients, respectively, and their left kidneys were harvested in situ. In the control rats (n = 30), the renal artery (RA) and RV anastomoses were performed end-to-end with interrupted sutures by means of the conventional technique. In the experimental animals (n = 30), revascularization of the RA was fashioned end-in-end with the use of a modified sleeve anastomosis, the RV was anastomosed end-to-end with the use of a modified stenting technique and interrupted sutures, and the ureter was anastomosed with the use of the end-to-end interrupted suture technique., Results: The arterial anastomosis time in the control group was 8.52 ± 1.1 minutes, and that in the experimental group was 4.7 ± 0.6 minutes (P < .05). The venous anastomosis time in the experimental group was 9.2 ± 1.3 minutes, which also was less than in the control group (11.19 ± 0.78 minutes; P < .05). The warm ischemia time decreased from 26.8 ± 1.3 minutes in the control group to 20.7 ± 0.5 minutes in the experimental group (P < .05). The success rate of 93% at 21 days after grafting was identical in the experimental and control groups., Conclusions: We developed a modified model of orthotopic kidney transplantation that can significantly reduce the warm ischemia time., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the potential of high-resolution magic angle spinning (HR-MAS) 1 H nuclear magnetic resonance (NMR) spectroscopy for metabolite characterization and the differentiation of acute rejection after heart transplantation in rat models., Methods: We transplanted syngeneic heart grafts from Lewis rats (n = 4) and allogeneic heart grafts from F344 rats (n = 4) heterotopically into Lewis recipients. On day 7 postoperatively, the transplanted hearts were harvested for ex vivo 1 H NMR spectroscopy and HR-MAS 1 H NMR spectroscopy. 1 H NMR spectroscopy and HR-MAS 1 H NMR spectroscopy were performed at 4.7 T and 11.7 T, respectively. Metabolomic profiles contributing to the differentiation of allogeneic and syngeneic graft groups were statistically assessed by orthogonal partial least squares discriminant analysis (OPLS/O2PLS-DA). Metabolite concentrations were normalized by total spectral intensities and were compared using Mann-Whitney U tests., Results: One allogeneic graft that showed extensive necrotic change suggesting graft failure was excluded from the statistical analysis of the NMR spectroscopy. In the 4.7-T 1 H NMR spectroscopy, the creatine peak was decreased in the allogeneic group. The PLS-DA and OPLS/O2PLS-DA score plot demonstrated good discrimination of the allogeneic graft group from syngeneic graft group. The concentrations of creatine, myo-inositol, glucose, niacinamide, hypoxanthine, inosine, and glutamine were significantly decreased in the allogeneic graft group, whereas the concentrations of glycine, phosphoethanolamine, xanthine, sn-glycero-3-phosphocholine, leucine, valine, and tyrosine were significantly increased (P < .05)., Conclusions: HR-MAS 1 H NMR spectroscopy can metabolically characterize the acute rejection of heart transplantation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Delayed xenograft rejection (DXR) is an insurmountable barrier for xenotransplantation. Previous studies reported that hamster hearts transplanted into rats undergo DXR and stop functioning after the cessation of immunosuppression. Herein, we investigated the effects of resveratrol on preventing DXR in a hamster-to-rat vascularized cardiac xenograft model., Methods: Hamster hearts were engrafted into Lewis rat recipients. Recipient rats received a graft acceptance-inducing treatment of leflunomide and FK506 for 2 weeks to prevent acute xenograft rejection (AXR). Then, all recipient rats were randomly divided into two groups: one group was treated with 1% carboxymethyl cellulose vehicle (control group) and one group was treated with resveratrol (RES group)., Results: After cessation of FK506 and Lef in recipient rats, treatment with resveratrol prolonged the mean survival time from 8.5 days in the control group to 23.5 days. Compared with the control group, neutrophil infiltration in both cardiac muscle and blood vessel tissues of the grafts after resveratrol treatment was significantly reduced. Meanwhile, resveratrol treatment also significantly inhibited T-cell response along with increased production of cytokines including interleukin (IL)-4, IL-10, and decreased interferon (IFN)-gamma of recipients. Furthermore, in the RES group, immunoglobulin (Ig)M antibody production was markedly inhibited, while the production of IgG antibody and the frequency of B cells did not vary., Conclusion: Administration of resveratrol can markedly delay the emergence of DXR by inhibiting IgM secretion., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: This study analyzed the effects of short hairpin RNA targeting interleukin-1 receptor-associated kinase-4 (IRAK-4-shRNA) via portal vein perfusion during different periods on ischemia/reperfusion injury after liver transplantation., Methods: Rats were randomly divided into 3 groups: the cold ischemia transfection group (CIT group, n = 18), in which graft livers were perfused with the plasmid of pSIIRAK-4 expressing IRAK-4-shRNA for 4 minutes (0.5 mL/min) via the portal vein during the cold ischemia period; the in vivo transfection group (IVT group, n = 18), in which equivalent volumes (2 mL) of IRAK-4-ShRNA plasmid (pSIIRAK-4) were injected during the operation; and the control group (n = 18), in which the rats received equivalent volumes of blank plasmid. At 0, 60, and 180 minutes after portal vein reperfusion, blood and liver tissues were collected for examination. IRAK-4 expression, nuclear factor kB (NF-kB) activity, tumor necrosis factor α, interleukin (IL)-1β, and IL-6 serum levels, as well as histologic changes, were detected., Results: At 0 minutes after reperfusion, IRAK-4 expression, NF-κB activity, and serum levels of tumor necrosis factor α, IL-1β, and IL-6 showed no significant differences among the 3 groups (P > .05). At 60 and 180 minutes after reperfusion, all indices of the IVT and control groups were significantly higher than those of the CIT group (P < .01). Meanwhile, all indices of the CIT group showed no significant differences at various time points (P > .05). Liver function and histologic changes exhibited less liver injury in the CIT group than in the other groups., Conclusions: IRAK-4 activity was suppressed by IRAK-4-shRNA through portal vein perfusion during the cold ischemia period, and IRAK-4-shRNA effectively prevented ischemia/reperfusion injury after liver transplantation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: The use of positron-emission tomography (PET) with (18)F-fluorodeoxyglucose (FDG) -labeled islets has been considered to be a potential modality to visualize and quantify early engraftment of islet transplantation. The objective of this study was to evaluate the early islets' survival of the FDG-labeled islets with or without warm ischemic stress in portal transplanted rats using PET and autoradiography., Methods: Islets were isolated from Lewis rat pancreata with or without 30-minute warm ischemia times (WITs). For islets' labeling, 300 islets were incubated with 3 MBq FDG for 60 minutes. FDG-labeled islets were transplanted into the liver via portal vein. In in vivo study, a PET study was scanned for 90 minutes and the FDG uptake was expressed as percentage of liver injection dose (ID). In ex vivo study, the liver was exposed for 30 minutes with single fluorescence autoradiography., Results: In the PET study, the percentage of liver ID of the islets without WIT was 27.8 and that of the WIT islets was 20.1 at the end of islet transplantation. At 90 minutes after transplantation, the percentage of liver ID was decreased to 14.7 in the islets without WIT and 10.1 in the WIT islets. In the autoradiogram, the number of hot spots was more obviously visualized in the liver transplanted without WIT islets than in the liver transplanted with WIT islets., Conclusion: Almost 50% of the islets were immediately lost in both the islets without WIT and those with WIT transplantation in the early period. However, islet survival was 1.4 times higher in the islets without WIT than that in those with WIT in the early engraftment phase., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Kupffer cell (KC), a kind of important antigen-presenting cell in liver, play an important role in the process of acute rejection after liver transplantation. The aim of this study was to investigate effect of suppression of donor KC B7 expression on recipient lymphocyte activation and secretion of interleukin-2 (IL-2) in vitro., Methods: Liver ex vivo perfusion with collagenase IV and density-gradient centrifugation were used to isolate donor Lewis rat KCs. The interference fragments of the B7 molecule were designed to construct RNA interference vector pSilencer 3.1H1-Neo-B7 that was transfected into KCs of donor rat. Reverse-transcription polymerase chain reaction was used to detect the changes in the expression of B7 molecules in KCs. The transfected KCs were divided into 3 groups: A, control group; B, empty vector group; and C, RNA interference group. The lymphocytes of recipient Brown Norway (BN) rats were isolated and cocultured with the cells in the 3 groups. Enzyme-linked immunosorbent assay was used to detect the content of IL-2 in the culture supernate. Methylthiazolyl tetrazolium assay was used to detect the proliferation of lymphocytes., Results: The yield rate of KCs was 5 × 10(7), and the cell viability was >98%. RNA interference vector had been successfully constructed and identified by means of enzyme digestion and sequencing. The expression of B7 in KCs decreased by 22% after RNA interference (P < .01). After coculturing with lymphocytes of BN rats, compared with the control group, the decreased expression of B7 significantly inhibited the activation and proliferation of lymphocytes as well as the secretion of IL-2 by lymphocytes. The proliferation of lymphocytes in recipient BN rats decreased by 49% (P < .01), and the secretion of IL-2 in the culture supernate decreased by 67% (P < .01)., Conclusions: This study successfully constructed a B7 RNA interference vector, and applied it to assessing reduction of B7 expression in donor KCs. RNA interference significantly suppressed the activation of recipient T lymphocytes and secretion of IL-2 via the CD28/B7 costimulatory pathway and may induce immune tolerance in liver transplants., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Limb allotransplantation is not a life-saving treatment. However, large doses of immunosuppressive agents are needed. There is an urgent need to increase the selectivity and targeting of drugs., Methods: We designed a rat model for intraarterial infusion of cyclosporine (CSA) based on the hindlimb replanted model to simulate the limb allotransplantation. To investigate whether intraartery infusion could improve the drug's distribution, we infused CSA 4.0 mg/kg per day continuously into either the superficial epigastric artery (IA group) or superficial epigastric vein (IV group) of Lewis rats., Results: On day 10, CSA concentrations were measured in skin, muscle, and bone tissues of hindlimb. Samples were taken from different parts of the bilateral hindlimbs in the IA group and right hindlimb only in the IV group. Tissue concentrations of the perfusion side were much higher in IA group. Systemic concentrations of IA group were higher than IV group., Conclusions: These results warrant further research in our next limb allotransplantation model., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Brain death (BD) and cold preservation are major risk factors for an unfavorable transplantation outcome. Although donor dopamine treatment in brain-dead rats improves renal function and histology in allogeneic recipients, it remains to be assessed if this also holds true for the combinations of BD and prolonged static cold preservation., Methods: BD was induced in F344 donor rats, which were subsequently treated with NaCl 1 mL/h (BD, n = 11), NaCl/hydroxy ethyl starch (BD-norm, n = 10), or 10 μg/min/kg dopamine (BD-dopa, n = 10). Renal grafts were harvested 4 h after BD and transplanted into bilateral nephrectomized Lewis recipients 6 h after cold preservation in University of Wisconsin solution. Renal function was evaluated by use of serum creatinine and urea concentrations at days 0, 1, 3, 5, and 10. Ten days after transplantation, recipients were killed and the renal allografts were processed for light microscopy and immune histology., Results: Serum urea concentrations at days 5 and 10 were significantly lower in recipients that received a renal graft from dopamine-treated rats; for serum creatinine, only a trend was observed at day 10. Immune histology revealed a lower degree of ED1-positive cells in the donor dopamine-treated group. Under light microscopy, Banff classification revealed significantly less intimal arteritis in these grafts (P < .05)., Conclusions: Although donor dopamine treatment clearly improves renal histology in this model, the beneficial effect on early renal function was marginal. It remains to be assessed if donor dopamine treatment has a beneficial effect on renal function in long-term follow-up., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A previous study showed that local gene transfer of cytotoxic T-lymphocyte-associated antigen-4 immunoglobulin (CTLA4Ig) significantly prolonged the survival time of rat flap allografts. However, the underlying mechanism is not fully understood. Indoleamine 2,3-dioxygenase (IDO) is considered to be able to modulate the unresponsiveness state of allografts. In this study, we tested the expression of the CD80 molecule, IDO mRNA, and the level of the tryptophan metabolite kynurenine with or without the application of the IDO blocker 1-methyl-tryptophan (1-MT) in a rat composite tissue allotransplantation model. CD80 expression could be detected in the allograft. The ration of IDO mRNA/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA and the level of kynurenine were both enhanced (46.3 ± 8.8 versus 4.6 ± 1.8 and 18.9 ± 1.3 μmol/L versus 2.1 ± 0.2 μmol/L separately) after adenovirus-mediated CTLA4Ig (AdCTLA4Ig) transduction. When 1-MT was applied to the AdCTLA4Ig perfusion recipients, the ration of IDO mRNA/GAPDH mRNA (5.2 ± 2.9) and the level of kynurenine (0.8 ± 0.5 μmol/L) were significantly reduced. Moreover, the allograft survival time was greatly reduced when 1-MT was applied to AdCTLA4Ig perfusion recipients compared to single AdCTLA4Ig perfusion therapy recipients (7.2 days versus 13.6 days). We showed that the inhibitory effect of locally delivered CTLA4Ig is dependent on IDO activities within the allograft., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Hepatocyte transplantation (HT) has been performed in patients with liver-based metabolic disease and acute liver failure as a potential alternative to liver transplantation in countries in which ethical regulations do not allow organ transplantation. One of the problems remains that substances normally secreted by the surviving hepatocytes to bile cannot be removed because of lack of bile canaliculi. We found that ligation of the recipient's common bile duct in hepatocyte transplantation recipients is followed by formation of bile canaliculi. The question arose as to whether the signal released from the obstructed bile vasculature activated the transplanted hepatocytes (HC) or cholangiocyte (CH) to form bile canaliculi., Methods: We transplanted separately isolated autologous HC and CH to spleens and observed the structural organization of the grafted cells., Results: HC formed glycogen-rich clusters but not cords usually not attached to the CH of the new bile canaliculi. Separate clusters of bile canaliculi with keratin 7 and 19-positive and gamma-glutamyl transpeptidase-positive cells were observed. Transplanted CH remained keratin 7 and 19-positive and gamma-glutamyl transpeptidase positive but did not form canaliculi., Conclusions: The transplanted HC fraction may contain hepatic progenitor cells for cholangiocytes, but they become activated only under the condition of bile stasis by an as-yet undefined factor.

Objective: We previously demonstrated that collagenase H (ColH) plays a crucial role in rat islet isolation, whereas collagenase G (ColG) plays only a supporting role. We also showed that collagen III appears to be one of the key targets of ColH based on a mass spectrometry analysis. In the present study, we investigated whether our novel findings in an islet isolation model are universally applicable for other types of cell isolation, such as a hepatocyte isolation, with the use of enzyme blends of recombinant collagenases., Methods: As the first step, the expression of one of the main matrix components, collagen III, on rat pancreatic and hepatic tissues was assessed with the use of immunohistochemical staining. ColG and ColH were expressed in recombinant E. coli carrying expression plasmids for each collagenase. Then the efficiency of the collagenase subtype on rat hepatocyte isolation was evaluated in terms of cell yield with the use of thermolysin combined with either ColG or ColH (n = 3, respectively)., Results: The expression of collagen III on rat hepatic tissues was dramatically lower than that of rat pancreatic tissues. In the rat hepatocyte isolation, a substantial amount of hepatocytes (0.81 ± 0.11 × 10(6)) were obtained in the ColG group, whereas almost no hepatocytes were retrieved in the ColH group, indicating that the influence of the collagenase subtypes in rat hepatocyte isolation are completely opposite to that observed in rat islet isolation., Conclusions: Considering that the expression of collagen III on hepatic tissues was relatively low and that almost no hepatocytes were retrieved when ColH and thermolysin were used, the present study supports our novel finding that collagen III appears to be one of the key targets of ColH in hepatocyte isolation. Therefore, the semiquantification of collagen III on the target tissues not only may positively contribute to efficient islet isolation, but also may affect other types of cell isolation by optimizing the ColH amount., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Acute rejection (AR) remains a challenge in organ transplantation. Preconditioning donor organs can reduce AR and prolong survival. Whether preconditioning with cromolyn (CRM), a mast cell (MC) stabilizer, or compound 48/80 (CMP 48/80), a MC degranulator, can alleviate AR and prolong survival has not been studied., Methods: We used the male-DA-to-female-Lewis-rat orthotopic liver transplantation (OLT) model. Donors were preconditioned with CRM in a MC stabilizing way (CRM group) or CMP 48/80 in a MC depleting way (CMP 48/80 group). Rats preconditioned with phosphate-buffered saline were used as controls (PBS group). After preconditioning, OLT surgeries were carried out. OLT male-Lewis-to-female-Lewis-rats were used as the syngeneic group (syngeneic group)., Results: Rats in the PBS group developed AR rapidly and died at 7.40 ± 1.14 days. Rats in the CRM and CMP 48/80 groups had significantly slower rejections and died at day 17.40 ± 1.67 or 14.20 ± 2.28, respectively (P < .05). Rats in the syngeneic group survived more than 60 days. Rejection activity indexes (RAIs) and liver functions were all alleviated through CRM or CMP 48/80 preconditioning. Interferon-γ messenger RNA (mRNA) expressions were reduced and interleukin-10 mRNA levels were higher in allografts in the CRM and CMP 48/80 groups, compared with the PBS group. These were confirmed by testing serum interferon-γ and interlerkin-10., Conclusion: Preconditioning donor livers with CRM or CMP 48/80 can reduce AR and prolong survival of recipients after OLT., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC administration in marginal donor grafts may help avoid the ischemia-reperfusion injury associated with solid organ transplants. Given the reports of side effects after intravenous MSC administration, local MSC administration to the target organ might be a better approach. We administered adipose tissue-derived MSCs (AT-MSCs) ex vivo to donor rat kidneys obtained after cardiac death (CD)., Methods: Using male Lewis rats (8-10 weeks), and a marginal transplant model of 1hr CD plus 1hr sub-normothermic ET-Kyoto solution preservation were conducted. AT-MSCs obtained from double-reporter (luciferase-LacZ) transgenic Lewis rats were injected either systemically (1.0 × 10(6) cells/0.5 mL) to bilaterally nephrectomized recipient rats that had received a marginal kidney graft (n = 6), or locally via the renal artery (500 μL ET-Kyoto solution containing the same number of AT-MSCs) to marginal kidney grafts, which were then preserved (1 hour; 22°C) before being transplanted into bilaterally nephrectomized recipient rats (n = 8). Serum was collected to assess the therapeutic effects of AT-MSC administration, and the recipients of rats surviving to Day 14 were separately evaluated histopathologically. Follow-up was by in vivo imaging and histological LacZ staining, and tumor formation was evaluated in MSC-injected rats at 3 months., Results: Systemic injection of MSC did not improve recipient survival. In vivo imaging showed MSCs trapped in the lung that later became undetectable. Ex vivo injection of MSCs did show a benefit without adverse effects. At Day 14 after RTx, 75% of the rats in the AT-MSC-injected group (MSC[+]) had survived, whereas 50% of the rats in the AT-MSC-non-injected group (MSC[-]) had died. Renal function in the MSC(+) group was improved compared with that in the MSC(-) group at Day 4. LacZ staining revealed AT-MSCs attached to the renal tubules at 24 hours after RTx that later became undetectable. Histopathologic examination showed little difference in fibrosis between the groups at Day 14. No teratomas or other abnormalities were seen at 3 months., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Adipose-derived mesenchymal stem cells (ASCs) have been considered to be attractive and readily available adult mesenchymal stem cells (MSCs) and are becoming increasingly popular for use in regenerating cell therapy. However, recent evidence attributed a fibrotic potential to MSCs which differentiated into myofibroblasts with highly increased α-smooth muscle actin (α-SMA) expression while transplanted into an injured/regenerating liver in mice. In this study, we studied the role of miR-27b in ASCs and their regenerative potential after partial liver resection in rats. ASCs transfected with control siRNA or miR-27b were intravenously injected into autologous rats undergoing 70% partial hepatectomy (PH). Our data showed that the regenerative capacities of ASCs with overexpressed miR-27b were significantly higher compared with control ASCs. However, the enhanced regeneration, hepatic differentiation, and suppressed liver inflammation, as well as fibrotic activity, were significantly reverted by ZnPP coadministration (heme oxygenase-1 [HO-1] inhibitor) indicating an important role of HO-1 in the regenerating and cytoprotective activities of miR-27b-transfected ASCs. We demonstrated that administration of autologous ASCs overexpressed with miR-27b enhances rapid and early liver regeneration and, importantly, preserves function after PH. The ASCs with miR-27b overexpression might offer a viable therapeutic option to facilitate rapid recovery after liver resection., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: The solution in which graft tissue is stored (that is, preservation solution) is an important component of liver transplantation technology. Its protective effect is induced by substances in the solution, including radical scavengers, buffers, and energy-giving substances. New preservation solutions have proven to be effective in preventing organ damage during cold ischemia and in extending the time limits for storage., Aim: This study determined the relationship between luminescence intensity and content of adenosine triphosphate (ATP) in liver tissue and proposes a new ex vivo screening system that uses Lewis rats transgenic for luciferase for evaluating the effectiveness of preservation solutions., Methods: Samples (diameter, 2 mm) of liver were obtained from transgenic rats. The viability of these tissues after storage for as long as 6 hours in University of Wisconsin (UW) solution, extracellular trehalose solution of Kyoto, Euro-Collins (EC) solution, histidine-tryptophan-ketoflutarate solution, low potassium dextran solution, or normal saline was assessed by determining ATP content and luminescence intensity., Results: Luminescence had a linear relationship (R = 0.88) with ATP levels. Regardless of the preservation solution used, the luminescence intensities of the liver tissue chips decreased linearly with time especially through a short span of time (0 to 2 hours; R(2) = 0.58-1.0). The luminescence of liver chip tissues maintained long term (2 to 6 hours) in UW solution tended to be higher than those of tissues stored in other solutions (P < .05; 6 hours). On the basis of luminescence intensity, EC might be preferable to the other solutions tested for ultra-short-term storage (0.5 to 2 hours)., Conclusion: Our model, which combines the use of the bioimaging system and Lewis rats transgenic for luciferase, effectively assessed the viability of liver tissue samples. We believe that this ex vivo screening system will be an effective tool for evaluating preservation solutions for liver grafts., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: This study aimed to investigate the relationship between the severity of ischemia/reperfusion (I/R) injury and the acute rejection (AR) of allogeneic liver transplants in rats., Methods: The experimental rats were divided in different groups: normal control group (sham group, group I); syngeneic liver transplant control group (similar gene group, group II); and allogeneic liver transplant groups (groups III to VI). The rats were humanely killed at 1, 3, 5, and 7 days after transplantation or sham operation to determine the severity of I/R injury, rejection classification, and hepatocyte apoptosis. Messenger RNA (mRNA) and protein expression levels of Fas, perforin, and granzyme B were assessed in the liver tissues using real-time polymerase chain reaction and immunohistochemistry, respectively., Results: The rejection scores of the transplanted liver tissues gradually increased until these scores were proportional to the severity of I/R injury in groups III, IV, and V. The maximum scores were reached at 7 days after transplantation as the duration of transplantation was extended. The mRNA and protein expression levels of Fas, perforin, and granzyme B were significantly increased at 1, 3, 3, 5, and 7 days after liver reperfusion in groups III, IV, and V compared with those in groups I, II, and VI (P < .05)., Conclusion: The occurrence of AR after allogeneic liver transplantation in rats was positively correlated with the severity of I/R injury. Given that I/R injury caused serious damage to the transplanted liver, the occurrence of AR consequently decreased., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Poor early graft function (EGF), a frequent complication of kidney transplantation, can be caused by many risk factors, including donor kidney and body weights., Methods: We studied the relationship to early graft function in a rat kidney transplantation model among 3 indices: ratio of graft to body weight; ratio of native kidney to body weight, and weight difference/body weight of the recipient. We categorized 2 groups based on contralateral nephrectomy at day 1 (G1) or day 3 (G2) after transplantation. EGF was evaluated by measuring serum creatinine levels at day 1 after bilateral nephrectomy., Results: The 3 indices, ie, weight difference/body weight of recipient (G1 and G2: P < .0001), ratio of native kidney to body weight (G1: P < .0001; G2: P = .0013), and ratio of graft to body weight (G1: P = .0064; G2: P = .014) strongly correlated with EGF regardless of the time of contralateral nephrectomy., Conclusions: The index of weight difference/body weight of recipient sensitively and predominantly influenced EGF, which probably reflects the systemic metabolic profile., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: This study was designed to evaluate the role of 0%, 3%, 6% hydroxyethyl starch (HES) and University of Wisconsin (UW) perfusion and preservation solutions on ischemia-reperfusion injury (IRI) of rat intestinal transplantations, solutions, respectively., Materials and Methods: Rats underwent orthotopic intestinal transplantation (Lewis to Lewis) after using perfusion and preservation saline (group l), 3% HES (group 2), 6% HES (group 3), or UW (group 4) solutions. The change in weight was recorded from preoperative to postoperative day (POD) 30. At 30 minutes after reperfusion, we harvested intestinal juice preoperatively as well as at 30 minutes after reperfusion and on POD 1 and 3 when recipients underwent open surgery for maltose absorption tests and sampling. The Park' scores of IRI were evaluated by light microscopy after hematoxylin and eosin (H&E) staining., Results: An increased weight was more evident in group 2 than the other groups, particularly the on POD 1 and POD 3 (P < .05). It was significantly greater than groups 1 and 3 on POD 7 (P < .05). Compared with the other groups, the 30-minute post-reperfusion. Park score and intestinal juice content in group 2 was decreased significantly (P < .01), while in group 3 the Park score was increased, and the maltose absorption level decreased significantly (P < .05)., Conclusion: Three percent HES solution attenuated IRI in rat intestinal transplantation. High-concentration HES solutions were unfit for intestinal preservation. Thus the adverse effects of UW solution may be attribute at least in part to its high HES, concentration., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: We sought to assess the expression of five cell stress molecules: Heat shock protein (HSP)-60, HSP-70, interferon (IFN)-α, rat retinoic acid early induible-1 like transcript (RRLT), and vasoactive intestinal peptide (VIP) during rat chronic small bowel allograft rejection., Methods: A rat model of small bowel allograft rejection was induced on postoperative day 90 using F344 rat as donor and Lewis rat as recipient., Results: We demonstrated increased expression of lamina propria HSP-60 (mean rank grade 8.3 and 3.2, respectively), graft RRLT (relative mRNA amount 5.1 ± 1.8 and 3.0 ± 1.3, respectively) and IFN-α (relative mRNA amount 108.3 ± 49.2 and 59.2 ± 22.0, respectively) in allograft group compared with the isograft group., Conclusion: These data suggest the presence of cell stress during chronic small bowel allograft rejection., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: In this study, we investigated the therapeutic efficacy and potential mechanisms of rhein to mitigate chronic allograft nephropathy (CAN) in rats., Materials and Methods: Fisher rat donors and Lewis rat recipients were used to establish the CAN model. Thirty rats with transplanted kidneys were randomly divided into two groups: 16 untreated and 14 rhein = treated rats. Five Lewis rat controls underwent removal of their right kidneys. The Intervention group was administered rhein oral solution (100 mg kg(-1) d(-1)) by gavage after transplantation. The untreated and control groups were given 0.5% sodium carboxymethyl cellulose. Blood and urine samples were collected at 4, 8, and 16 weeks to examine renal function and total urine protein. Half of the rats in each group were sacrifice at 8 or 16 weeks to examine renal pathology. Immunohistochemical examination and real-time polymerase chain reaction of renal tissues were performed to detect expressions of transforming growth-β1(TGF-β1), hepatic growth factor (HGF), bone morphogenetic protein 7 (BMP7), frobronectin, and collgen IV., Results: Rhein improved renal function and significantly reduced renal fibrosis and interstitial inflammation. The levels of BMP7 and HGF were significantly elevated in the renal tissues of the rhein intervention group. In the meantime, fibronectin and collagen IV were decreased in the extracellular matrix. The expression of TGF-β1 was similar between these two groups., Conclusion: Rhein improved renal function and reduced renal fibrosis and interstitial inflammation by inducing production of HGF and BMP7., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: The study was designed to evaluate the role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in ischemia-reperfusion injury (IRI) and acute rejection (AR) in rat intestinal transplantation, by administration of nitric oxide inhibitor N(G)-nitro-L-arginine methyl ester (LNAME)., Materials and Methods: Rats that underwent orthotopic intestinal transplantation were assigned to 2 sets of groups: (1) iso-geneic group (Lewis-Lewis), L-NAME 0 mg/kg/d group (1-1), 4 mg/kg/d (group 1-2), or 8 mg/kg/d (group 1-3) injected intraperitoneally or (2) allogeneic group (Dark Agouti-Lewis), L-NAME 0 mg/kg/d (group 2-1) or 8 mg/kg/d (group 2-2) injected intraperitoneally. We examined survival times, light microscopy as well as maltose absorption tests. The nNOS and iNOS activities were measured by immunohistochemical methods., Results: Histologic examination showed inhibited iNOS activity compared with group l-l, and Park scores decreased significantly in group 1-2 at 30 minutes after reperfusion (1.42 ± 0.38 vs 2.58 ± 0.49, P < .01). Both iNOS and nNOS activities were inhibited and Park scores increased significantly in group 1-3 from 30 minutes to day 3 after reperfusion (P < .0l). nNOS activity decreased and iNOS activity increased among group 2-1 during AR. Compared with group 2-1, iNOS activity was inhibited, progression of AR delayed, and survival significantly prolonged in group 2-2 (10.17 ± 0.98 vs 6.83 ± 0.75, P < .01)., Conclusion: This study suggested that decreased nNOS and increased iNOS activity both contributed to IRI and AR. More importantly, nNOS more importantly than iNOS activity was closely related to graft structure and function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: NKG2D (natural killer group 2 member D), are activating or coactivating receptor on NK cells, γδ T, and CD8(+) T cells, stimulates cytokine secretion by the former two and plays a costimulatory role for the last CD8(+) T cells., Methods: Male Lewis rat hearts were flushed and stored in cold Bretschneider preservation solution for 8 hours. Anti-NKG2D monoclonal antibody (mAb) was administered before transplantation into syngeneic recipients. Expressions of Troponin-T, myeloperoxidase (MPO), tumor necrosis factor (INF), (ICAM) and interleukin (IL)-17 were examined on days 1, 3, and 7 after reperfusion., Results: We observed that isografts from anti-NKG2D mAb-treated animals showed decreased cardiac troponin-T, low expression of MPO, TNF, and ICAM, and superior cardiac output. Furthermore, blockade of NKG2D significantly reduced the number of γδ T cells, which are the main source of IL-17 production., Conclusion: Blockade of NKG2D significantly attenuated ischemia-reperfusion injury in a cardiac transplantation model. The effect coincided with a low expression of TNFα, ICAM and a reduced number of infiltrating IL-17-producing γδ T cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Hepatic artery (HA) reconstruction is performed in the clinical liver transplantation., Methods: We assessed the importance of HA reconstruction in the success of liver transplantation. Orthotopic liver transplantation was performed without immunosspression from Lewis (RT1l) to Lewis rats (syngeneic transplantation) as well as Lewis to BN (RT1n) rats (allogeneic transplantation) with or without HA reconstruction. We examined graft function, pathology, and mRNA levels using DNA arrays in both arterialized and nonarterialized liver grafts., Results: In Lewis-to-Lewis syngeneic grafts, both the arterialized and nonarterialized grafts survived >120 days with normal graft function. lnfiltration of CD3(+) T cells and CD68(+) macrophages, marked bile duct proliferation with apoptotic epithelial cells, and expansion and increasing fibrosis of portal areas were evident in the nonarterialized grafts at day 120, although preservation of architecture was noted in the arterialized grafts. DNA array analysis of nonarterialized syngeneic grafts demonstrated the upregulation of mRNA of cell death-related proteins, cell cycle-related proteins, and inflammation-related proteins than those in arterialized grafts. Moreover, the arterialized Lewis-to-BN allogeneic grafts could survive for a long time with less severe graft dysfunction than those in non-arterialized allogeneic grafts., Conclusions: HA reconstruction in liver transplantation inhibited hypoxic injury and subsequent inflammation and bile duct proliferation, prevented the augmentation of T-cell-and antibody-mediated rejection, and mediated long-term graft acceptance. HA reconstruction is essential factor in the success of liver transplantation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: We established a procedure to engineer therapeutic neo-islets in subcutaneous spaces in mice by transplanting contiguous layers of islet cell sheets. In this study, we investigated the cellular arrangements of α and β within these engineered neo-islets in vivo as a function of time after sheet transplantation., Methods and Results: Temperature-responsive culture dishes optimized for dispersed islet cell culture were prepared by covalently immobilizing a temperature-responsive polymer poly(N-isopropylacrylamide) (PIPAAm) on plastic dishes followed by laminin-5 coating. Dispersed islet cells obtained from Lewis rats were plated onto the PIPAAm dishes. After reaching confluence at day 2, islet cells were harvested as uniformly spread islet cell sheets by lowering the culture temperature from 37°C to 20°C for 20 minutes. Islet sheet transplantation was performed to creat neo-islet tissues in the subcutaneous spaces of SCID mice with streptozotocin-induced diabetes. This neo-islet engineering approach successfully lowered mouse blood glucose levels, achieving euglycemia at day 5 and thereafter. Histologic analyses of samples obtained at day 4 revealed that neo-islet tissues in the subcutaneous spaces showed heterogeneous cellular alignment of α and β cells. In contrast, analyses of samples at days 14 and 60 revealed α and β cells predominantly located at the peripheral and central parts of the engineered tissues, respectively., Conclusions: Reassembly of α and β cells occurred in neo-islet tissues engineered by sheet transplantation. The unique cellular arrangements in neo-islet tissues, which were similar to those in naïve pancreatic islets, may contribute to their longevity and long-term function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chronic allograft vasculopathy (CAV) is a typical feature of chronic rejection of small bowel transplantations (SBTx). Our previous studies revealed that feeding fish oil, a natural source of n-3 polyunsaturated fatty acids (PUFAs), protected against CAV. The underlying mechanism remains to be clarified. The pathway mediated by the receptor for advanced glycation end products (RAGE) and its ligand, high mobility group box-1 (HMGB1), which may contribute to the pathogenesis of CAV, is potentially regulated by n-3 PUFAs. Using a chronic rejection model of rat SBTx, the present study investigated whether amelioration of CAV by fish oil feeding was associated with regulation of the RAGE signaling pathway. Moreover, our previous studies also showed that feeding low-dose fish oil for 3 months had no effect. Since an relatively short duration of treatment might fail to produce a visible response, we fed low-dose fish oil for 190 postoperative days. Male inbred Lewis rats and F344 rats were used to establish a chronic rejection model of SBTx. The recipient rats were administered phosphate-buffered saline or fish oil at a daily dose of 3 mL/kg. All rats survived over 190 postoperative days. The expression of HMGB1 and RAGE increased in CAV-bearing vessels. Feeding low-dose fish oil for 6 months attenuated CAV, and significantly reduced HMGB1 and RAGE expressions, indicating beneficial effects of low-dose fish oil on CAV may occur via down-regulation of the HMGB1-RAGE pathway., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: To establish 2 models of long-term chronic allograft rejection (CR) of orthotopic small bowel transplantation (OSBT) using cyclosporine (CsA) or tacrolimus (FK506)., Methods: F344 and Lewis rats served as donors and recipients respectively for OSBT. In the study 1, the rat was administered CsA (5 mg/kg/d) intramuscularly from postoperative day (POD) 0 to 13. In study 2, intramuscular FK 506 (0.3, 0.5, or 1.0 mg/kg/d) was delivered on POD 0-13, 20 and 27. We observed body weight gain, survival rate, and histology., Results: In study 1, all allografts demonstrated one or more histological features of CR but no serious villous blunting. In addition, there was no significant difference in the features of chronic allograft rejection between POD 60 and POD 90. In study 2, the rats that received FK506 at doses of 0.3 or 0.5 mg/kg/day survived to POD 126 but all died before OD 156. Recipients that were administered FK506 at a dose of 1.0 mg/kg/d survived beyond POD 180 showing the same body weight gain as the isogeneic group. Histopathologic analysis revealed distinctive features of CR in the rat models of OSBT induced by CsA or FK506., Conclusion: Using CsA or FK506, we developed a rat CR model of OSBT; recipients administered FK506 survived longer and showed more classic characteristics of CR compared with those treated with CsA., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Many obstacles beset islet transplantation, particularly insufficient tissue mass. Previously, we reported production of pseudo-islets. In addition, there have been reports in which coculture with pancreatic islet and bone marrow mesenchymal stem cells (BMSCs) demonstrated positive effects on pancreatic islet function. The purpose of this study was to perform morphologic and functional evaluations of pancreatic pseudo-islets cocultured with BMSCs. Pancreatic endocrine cells (PECs) were collected with a previously reported method; bone marrow was aspirated from the rat femur. Subsequently, PECs and BMSCs cocultured at high density on low-cell-binding culture dishes kept suspended by shaking. The functionality and characteristics of the mixed cell complexes were evaluated by glucose challenge, insulin enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, and immunohistochemistry. Through expansion for 2 weeks in continuous culture passages, ∼1 million PECs were recovered after aggregation. They presented spherical shapes and sizes similar to naïve islets, according to phase-contrast microscopy. The spheroid aggregates of pancreatic islet cells and BMSCs showed fortified functions and maintained viability. In conclusion, PECs served as a cell source for pseudo-islets, which were both morphologically and genetically similar to naïve islets. We also suggest a manufacturing method for mixed cellular complexes from 2 different origins that can improve secretion ability and cell differentiation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The category of chronic antibody-mediated rejection (AMR) is not included in Banff schema for liver allograft rejection. In the present study, we examined the pathology of chronic rejection using rat liver transplantation. Orthotopic liver transplantation from Lewis to BN rats was performed without immunosuppression, and with or without HA reconstruction. We studied grafts at day 120 for arterialized and day 39 for nonarterialized transplants focusing on the immunoglobulin G (IgG) deposition and the pathologic characteristics of rejection. About 20% of arterialized grafts survived more than 120 days. Between day 7 and day 120, T-cell infiltration to arterialized grafts was accompanied by IgG deposition in portal veins, hepatic arteries, and bile ducts in portal areas, sinusoids and hepatocytes. At day 120, arterialized grafts were morphologically characterized by late chronic rejection with IgG deposition, intraluminal portal veins fibrosis, intimal fibrous thickening of hepatic arteries, diffuse sinusoidal fibrosis, as well as injury and loss of bile ducts due to fibrosis. The severities of T cell-mediated rejection and AMR were higher in nonarterialized than arterialized grafts. Nonarterialized Lewis liver grafts in BN rats were rejected by day 39, as characterized by late chronic rejection with IgG deposition and cellular infiltration. In conclusion, chronic AMR may be involved in chronic rejection of liver transplantations. When chronic AMR was involved in chronic liver graft rejection, typical late morphological changes emerged within a short period., (Copyright © 2013 Elsevier Inc. All rights reserved.)