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Objective Ex vivo lung perfusion has emerged as a novel technique to safely preserve lungs before transplantation. Recent studies have demonstrated an accumulation of inflammatory molecules in the perfusate during ex vivo lung perfusion. These proinflammatory molecules, including damage-associated molecular patterns and inflammatory cytokines, may contribute to acute and chronic allograft dysfunction. At present, ex vivo lung perfusion is performed clinically at normothermic temperature (37°C). The effect of lowering temperature to the subnormothermic range during ex vivo lung perfusion has not been reported. In this study, we hypothesized that lower ex vivo lung perfusion temperature will lead to a reduction in allograft inflammation and result in improved post-transplant graft function. Methods Lewis rat heart-lung blocs underwent 4 hours of ex vivo lung perfusion in 3 temperature groups: 37°C (MP37), 30°C (MP30), and 25°C (MP25). In the control group, lung grafts were preserved by static cold storage before transplantation. After ex vivo lung perfusion or static cold storage, the left lung was transplanted for 2 hours before the animal was killed. Sera and tissue were collected and analyzed. Results There were no differences in partial pressure of arterial oxygenation to fraction of inspired oxygen ratios during 4 hours of ex vivo lung perfusion between temperature groups. Tumor necrosis factor α significantly increased in the MP37 group during ex vivo lung perfusion, whereas this was not seen at lower temperatures. Extracellular DNA and high-mobility group box 1 perfusate concentrations increased significantly during ex vivo lung perfusion in all groups, but the rate of increase was diminished at lower temperature. Two hours post-transplant, there were no significant differences in partial pressure of arterial oxygenation to fraction of inspired oxygen ratios of the lung graft or serum damage-associated molecular pattern levels among groups. On histologic grading after transplantation, greater injury was observed in the MP30 and MP37 groups, but not MP25, when compared with static cold storage. Conclusions Subnormothermic ex vivo lung perfusion at 25°C reduces the production of inflammatory mediators during ex vivo lung perfusion and is associated with reduced histologic graft injury after transplantation.

Objective Organ donation after circulatory death (DCD) is a potential solution for the shortage of suitable organs for transplant. Heart transplantation using DCD donors is not frequently performed due to the potential myocardial damage following warm ischemia. Heat shock protein (HSP) 90 has recently been investigated as a novel target to reduce ischemia/reperfusion injury. The objective of this study is to evaluate an innovative HSP90 inhibitor (HSP90i) as a cardioprotective agent in a model of DCD heart. Methods A DCD protocol was initiated in anesthetized Lewis rats by discontinuation of ventilation and confirmation of circulatory death by invasive monitoring. Following 15 minutes of warm ischemia, cardioplegia was perfused for 5 minutes at physiological pressure. DCD hearts were mounted on a Langendorff ex vivo heart perfusion system for reconditioning and functional assessment (60 minutes). HSP90i (0.01 μmol/L) or vehicle was perfused in the cardioplegia and during the first 10 minutes of ex vivo heart perfusion reperfusion. Following assessment, pro-survival pathway signaling was evaluated by western blot or polymerase chain reaction. Results Treatment with HSP90i preserved left ventricular contractility (maximum + dP/dt, 2385 ± 249 vs 1745 ± 150 mm Hg/s), relaxation (minimum –dP/dt, –1437 ± 97 vs 1125 ± 85 mm Hg/s), and developed pressure (60.7 ± 5.6 vs 43.9 ± 4.0 mm Hg), when compared with control DCD hearts (All P = .001). Treatment abrogates ischemic injury as demonstrated by a significant reduction of infarct size (2,3,5-triphenyl-tetrazolium chloride staining) of 7 ± 3% versus 19 ± 4% (P = .03), troponin T release, and mRNA expression of Bax/Bcl-2 (P Conclusions The cardioprotective effects of HSP90i when used following circulatory death might improve transplant organ availability by expanding the use of DCD hearts.

Osteoporosis is a systemic bone metabolism disorder, which increases the risk of fractures, and in severe cases it may cause disability or even death. An important factor contributing to osteoporosis is the imbalance between bone formation and resorption. Naringin was reported to promote osteoblast differentiation, thus enhancing bone formation and alleviating osteoporosis development. However, the signalling pathways related to the regulatory mechanism of naringin in osteoporosis development are not clear. Proliferation of bone mesenchymal stem cells (BMSCs) treated with naringin in vitro was detected by CCK-8. An osteogenesis differentiation medium supplemented with naringin was applied to explore the effects of naringin on BMSC osteogenic differentiation, as detected by Alizarin red staining. Ovariectomy (OVX)-induced postmenopausal osteoporosis (PMOP) rats were orally administered with naringin. Dual-energy X-ray absorptiometry (DEXA) and micro-CT were applied to measure bone mineral density (BMD), bone volume/total volume (BV/TV), trabecula thickness (Tb.Th), trabecula number (Tb.N), trabecular separation (Tb.Sp) and bone surface/bone volume (BS/BV). H&E staining was performed to show pathological changes of the femur in PMOP rats after naringin treatment. Bone metabolism indicators were assessed by ELISA. We found that naringin suppressed the activation of the JAK2/STAT3 pathway. Naringin promoted BMSC proliferation and osteogenic differentiation. Furthermore, naringin alleviates bone loss and improves abnormal bone metabolism of PMOP rats. Collectively, naringin promotes BMSC osteogenic differentiation to ameliorate osteoporosis development by targeting JAK2/STAT3 signalling.

Ventral hernia repair (VHR) with acellular dermal matrix (ADM) has high rates of recurrence that may be improved with allogeneic growth factor augmentation such as amniotic fluid allograft (AFA). We hypothesized that AFA would modulate the host response to improve ADM incorporation in VHR. Lewis rats underwent chronic VHR with porcine ADM alone or with AFA augmentation. Tissue harvested at 3, 14, or 28 days was assessed for region-specific cellularity, and a validated histomorphometric score was generated for tissue incorporation. Expression of pro-inflammatory (Nos1, Tnfα), anti-inflammatory (Arg1, Il-10, Mrc1) and tissue regeneration (Col1a1, Col3a1, Vegf, and alpha actinin-2) genes were quantified using quantitative reverse-transcription polymerase chain reaction. Amniotic fluid allograft treatment caused enhanced vascularization and cellularization translating to increased histomorphometric scores at 14 days, likely mediated by upregulation of pro-regeneration genes throughout the study period and molecular evidence of anti-inflammatory, M2-polarized macrophage phenotype. Collectively, this suggests AFA may have a therapeutic role as a VHR adjunct.

INTRODUCTION The medical demand for lymphedema treatment is huge since the disease mechanism remains unclear, and management are difficult. Our purpose was to develop a reliable lymphedema model mimicking the clinical scenario and allows a microsurgical approach. MATERIALS AND METHODS Male Lewis rats weighing 400 to 450 g were used to create lymphedema with groin and popliteal lymph node dissection and creation of 5 mm circumferential skin defect (n = 6). A skin incision was made and closed primarily for control group (n = 5). Evaluation included indocyanine green (ICG) lymphangiography 1 and 2 months postoperatively, volume difference between bilateral hindlimbs measured using micro-CT, and the skin was harvested for histological evaluation 2 months postoperatively. RESULTS Larger volume differences present in the lymphedema group (17.50 ± 7.76 vs. 3.73 ± 2.66%, p

BACKGROUND Exosomes participate in intercellular communication and act as important molecular vehicles in the regulation of numerous physiological and pathological processes, including autoimmune development. The role of circulating exosomes in the development of autoimmune uveitis is unknown. In this study, using the rat model of experimental autoimmune uveitis, which has clinical and histological features of pan uveitis in man, we evaluated the immunoregulatory function of circulating exosomes. METHODS Experimental autoimmune uveitis was induced in Lewis rats either immunised with interphotoreceptor retinoid-binding protein R16 peptides or injected with activated R16-specific T cells. The disease incidence and severity were examined by indirect fundoscopy and flow cytometry. Circulating exosomes were isolated from peripheral blood of naive and Day 14 R16 immunised Lewis rats. The effect of exosomes on specific T cells was evaluated by R16-specific T cell proliferation, cytokine production and recurrent uveitis induction. RESULTS Circulating exosomes derived from active immunised uveitis rats selectively inhibited immune responses of R16-specific T cells in vitro. Vaccination of naive rats with these exosomes reduced the incidence of recurrent uveitis in an antigen-specific manner. Antigen-specific uveitogenic T cells reduced IFN-γ production and increased IL-10 after vaccination. CONCLUSIONS Circulating exosomes in autoimmune uveitis have the potential to be a novel treatment for recurrent autoimmune uveitis.

OBJECTIVES Orthotopic liver transplant remains technically challenging. MATERIALS AND METHODS We performed whole graft orthotopic liver transplants with different anhepatic times (≤20 min, n = 19; vs 30 min, n = 9) and partial orthotopic liver transplants in rats including a male-to-male Sprague-Dawley group (n = 15), a male-to-male Lewis-to-Brown Norway group (n = 20), and a male-to-male Sprague-Dawley-to-Lewis group (n = 20); there was also a female-to-male SpragueDawley group (n = 19). RESULTS For the groups with ≤20-minute or 30-minute anhepatic time, 14-day and 30-day survival rates were 94.7%, 89.5%, 88.9%, and 88.9%, respectively, and there was no difference in survival (P = .716). For 50% orthotopic liver transplants from the male-tomale Sprague-Dawley group, 14-day and 30-day survival rates were 93.3% and 86.7%, respectively, with no difference between whole and 50% graft orthotopic liver transplant. The 14-day and 30-day survival rates were, respectively, 30% and 10% for the Lewis-to-Brown Norway group and 30% and 6.6% for the Sprague-Dawley-to-Lewis group, with no differences between the 2 groups (P = .564). Most of the recipient rats died within 72 hours. Acute rejections and wound dehiscence were the causes of death. Recipients from the female-to-male SpragueDawley orthotopic liver transplant group died shortly after surgery. CONCLUSIONS Orthotopic liver transplants can be performed to achieve high success rates in the extended anhepatic time; however, orthotopic liver transplants from female Sprague-Dawley donor rats have a high risk of failure.

BACKGROUND The aim of this study was to investigate the combined effect of mesenchymal stem cells (MSC) and local delivery of tacrolimus (FK506) on nerve regeneration when applied to nerve autografts and decellularized allografts. METHODS A three-dimensional in vitro compartmented cell culture system consisting of a neonatal dorsal root ganglion adjacent to a nerve graft was used to evaluate the regenerating neurites into the peripheral nerve scaffold. Nerve autografts and allografts were treated with (i) undifferentiated MSCs, (ii) FK506 (100 ng/mL) or (iii) both (N = 9/group). After 48 hours, neurite extension was measured to quantify nerve regeneration and stem cell viability was evaluated. RESULTS Stem cell viability was confirmed in all MSC-treated grafts. Neurite extension was superior in autografts treated with FK506, and MSCs and FK506 combined (p

Background and Objectives: Transfusion-associated circulatory overload (TACO) is the primary cause of transfusion-related mortality. Speed and volume of transfusion are major risk factors. The aim of this study was to investigate the interaction of red blood cell (RBC) transfusion speed and volume on the development of TACO. Materials and Methods: A validated model for TACO in anaemic Lewis rats with an acute myocardial infarction was used. The effect on pulmonary hydrostatic pressure of one, two or four units of packed RBCs transfused in either 30 or 60 min was evaluated (3.3–26.6 ml·kg −1·hr −1). Pulmonary capillary pressure was measured as left ventricular end-diastolic pressure (LVEDP). Cardiac stress biomarkers atrial natriuretic-peptide (ANP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured 1-h post-transfusion. Results: Thirty animals were included (n = 5 per group). Transfusion of RBCs increased LVEDP in a volume-dependent manner (ΔLVEDP [mmHg]: −0.95, +0.50, +6.26, p < 0.001). Fast transfusion increased overall ΔLVEDP by +3.5 mmHg and up to +11.8 mmHg in the four units' group (p = 0.016). Doubling transfusion speed increased ΔLVEDP more than doubling volume in the larger volume groups. No difference in ANP or NT-proBNP were seen in high transfusion volume or groups. Conclusion: Transfusion volume dose-dependently increased LVEDP, with speed of transfusion rapidly elevating LVEDP at higher transfusion volumes. ANP and NT-proBNP were not impacted by transfusion volume or speed in this model. TACO is seen as purely volume overload, however, this study emphasizes that limiting transfusion speed, as a modifiable risk factor, might aid in preventing TACO.

BACKGROUND: Decellularized nerve allografting is one of promising treatment options for nerve defect. As an effort to develop more efficient nerve graft, recently we have developed a new decellularization method for nerve allograft. The aim of this study was to evaluate the effectiveness and biocompatibility of nerve graft decellularized by our newly developed method. METHODS: Forty-eight inbred male Lewis rats were divided into two groups, Group I (autograft group, n = 25), Group II (decellularized isograft group, n = 23). Decellularized nerve grafts were prepared with our newly developed methods using amphoteric detergent and nuclease treatment. Serum cytokine level measurements at 0, 2, and 4 weeks and histologic evaluation for inflammatory cell infiltration at 6 and 16 weeks after nerve graft. RESULTS: There was no significant difference in mean maximum isometric tetanic force and weight of tibialis anterior muscle or ankle angle at toe-off phase between two groups at 6 and 16 weeks survival time points (p > 0.05). There was no inflammatory cell infiltration in either group and histomorphometric assessments of 6- and 16-week specimens of the isograft group did not differ from those in the autograft group with regard to number of fascicle, cross sectional area, fascicle area ratio, and number of regenerated nerve cells. CONCLUSION: Based on inflammatory reaction, axonal regeneration, and functional outcomes, our newly developed decellularized nerve grafts were fairly biocompatible and had comparable effectiveness to autografts for nerve regeneration, which suggested it would be suitable for nerve reconstruction as an alternative to autograft.

Kidney resident macrophages (KRMs) are involved in maintaining renal homeostasis and in controlling the pathological outcome of acute kidney injury and cystic kidney disease in mice. In adult mice, KRMs maintain their population through self-renewal with little or no input from the peripheral blood. Despite recent data suggesting that a transcriptionally similar population of KRM-like cells is present across species, the idea that they are self-renewing and minimally dependent on peripheral blood input in other species has yet to be proven due to the lack of an appropriate model and cross-species expression markers. In this study, we used our recently identified cross-species KRM cell surface markers and parabiosis surgery in inbred Lewis rats to determine if rat KRMs are maintained independent of peripheral blood input, similar to their mouse counterparts. Flow cytometry analysis indicated that parabiosis surgery in the rat results in the establishment of chimerism of T/B cells, neutrophils, and monocyte-derived infiltrating macrophages in the blood, spleen, and kidney 3 wk after parabiosis surgery. Analysis of KRMs using the cell surface markers CD81 and C1q indicated that these cells have minimal chimerism and, therefore, receive little input from the peripheral blood. These data indicate that KRM properties are conserved in at least two different species. NEW & NOTEWORTHY In this report, we performed parabiosis surgery on inbred Lewis rats and showed that rat kidney resident macrophages (KRMs), identified using our novel cross-species markers, are minimally dependent on peripheral blood input. Thus, for the first time, to our knowledge, we confirm that a hallmark of mouse KRMs is also present in KRMs isolated from another species.

BACKGROUND Transplant glomerulopathy (TG) is a pathological feature of chronic active antibody-mediated rejection (cAMR) and is associated with renal allograft failure. The specific role of B cells in the pathogenesis of TG is unclear. METHODS We used a minor mismatched rat kidney transplant model with B cell-deficient recipients, generated by clustered regularly interspaced short palindromic repeats/Cas9 technology, to investigate the impact of B-cell depletion on the pathogenesis of TG. We hypothesized that B-cell deficiency would prevent TG in the rat kidney transplant model of cAMR. Treatment groups included syngeneic, allogeneic, sensitized allogeneic, and B cell-deficient allogeneic transplant recipients. RESULTS B cell-deficient recipients demonstrated reduced TG lesions, decreased microvascular inflammation, reduced allograft infiltrating macrophages, and reduced interferon gamma transcripts within the allograft. Allograft transcript levels of interferon gamma, monocyte chemoattractant protein-1, and interleukin-1β correlated with numbers of intragraft macrophages. B cell-deficient recipients lacked circulating donor-specific antibodies and had an increased splenic regulatory T-cell population. CONCLUSIONS In this model of cAMR, B-cell depletion attenuated the development of TG with effects on T cell and innate immunity.

While bone marrow-derived mesenchymal stem cells (BMCs) have been widely used in spinal fusion procedures, adipose-derived stem cells (ASCs) offer a number of advantages as an alternative clinical cell source. This study directly compares the efficacy of ASCs and BMCs from the same donor animals to achieve successful fusion when combined with a clinical-grade bone graft substitute in a rat lumbar fusion model. ASCs and BMCs were isolated from the same Lewis donor rats and grown to passage 2 (P2). Single-level bilateral posterolateral intertransverse process lumbar fusion surgery was performed on syngeneic rats divided into three experimental groups: clinical-grade bone graft substitute alone (CBGS); CBGS + rat ASCs (rASC); and, CBGS + rat BMCs (rBMC). Eight weeks postoperatively, fusion was evaluated via microCT, manual palpation and histology. In vitro analysis of the osteogenic capacity of rBMCs and rASCs was also performed. Results indicated that the average fusion volume in the rASC group was the largest and was significantly larger than the CBGS group. Although the rASC group displayed the highest fusion rates via microCT and manual palpation, this difference was not statistically significant. Cell-seeded grafts showed more histological bone formation than cell-free grafts. P2 rASCs and rBMCs displayed similar in vitro osteogenic differentiation capacities. Overall, this study showed that, when combined with a clinical-grade bone graft substitute in a rat model, rASCs cells yielded the largest fusion masses and comparable fusion results to rBMCs. These results add to growing evidence that ASCs provide an attractive alternative to BMCs for spinal fusion procedures. This article is protected by copyright. All rights reserved.

Aberrant complement activation leads to tissue damage during kidney transplantation, and it is recognized as an important target for therapeutic intervention. However, it is not clear whether cold storage (CS) triggers the complement pathway in transplanted kidneys. The goal of the present study was to determine the impact of CS on complement activation in renal transplants. Male Lewis and Fischer rats were used, and donor rat kidneys were exposed to 4 h or 18 h of CS followed by transplantation (CS + transplant). To study CS-induced effects, a group with no CS was included in which the kidney was removed and transplanted back to the same rat [autotransplantation (ATx)]. Complement proteins (C3 and C5b-9) were evaluated with Western blot analysis (reducing and nonreducing conditions) and immunostaining. Western blot analysis of renal extracts or serum indicated that the levels of C3 and C5b-9 increased after CS + transplant compared with ATx. Quite strikingly, intracellular C3 was profoundly elevated within renal tubules after CS + transplant but was absent in sham or ATx groups, which showed only extratubular C3. Similarly, C5b-9 immunofluorescence staining of renal sections showed an increase in C5b-9 deposits in kidneys after CS + transplant. Real-time PCR (SYBR green) showed increased expression of CD11b and CD11c, components of complement receptors 3 and 4, respectively, as well as inflammatory markers such as TNF-α. In addition, recombinant TNF-α significantly increased C3 levels in renal cells. Collectively, these results demonstrate that CS mediates aberrant activation of the complement system in renal grafts following transplantation. NEW & NOTEWORTHY This study highlights cold storage-mediated aberrant activation of complement components in renal allografts following transplantation. Specifically, the results demonstrate, for the first time, that cold storage functions in exacerbation of C5b-9, a terminal cytolytic membrane attack complex, in renal grafts following transplantation. In addition, the results indicated that cold storage induces local C3 biogenesis in renal proximal cells/tubules and that TNF-α promotes C3 biogenesis and activation in renal proximal tubular cells.

The aim of this study was to determine that low-intensity pulsed ultrasound (LIPUS) at an intensity of 140 mW/cm² promotes functional and histologic improvements in sciatic nerve crush injury in a rat model and to investigate changes over time in relevant growth factors and receptors, exploring the mechanism of LIPUS in the recovery process after injury. Toe angle in the toe-off phase, regenerative axonal length, myelinated nerve fiber density, diameter of myelinated nerve fiber, axon diameter and myelin sheath thickness were significantly higher in the LIPUS group than in the sham group. Gene and protein expression of brain-derived neurotrophic factor (BDNF) was upregulated in the LIPUS group. In conclusion, LIPUS contributed to rapid functional and histologic improvement and upregulated BDNF expression after sciatic nerve crush injury in rats.

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.

BACKGROUND Recent studies have demonstrated that inhibition of CD26 potentiates stromal cell-derived factor-1α (SDF-1α), promotes tissue regeneration, and suppresses the rejection of organ transplants. This study investigated whether the combination of a CD26 inhibitor (CD26i) with granulocyte colony-stimulating factor (G-CSF) and short-term immunosuppressants modulates vascularized composite tissue allotransplant survival in a rodent orthotopic hindlimb allotransplant model. METHODS The hindlimb allotransplantation from Brown-Norway to Lewis rats was divided into 4 groups. Group 1 (controls) did not receive any treatment. Group 2 was treated with short-term antilymphocyte serum (ALS) and cyclosporine-A (CsA). Group 3 was administrated CD26i and G-CSF. Group 4 received a combination of CD26i/G-CSF/ALS/CsA. Each subgroup comprised 10 rats. Peripheral blood and sampling of transplanted tissues were collected for immunological and histological analysis. RESULTS The results revealed that allotransplant survival was found to be significantly prolonged in group 4 with CD26i/G-CSF/ALS/CsA treatment compared with those in the other groups. The interleukin-10 and transforming growth factor-βl levels, the percentage of CD4+/CD25+/FoxP3+ T cells, as well as the levels of SDF-1α expressions were significantly increased in group 4 compared with those in the other groups. Group 4 revealed a statistical increase in the percentage of donor cells (RT1n) expression in the recipient peripheral blood, and the mixed lymphocyte reaction showed hyporesponsiveness of the T cells to donor alloantigens. CONCLUSION The combination of CD26i/G-CSF and short-term immunosuppressants prolongs allotransplant survival by inducing immunoregulatory effects and enhancing the percentage of SDF-1α expression. This immunomodulatory approach has great potential as a strategy to increase vascularized composite allotransplantation survival.

Background The neuropsychiatric disorders due to post‐streptococcal autoimmune complications such as Sydenham's chorea (SC) are associated with acute rheumatic fever and rheumatic heart disease (ARF/RHD). An animal model that exhibits characteristics of both cardiac and neurobehavioral defects in ARF/RHD would be an important adjunct for future studies. Since age, gender, strain differences, and genotypes impact on the development of autoimmunity, we investigated the behavior of male and female Wistar and Lewis rat strains in two age cohorts (12 weeks) under normal husbandry conditions and following exposure to group A streptococcus (GAS). Methods Standard behavioral assessments were performed to determine the impairments in fine motor control (food manipulation test), gait and balance (beam walking test), and obsessive‐compulsive behavior (grooming and marble burying tests). Furthermore, electrocardiography, histology, and behavioral assessments were performed on male and female Lewis rats injected with GAS antigens. Results For control Lewis rats there were no significant age and gender dependent differences in marble burying, food manipulation, beam walking and grooming behaviors. In contrast significant age‐dependent differences were observed in Wistar rats in all the behavioral tests except for food manipulation. Therefore, Lewis rats were selected for further experiments to determine the effect of GAS. After exposure to GAS, Lewis rats demonstrated neurobehavioral abnormalities and cardiac pathology akin to SC and ARF/RHD, respectively. Conclusion We have characterised a new model that provides longitudinal stability of age‐dependent behavior, to simultaneously investigate both neurobehavioral and cardiac abnormalities associated with post‐streptococcal complications.

Objective There is a critical need for development of biomarkers to noninvasively monitor for lung transplant rejection. We investigated the potential of circulating donor lung-specific exosome profiles for time sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. Methods Left lungs from Wistar transgenic rats expressing human CD63-GFP, an exosome marker, were transplanted into fully MHC-mismatched Lewis recipients or syngeneic controls. Recipient blood was collected between 4 hours and 10 days after transplantation and plasma was processed for exosome isolation by size exclusion column chromatography and ultracentrifugation. Circulating donor exosomes were profiled using anti-human CD63 antibody quantum dot on the nanoparticle detector, and via GFP trigger on the nanoparticle flow cytometer (FACS). Results In syngeneic controls, steady state levels of circulating donor exosomes were detected at all post-transplant time points. Allogeneic grafts lost perfusion by day 8, consistent with acute rejection. Levels of circulating donor exosomes peaked on day 1, decreased significantly by day 2, and then reached baseline levels by day 3. Notably, decrease in peripheral donor exosome levels occurred before grafts had histological evidence of acute rejection. Conclusions Circulating donor lung-specific exosome profiles enable an early detection of acute rejection before histologic manifestation of injury to the pulmonary allograft. As acute rejection episodes are a major risk factor for the development of chronic lung allograft dysfunction, this biomarker may provide a novel noninvasive diagnostic platform that can translate into earlier therapeutic intervention for lung transplant patients.

After spinal cord injury (SCI) in mammals, there is only limited repair and, consequently, only moderate recovery. One mechanism frequently discussed to be involved in this recovery is plasticity (i.e., adaptations in spared neuronal circuitries). In the current study, we tested the effect of an intrathecal application of the TrkB agonist antibody, 29D7, on plasticity after cervical SCI in adult rats. Treatment with 29D7 for 4 weeks resulted in an ∼50% increase in collateral sprouting of severed corticospinal tract fibers above the lesion compared to the control group and enhanced branching in the gray matter rostral to the injury. Growth-associated protein 43 immunoreactivity in the spinal cord rostral to the level of the injury as well as contralateral to the lesion was also increased. These indications of enhanced plasticity by 29D7 were paralleled by improved performances of the mildly affected paw, as assessed by Montoya and tray reaching tasks. The reaching behaviors of the paw ipsilateral to the side of severe injury to the cortico- and rubrospinal tract were not altered by the treatment. The present study suggests that 29D7 may be a potential candidate to promote plasticity and functional recovery, especially after moderate SCI. Future studies confirming these results, along with a potential combinatory therapy including rehabilitative training, will be needed to evaluate the clinical value of such a treatment.

The present study was conducted to clarify the therapeutic effect of cornuside on experimental autoimmune encephalomyelitis (EAE) and its influence on T helper 17 (Th17) cell and regulatory T (Treg) cell infiltration into the central nervous system. Rats were randomly placed into four treatment groups: control, EAE, EAE+cornuside, and EAE+prednisolone. The neurological function scores of rats were assessed daily. On the second day after EAE rats began to show neurological deficit symptoms, the four groups were treated with normal saline, normal saline, cornuside (150 mg/kg), and prednisolone (5 mg/kg), respectively. The treatment was discontinued after two weeks, and the spinal cord was obtained for hematoxylin and eosin (H&E) and luxol fast blue staining, as well as retinoic acid receptor-related orphan receptor γ (RORγ) and forkhead box protein P3 (Foxp3) immunohistochemical staining. Blood was collected for Th17 and Treg cell flow cytometry testing, and the serum levels of interleukin (IL)-17A, IL-10, transforming growth factor-β (TGF-β), IL-6, IL-23, and IL-2 were measured via enzyme-linked immunosorbent assay (ELISA). Compared with rats in the EAE group, rats in the EAE+cornuside and EAE+prednisolone groups began to recover from neurological deficits earlier, and had a greater degree of improvement of symptoms. Focal inflammation, demyelination, and RORγ-positive cell infiltration were reduced by cornuside or prednisolone treatment, whereas the Foxp3-positive cell numbers were not significantly different. Meanwhile, the number of Th17 cells and the IL-17A, IL-6, and IL-23 levels were lower in the blood after cornuside or prednisolone treatment, whereas the number of Treg cells or the levels of IL-10, TGF-β, and IL-2 were not markedly different. Cornuside can alleviate symptoms of EAE neurological deficits through its anti-inflammatory and immunosuppressive effects, and Th17 cells may be one of its therapeutic targets.

BACKGROUND AND OBJECTIVES Chronic cocaine exposure has differential neural effects in Fischer 344 (F344) vs Lewis inbred rats that may explain strain-dependent differences during acquisition vs maintenance of cocaine self-administration. We assessed whether prior cocaine exposure alters operant responding for food across various phases (acquisition, maintenance, extinction, spontaneous recovery, reinitiation) in these strains. METHODS Lewis and F344 rats (N = 12) were administered three cocaine (15 mg/kg) or saline injections at hourly intervals for 3 consecutive days. Beginning the next day for 24 days, rats had access to operant chambers in which one lever depression resulted in the delivery of a food pellet. Then, four extinction sessions were conducted in which food was no longer available, but other stimulus conditions remained the same. After a 2-day break, spontaneous recovery was assessed over four sessions. Food delivery was then restored for 3 days to test reinitiation followed by a progressive ratio session. RESULTS Lewis rats acquired the operant faster than F344 rats. F344 rats showed lower maintenance rates than Lewis rats but higher spontaneous recovery responding. Cocaine exposure caused persistence of responding during extinction in F344 but not Lewis rats. All groups reinitiated responding when food was available again and did not differ in final ratios completed under the progressive ratio schedule. DISCUSSION AND CONCLUSIONS That prior cocaine exposure led to persistence of responding in F344 rats during extinction may reflect heightened contextual conditioning that interferes with the ability to extinguish responding. SCIENTIFIC SIGNIFICANCE Results have implications for the genetic contribution to relapse-like behaviors. (Am J Addict 2021;00:00-00).

BACKGROUND Cell-based treatments have demonstrated the capacity to enhance reconstructive outcomes in recent decades but are hindered in clinical utility by regulatory hurdles surrounding cell culture. This investigation examines the ability of a noncultured stromal vascular fraction derived from lipoaspirate to enhance bone healing during fracture repair to further the development of translatable cell therapies that may improve outcomes in irradiated reconstruction. METHODS Isogenic male Lewis rats were divided into three groups: fracture, irradiated fracture, and irradiated fracture with stromal vascular fraction treatment. Irradiated groups received a fractioned dose of 35 Gy before mandibular osteotomy. Stromal vascular fraction was harvested from the inguinal fat of isogenic donors, centrifuged, and placed intraoperatively into the osteotomy site. All mandibles were evaluated for bony union and vascularity using micro-computed tomography before histologic analysis. RESULTS Union rates were significantly improved in the irradiated fracture with stromal vascular fraction treatment group (82 percent) compared to the irradiated fracture group (25 percent) and were not statistically different from the fracture group (100 percent). Stromal vascular fraction therapy significantly improved all metrics of bone vascularization compared to the irradiated fracture group and was not statistically different from fracture. Osteocyte proliferation and mature bone formation were significantly reduced in the irradiated fracture group. Bone cellularity and maturity were restored to nonirradiated levels in the irradiated fracture with stromal vascular fraction treatment group despite preoperative irradiation. CONCLUSIONS Vascular and cellular depletion represent principal obstacles in the reconstruction of irradiated bone. This study demonstrates the efficacy of stromal vascular fraction therapy in remediating these damaging effects and provides a promising foundation for future studies aimed at developing noncultured, cell-based therapies for clinical implementation.

Sulfonylureas, widely used as hypoglycemic agents in adults with type 2 diabetes, have neuroprotective effects in preclinical models of central nervous system injury, and in children with neuropsychomotor impairments linked to neonatal diabetes secondary to ATP-sensitive potassium channel mutations. In the human and rodent retina, we show that the glibenclamide-activated channel sulfonylurea receptor 1 (SUR1) is expressed in the retina and enriched in the macula; we also show that it colocalizes with the potassium channel Kir6.2, and with the cation channel transporter TRPM4. Glibenclamide (glyburide), administered at doses that did not decrease the glycemia, or injected directly into the eye, protected the structure and the function of the retina in various models of retinal injury that recapitulate the pathogenic neurodegenerative events in the diabetic retina. The downregulation of SUR1 using a siRNA suppressed the neuroprotective effects of glibenclamide on excitotoxic stress-induced cell death. The glibenclamide effects include the transcriptional regulation of antioxidant and neuroprotective genes. Ocular glibenclamide could be repurposed for diabetic retinopathy.

One of the main challenges to understand drug addiction is defining the biological mechanisms that underlie individual differences in recidivism. Studies of these mechanisms have mainly focused on the brain, yet we demonstrate here a significant influence of the peripheral immune system on this phenomenon. Lewis (LEW) and Fischer 344 (F344) rats have different immunological profiles and they display a distinct vulnerability to the reinforcing effects of cocaine, with F344 more resistant to reinstate cocaine-seeking behavior. Bone marrow from male LEW and F344 rats was transferred to male F344 rats (F344/LEW-BM and F344/F344-BM, respectively), and these rats were trained to self-administer cocaine over 21 days. Following extinction, these animals received a sub-threshold primer dose of cocaine to evaluate reinstatement. F344/LEW-BM but not F344/F344-BM rats reinstated cocaine-seeking behavior, in conjunction with changes in their peripheral immune cell populations to a profile that corresponded to that of the LEW donors. After cocaine exposure, higher CD4+ T-cells and lower CD4+CD25+ T-cells levels were observed in F344/LEW-BM rats referred to control, and the splenic expression of Il-17a, Tgf-β, Tlr-2, Tlr-4 and Il-1β was altered in both groups. We propose that peripheral T-cells respond to cocaine, with CD4+ T-cells in particular undergoing Th17 polarization and generating long-term memory, these cells releasing mediators that trigger central mechanisms to induce reinstatement after a second encounter. This immune response may explain the high rates of recidivism observed despite long periods of detoxification, shedding light on the mechanisms underlying the vulnerability and resilience of specific individuals, and opening new perspectives for personalized medicine in the treatment of relapse.

PURPOSE/AIM: Volumetric muscle loss (VML) is a devastating orthopaedic injury resulting in chronic persistent functional deficits, loss of joint range of motion, pathologic fibrotic deposition and lifelong disability. However, there is only limited mechanistic understanding of VML-induced fibrosis. Herein we examined the temporal changes in the fibrotic deposition at 3, 7, 14, 28, and 48 days post-VML injury. MATERIALS AND METHODS: Adult male Lewis rats (n=39) underwent a full thickness ~20% (~85mg) VML injury to the tibialis anterior (TA) muscle unilaterally, the contralateral TA muscle served as the control group. All TA muscles were harvested for biochemical and histologic evaluation. RESULTS: The ratio of collagen I/III was decreased at 3, 7, and 14 days post-VML, but significantly increased at 48 days. Decorin content followed an opposite trend, significantly increasing by day 3 before dropping to below control levels by 48 days. Histological evaluation of the defect area indicates a shift from loosely packed collagen at early time points post-VML, to a densely packed fibrotic scar by 48 days. CONCLUSIONS: The shift from early wound healing efforts to a fibrotic scar with densely packed collagen within the skeletal muscle occurs around 21 days after VML injury through dogmatic synchronous reduction of collagen III and increase in collagen I. Thus, there appears to be an early window for therapeutic intervention to prevent pathologic fibrous tissue formation, potentially by targeting CCN2/CTGF or using decorin as a therapeutic.

Spinal cord injury (SCI) induces haemodynamic instability that threatens survival1–3, impairs neurological recovery4,5, increases the risk of cardiovascular disease6,7, and reduces quality of life8,9. Haemodynamic instability in this context is due to the interruption of supraspinal efferent commands to sympathetic circuits located in the spinal cord10, which prevents the natural baroreflex from controlling these circuits to adjust peripheral vascular resistance. Epidural electrical stimulation (EES) of the spinal cord has been shown to compensate for interrupted supraspinal commands to motor circuits below the injury11, and restored walking after paralysis12. Here, we leveraged these concepts to develop EES protocols that restored haemodynamic stability after SCI. We established a preclinical model that enabled us to dissect the topology and dynamics of the sympathetic circuits, and to understand how EES can engage these circuits. We incorporated these spatial and temporal features into stimulation protocols to conceive a clinical-grade biomimetic haemodynamic regulator that operates in a closed loop. This ‘neuroprosthetic baroreflex’ controlled haemodynamics for extended periods of time in rodents, non-human primates and humans, after both acute and chronic SCI. We will now conduct clinical trials to turn the neuroprosthetic baroreflex into a commonly available therapy for people with SCI. An epidural spinal cord stimulation system regulates blood pressure in the acute and chronic phases of spinal cord injury.

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.

Chronic allograft dysfunction is a major cause of late graft failure after kidney transplantation. One of the histological changes is interstitial fibrosis, which is associated with epithelial–mesenchymal transition. Bortezomib has been reported to prevent the progression of fibrosis in organs. We used rat renal transplantation model and human kidney 2 cell line treated with tumor necrosis factor-α (TNF-α) to examine their response to bortezomib. To explore the mechanism behind it, we assessed the previously studied TNF-α/protein kinase B (Akt)/Smad ubiquitin regulatory factor 2 (Smurf2) signaling and performed RNA sequencing. Our results suggested that bortezomib could attenuate the TNF-α-induced epithelial–mesenchymal transition and renal allograft interstitial fibrosis in vitro and in vivo. In addition to blocking Akt/mammalian target of rapamycin (mTOR)/p70S6 kinase/Smurf2 signaling, bortezomib’s effect on the epithelial–mesenchymal transition was associated with inhibition of nuclear factor kappa B (NF-κB) pathway by stabilizing inhibitor of NF-κB. The study highlighted the therapeutic potential of bortezomib on renal allograft interstitial fibrosis. Such an effect may result from inhibition of NF-κB/TNF-α/Akt/mTOR/p70S6 kinase/Smurf2 signaling via stabilizing protein of inhibitor of NF-κB.

Purpose of review Experimental intestinal transplantation (ITx ) has generated invaluable knowledge that has ultimately benefited the clinical activity. Herein, we appraise the recent publications pertaining to experimental ITx and highlight the main current research topics. Recent findings During the recent years, ischemia-reperfusion injury (IRI) and Graft-versus-host disease (GVHD) have gradually replaced acute rejection as the main research topic. New additives to established preservation solutions and relatively novel approaches such as luminal interventions during cold storage may prolong the storage time and alleviate IRI. High donor age does not seem to worsen preservation injury. The ischemic susceptibility seems to differ between species, which may impact the translatability of the experimental findings. A new experimental model of modified multivisceral transplantation including the donor spleen may offer a new tool with which to study GVHD, besides the classical Lewis-Brown Norway rat combination. Flushing the graft with fludarabine may mitigate GVHD in rats. T-cell activation inhibitor-mitochondrial was downregulated in the peripheral blood leukocytes before other signs of acute and severe chronic rejection could be observed. Summary Experimental research in ITx has largely shifted focus from acute rejection to IRI and GVHD. Several lines of research have matured toward clinical translation, yet no breakthrough is imminent.

Summary Despite undeniable improvement in the management of rheumatoid arthritis (RA), the discovery of more effective, less toxic and, ideally, less immune suppressive drugs are much needed. In the current study, we set to explore the potential anti-rheumatic activity of the non-toxic, tellurium-based immunomodulator, AS101 in an experimental animal model of RA. The effect of AS101 was assessed on adjuvant-induced arthritis (AIA) rats. Clinical signs of arthritis were assessed. Histopathological examination was used to assess inflammation, synovial changes and tissue lesions. Very late antigen-4 (VLA-4)+ cellular infiltration was detected using immunohistochemical staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure circulating anti-cyclic citrullinated-peptide autoantibody (ACPA) and real-time polymerase chain reaction (PCR) was used to measure the in-vitro effect of AS101 on interleukin (IL)-6 and IL-1β expression in activated primary human fibroblasts. Prophylactic treatment with intraperitoneal AS101 reduced clinical arthritis scores in AIA rats (P

The recurrence of ventral hernias continues to be a problem faced by surgeons, in spite of efforts toward implementing novel repair techniques and utilizing different materials to promote healing. Cadaveric acellular dermal matrices (Alloderm) have shown some promise in numerous surgical subspecialties, but these meshes still suffer from subsequent failure and necessitation of re-intervention. Here, it is demonstrated that the addition of platelet rich plasma to Alloderm meshes temporally modulates both the innate and cytotoxic inflammatory responses to the implanted material. This results in decreased inflammatory cytokine production at early time points, decreased matrix metalloproteinase expression, and decreased CD8+ T cell infiltration. Collectively, these immune effects result in a healing phenotype that is free from mesh thinning and characterized by increased material stiffness.

Background Vein graft stenosis is a major complication of coronary artery bypass surgery and peripheral arterial bypass procedures. Experimental models of this clinical complication have used in vivo grafting procedures, relying on the relatively modest neointimal thickening in these models as a surrogate for clinical graft stenosis without regard to the donor site origin of the vein graft. Materials and methods In a standard rat model of vein grafting, three different donor sites were used to supply veins used as interpositional grafts to the femoral artery: the superficial inferior epigastric vein, the common femoral vein, and the posterior facial vein (distal branch of the jugular vein). Grafts were harvested as 4 wk and histomorphometrically evaluated for the extent of neointimal formation and lumen narrowing. Results The posterior facial vein showed significantly thicker neointima and a greater extent of lumen narrowing than the other two graft sources, despite having a similar diameter to the femoral vein and nearly twice the initial diameter of the epigastric vein. Conclusions The source of donor graft material can greatly influence the extent of neointimal response after interpositional vein grafting to arterial flow. These findings support use of the posterior facial vein graft over other more standard donor vein grafts in research directed at understanding the causes and prevention of vein graft stenosis.

Nerve demyelination or axonal lesions are characteristic of experimental autoimmune neuritis (EAN). Previous studies have demonstrated that microRNA-338 can regulate the differentiation and maturation of oligodendrocytes and Schwann cells and promote injured peripheral nerves in rats. In this study, we used microRNA-338 coded lentivirus vector (miR-338-LV) in a Lewis rat EAN model, in with the conjunction P0 peptide 180–199 which was injected into the footpads of animals to induce immunization. The clinical scores of miR-338-LV and intravenous immunoglobulin (IVIg) (positive drug) groups were significantly superior to those of untreated group at disease peak and disease plateau (p

BACKGROUND Chronic renal allograft dysfunction (CRAD) is a major condition that impedes the long-term survival of renal allografts. However, the mechanism of CRAD is obscure, and the effective strategies for controlling the progression of CRAD are lacking. The present study used a CRAD rat model to assess the effect of glycogen synthase kinase 3β (GSK-3β) inhibition on the development of CRAD. METHODS A classical F334-to-LEW orthotopic renal transplantation was performed on the CRAD group. The treatment group was treated with the GSK-3β inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione for 12 consecutive weeks following renal transplantation. The study included uninephrectomized F344 and Lewis rats as control subjects. Twelve weeks post surgery, the rats were retrieved for analysis of renal function, urine protein levels, histological, immunohistochemical, and molecular biological parameters. RESULTS Administration of 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione inactivated GSK-3β and thereby improved renal function, attenuated proteinuria, and reduced renal tissue damage in CRAD rats. Besides, inactivation of GSK-3β inhibited nuclear factor-κB activation, macrophage infiltration, and expression of multiple proinflammatory cytokines/chemokines. Inhibition of GSK-3β also decreased the levels of malondialdehyde, increased superoxide dismutase levels, upregulated the expression of heme oxygenase-1 and NAD(P)H quinone oxidoreductase-1, and enhanced nuclear translocation of nuclear factor erythroid 2-related factor 2 in the kidneys of CRAD rats. CONCLUSIONS Inhibition of GSK-3β attenuates the development of CRAD by inhibiting inflammation and oxidant stress. Thus, GSK-3β inhibition may represent a potential therapeutic strategy for the prevention and treatment of CRAD.

QiShenYiQi pill (QSYQ), a traditional Chinese medicine, is well known for improving the myocardial remodelling, but the dose‐effect relationship of its intervention in the reparative myocardial fibrosis is still unclear. We investigated the effect of QSYQ on the reparative myocardial fibrosis in cardiac myosin‐induced rats and explored its mechanism of action by regulating autophagy. The results indicated that QSYQ increased LVEF and LVFS, and decreased the LVEDD, LVESD, HMI, LVMI, myocardial inflammation histology score, and collagen volume fraction in a dose‐dependent manner. In addition, QSYQ declined the number of autophagosomes, down‐regulated the expression of myocardial Beclin‐1 and LC3B, up‐regulated the expression of myocardial p62 and increased the ratios of myocardial p‐PI3K/PI3K, p‐Akt/Akt and p‐mTOR/mTOR. We provided evidence for that QSYQ could inhibit excessive myocardial autophagy by regulating the PI3K/Akt‐mTOR pathway and can be a potential therapeutic approach in treating the cardiovascular diseases such as myocarditis and dilated cardiomyopathy.

Intestinal passenger T leukocytes are responsible for graft-versus-host disease (GvHD) in intestinal transplantation (ITx). We hypothesized that ex vivo fludarabine treatment of the bowel graft would diminish the risk of GvHD and improve overall survival post-transplant. We performed isolated heterotopic small bowel transplantations from Lewis (LEW) to Brown Norway (BN) rat strains, which generated GvHD signs from the fourth day post-transplant. These symptoms included rash, weight loss, piloerection, and diarrhea. The grafts of one of the experimental groups were immersed and sealed in cold Celsior preservation solution with 1000 µm fludarabine for 1 h, prior to its implantation into recipient animals. No histological signs of intestinal tissue alterations were observed after fludarabine treatment. Fludarabine-treated bowel recipients showed significantly later and milder clinical signs of GvHD and reduced total donor cell chimerism, as determined by flow cytometry using strain-specific anti-HLA antibodies. Additionally, fludarabine treatment prolonged recipients' overall survival (13.5 days ± 0.3 days vs. 9.2 days ± 0.5). We conclude that active modification of the intestinal leukocyte composition is advantageous in our ITx animal model. Immunosuppression with fludarabine during the surgical procedure, which could be translated directly to the clinic, protects bowel recipients from GvHD and improves overall post-transplant survival.

Purpose Ileal transposition (IT) allows exploration of hindgut effects of bariatric procedures in inducing weight loss and reducing adiposity. Here we investigated the role of dietary macronutrient content on IT effects in rats. Methods Male Lewis rats consuming one of three isocaloric liquid diets enriched with fat (HF), carbohydrates (HC), or protein (HP) underwent IT or sham surgery. Body weight, energy intake, energy efficiency, body composition, and (meal-induced) changes in plasma GIP, GLP-1, PYY, neurotensin, and insulin levels were measured. Results Following IT, HC intake remained highest leading to smallest weight loss among dietary groups. IT in HF rats caused high initial weight loss and profound hypophagia, but the rats caught up later, and finally had the highest body fat content among IT rats. HP diet most efficaciously supported IT-induced reduction in body weight and adiposity, but (as opposed to other diet groups) lean mass was also reduced. Energy efficiency decreased immediately after IT irrespective of diet, but normalized later. Energy intake alone explained variation in post-operative weight change by 80%. GLP-1, neurotensin, and PYY were upregulated by IT, particularly during (0–60 min) and following 17-h post-ingestive intake, with marginal diet effects. Thirty-day post-operative cumulative energy intake was negatively correlated to 17-h post-ingestive PYY levels, explaining 47% of its variation. Conclusion Reduction in energy intake underlies IT-induced weight loss, with highest efficacy of the HP diet. PYY, GLP-1, and neurotensin levels are upregulated by IT, of which PYY may be most specifically related to reduced intake and weight loss after IT.

Outcome assessments that evaluate post-transection nerve repair do not often correlate with one another. The aims of this study were twofold: to compare four nerve repair techniques with each other and incorporate both negative and positive control groups and to identify possible correlations between outcome assessments.Sciatic nerve transection and repair was performed in Lewis rats using one of the following techniques: interrupted epineural, running epineural, grouped fascicular, epineural with absorbable type I collagen wrap, and high tension for incorporation of a negative control. A sham surgery group was also included as a positive control group. Outcomes were compared using assessments of functional recovery (behavior and electrophysiology) and nerve regrowth (imaging and histomorphometry). Three-dimensional printed custom electrode stabilization and imaging devices were designed and fabricated to provide standardization in assessment between subjects.Nerve repair was performed in 48 male Lewis rats. In all animals, functional testing was performed at week 13. The sham group (n = 7) performed the best on both behavioral assays (P 0.001) and electrophysiology assessments (P 0.001). The negative control group (high tension) performed poorest on multiple assessments, and there were no significant differences observed for any of the four repair types. Positive correlations were observed between behavioral and histomorphometric tests.There was no difference in outcome between the four types of nerve repair. High-tension nerve repair represents an ideal negative control. Not all assessment methods correlate equally, and consistent use of complimentary outcome assessments could allow for improved comparison between studies.

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.

Acutely increased renal venous pressure (RVP) impairs renal function, but the long-term impact is unknown. We investigated whether chronic RVP elevation impairs baseline renal function and prevents exacerbation of renal dysfunction and cardiovascular instability upon further RVP increase. RVP elevation (20–25 mmHg) or sham operation (sham) was performed in rats. After 1 wk ( n = 17) or 3 wk ( n = 22), blood pressure, RVP, renal blood flow (RBF), renal vascular conductance (RVC), and glomerular filtration rate (GFR) were measured at baseline and during superimposed RVP increase. Chronic RVP elevation induced extensive renal venous collateral formation. RVP fell to 6 ± 1 mmHg at 1 wk and 3 ± 1 mmHg at 3 wk. Baseline blood pressure and heart rate were unaltered compared with sham. RBF, RVC, and GFR were reduced at 1 wk but normalized by 3 wk. Upon further RVP increase, the drop in mean arterial pressure was attenuated at 3 wk compared with 1 wk ( P < 0.05), whereas heart rate fell comparably across all groups; the mean arterial pressure-heart rate relationship was disrupted at 1 and 3 wk. RBF fell to a similar degree as sham at 1 wk (−2.3 ± 0.7 vs. −3.9 ± 1.2 mL/min, P = 0.066); however, at 3 wk, this was attenuated compared with sham (−1.5 ± 0.5 vs. −4.2 ± 0.7 mL/min, P < 0.05). The drop in RVC and GFR was attenuated at 1 and 3 wk ( P < 0.05). Thus, chronic RVP elevation induced by partial renal vein ligation elicits extensive renal venous collateral formation, and although baseline renal function is impaired, chronic RVP elevation in this manner induces protective adaptations in kidneys of healthy rats, which attenuates the hemodynamic response to further RVP increase.

Glaucoma is characterized by a progressive damage of the retina and the optic nerve. Despite a huge research interest, the exact pathomechanisms are still unknown. In the experimental autoimmune glaucoma model, rats develop glaucoma-like damage of the retina and the optic nerve after immunization with an optic nerve antigen homogenate (ONA). An early activation of the complement system, even before optic nerve degeneration, was reported in this model. Here, we investigated the effects of a monoclonal antibody against complement factor C5 on optic nerves. Rats were immunized with ONA and compared to controls. In one eye of some ONA animals, the antibody against C5 was intravitreally injected (15 μmol: ONA + C5-I or 25 μmol: ONA + C5-II) before immunization and then every 2 weeks. After 6 weeks, optic nerves were processed for histology (n = 6/group). These analyses demonstrated that the intravitreal therapy reduced the depositions of the membrane attack complex compared to ONA animals (ONA + C5-I: p = 0.005; ONA + C5-II: p = 0.002). Cellular infiltration was significantly reduced in the ONA + C5-I group (p = 0.003), but not in ONA + C5-II tissues (p = 0.41). Furthermore, SMI-32 staining revealed that neurofilament was preserved in both treatment groups compared to ONA optic nerves (both p = 0.002). A decreased amount of microglia was found in treated animals in comparison to the ONA group (ONA + C5-I: p = 0.03; ONA + C5-II: p = 0.009). We observed, for the first time, that a complement system inhibition could prevent optic nerve damage in an autoimmune glaucoma model. Therefore, complement inhibition could serve as a new therapeutic tool for glaucoma.

Myocarditis is an inflammatory disease of the myocardium that is associated with immune dysfunction. Earlier studies have suggested that T helper 1/2 cell imbalance plays an important role in the development of myocarditis, but the role of T follicular helper (Tfh) cells in the development of autoimmune myocarditis has not previously been reported. Here, we investigated this involvement by using a rat model of experimental autoimmune myocarditis (EAM). Inflammatory cell infiltration, myocardial structure destruction and tissue necrosis were observed in EAM myocardial tissues, and the percentages of CD4+CXCR5+ Tfh cells and CD19+ B cells were both significantly higher in spleen and myocardial tissues of the EAM model as compared with the control group. Furthermore, the expression levels of interleukin‐21, CXCL13 and myosin antibody were significantly higher in the serum of rats with EAM compared with the control group on days 14 and 35 after immunization. Fourteen or 35 days after immunization, the expression levels of interleukin‐21 and CXCL13 were both significantly higher in myocardial tissues of rats with EAM as compared with the control group. Our findings suggest that Tfh cell balance is disrupted during the pathological process of autoimmune myocarditis., We constructed a model of experimental autoimmune myocarditis, which was used to show that T follicular helper cells can migrate to nonlymphoid tissues. The expression levels of interleukin‐21 and CXCL13 were elevated in serum and spleen of rats with autoimmune myocarditis. T follicular helper cells might play a key role in the pathogenesis of autoimmune myocarditis, and this may provide a basis for identification of new therapeutic targets.

Rat models of lumbar intervertebral disc (IVD) degeneration are widely employed to characterize biologic-based therapeutics, but their anatomy and small size preclude consistent delivery of injectable therapeutics to the lumbar spine via the traditional posterolateral approach. Here, we describe our experience with a repeat ventral transperitoneal approach in female Lewis rats, enabling induction of IVD degeneration and later intervention via an injectable therapeutic. In the initial surgery, the ventral aspect of the L5/L6 IVD was accessed, and an annular defect was created using a #11 scalpel blade. Eight weeks after the initial surgery, follow-up surgery was performed via the same approach, and an injectable gelatin hydrogel was delivered using a 31G needle. A custom injection guard was developed to control injection depth, ensuring consistent delivery to the nucleus pulposus. Notable challenges associated with repeat surgery were increased tissue adhesion, intraoperative bleeding, and difficulty placing the injection guard due to mobile gastrointestinal tissues. Complication rates were 9.4% and 15.6% for the initial and repeat surgeries, respectively. The most frequent complications associated with repeat surgery were transient neuropraxia and significant intraoperative bleeding (6.3% each). The repeat transperitoneal approach is a reproducible method to facilitate both injury and later intervention in a female rat model of lumbar IVD degeneration.

Background Localization of inflammatory stimuli may direct lung allografts to different phenotypes of chronic dysfunction, such as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). We hypothesized that airway stimulation with lipopolysaccharide (LPS) in rats leads to airway-centered inflammation similar to human BOS. Methods Rat left lung transplantation was conducted (donor: Brown Norway, recipient: Lewis). Allotransplant recipients received cyclosporine A (CsA) until postoperative day 56 with airway instillation of LPS (Allo-LPS, n = 8), phosphate buffered saline (Allo-PBS, n = 5) from days 35 to 46 (3 times a wk), or no further treatment (n = 4). Some allotransplant recipients received CsA until day 14 and were immunosuppression free after day 15 until day 56. Bronchial and pleural fibrosis were semiquantified; alveolar fibrosis was evaluated with a histological scale. Results The Allo-LPS group had significantly increased International Society for Heart and Lung Transplantation rejection grades (grade A, P = 0.005; grade B, P = 0.004), bronchial obstructive proportion (0.34 ± 0.04% [Allo-LPS] versus 0.11 ± 0.04% [Allo-PBS], P = 0.006), and airway resistance (3.05 ± 1.78 cm H2O·s/mL [Allo-LPS] versus 0.83 ± 0.58 cm H2O·s/mL [Allo-PBS], P = 0.007) compared with other groups. Allotransplant recipients that underwent a short course of CsA developed RAS-like fibrosis involving the airways, alveoli, and pleura. Conclusions Airway instillation of LPS in allografts under immunosuppression resulted in BOS-like airway-centered inflammation and fibrosis distinct from RAS-like diffuse fibrosis, which was induced by a shortened course of immunosuppression. We propose novel animal models for BOS and RAS after lung transplantation.

Gentle tactile stimuli have been shown to play an important role in the establishment and maintenance of affiliative social interactions. Oxytocin has also been shown to have similar actions. We investigated the effects of gentle stroking on affiliative relationships between humans and rats and the effects of gentle stroking on activation of oxytocin neurons. Male rats received 5-min stroking stimuli from an experimenter every other day for 4 weeks between 3 and 6 weeks of age (S3–6 group), for 4 weeks between 7 and 10 weeks of age (S7–10 group), or for 8 weeks between 3 and 10 weeks of age (S3–10 group). Control rats did not receive stroking stimuli. Rats in the S7–10 and S3–10 groups emitted 50-kHz calls, an index of positive emotion, more frequently during stroking stimuli. Rats in the S3–6, S7–10, and S3–10 groups showed affiliative behaviors toward the experimenter. Oxytocin neurons in the hypothalamic paraventricular nucleus of rats in the S3–6, S7–10, and S3–10 groups were activated following stroking stimuli. These findings revealed that post-weaning repeated stroking stimuli induce an affiliative relationship between rats and humans and activation of oxytocin neurons.

Cell therapy has shown potential in the field of peripheral nerve repair, and research using rodents is a critical and essential step toward clinical development of this approach. Traditionally, most experimental peripheral nerve injuries are conducted in inbred Lewis or outbred Sprague–Dawley strains. However, transplantation of xenogeneic cells such as human-derived cells typically triggers rejection in these animals. An alternative approach is to use immunodeficient animals, such as athymic nude rats. The lack of functional T cells in these animals renders them more accommodating to foreign cells from a different host. Currently, no literature exists regarding sensorimotor behavioral assessment of nude rats after peripheral nerve injury. To this end, we compared the functional recovery during a 6-wk period of behavioral testing of Lewis and nude rats after unilateral sciatic nerve crushing injury. Three sensorimotor behavioral assessments were performed weekly: a ladder rungwalking task to assess slip ratio and cross duration, von Frey nociception testing to determine the paw withdrawal threshold thus monitoring the regaining of sensory function, and sciatic functional index evaluation to monitor the recovery of integrated motor function. Both strains demonstrated significant sensory and motor deficits in the first week after injury, with a slight regain of sensory function, reduced slip ratio, and increased sciatic functional index starting at 2 wk. No significance difference existed between nude and Lewis rats in their recovery courses. We conclude that nude rats are a suitable model for behavioral training and assessment for cell transplantation studies in peripheral nerve injury and repair.

Purpose We sought to examine donor mesenchymal stem cell (MSC) fate after birth following transamniotic stem cell therapy (TRASCET) in a healthy model. Methods Lewis rat fetuses (n = 91) were divided into two groups based on the content of volume-matched intraamniotic injections performed on gestational day 17 (term = 21–22 days): either a suspension of amniotic fluid-derived MSCs (afMSCs) labeled with luciferase (n = 38) or acellular luciferase only (n = 53). Infused afMSCs consisted of syngeneic Lewis rat cells phenotyped by flow cytometry. Samples from 14 anatomical sites (heart, lung, brain, liver, spleen, pancreas, bowel, kidney, thyroid, skin, skeletal muscle, thymus, peripheral blood and bone marrow) from survivors were screened for luciferase activity 16 days after birth. Statistical analysis was by logistic regression and the Wald test (p Results Overall survival was 32% (29/91). When controlled by the acellular luciferase injections, donor afMSCs were not identified at any anatomical site in any neonate as measured by relative light units (all p > 0.05). Donor afMSC viability was confirmed in term placentas. Conclusions Donor mesenchymal stem cells are not detectable in the neonate after intraamniotic injection in a normal syngeneic rodent model. This finding suggests that clinical trials of transamniotic stem cell therapy may be amenable to regulatory approval. Level of evidence N/A (animal and laboratory study).

Myasthenia gravis (MG) is an antibody-mediated, autoimmune neuromuscular disease. Reports have indicated that the CD28/B7 ligand interactions play a crucial role during primary immune responses. Hence, the aim of the present study was to investigate the possible effects of the CD28/B7 pathway on the occurrence and development of MG and its associated cytokine factors.An experimental autoimmune myasthenia gravis (EAMG) was initially established by immunization of Lewis rats with acetylcholine receptor (AChR) α97-116 peptide. Then the rats were treated with dexamethasone and CTLA4-Ig (used for inhibiting the CD28/B7 pathway). Serum levels of AChR IgG and AChR IgG2b were then detected using ELISA. The clinical features, muscle contraction function, AChR content, expression of CD28, CTLA4, B7.1 and B7.2 in mononuclear cells of peripheral blood and the secretion of cytokines (INF-γ, IL-2, IL-10 and IL-12) in serum of rats were measured. Finally, lymphocyte proliferation upon CTLA4 IgG treatment was examinedInhibition of the CD28/B7 pathway and dexamethasone were found to significantly improve clinical symptoms of EAMG rats, reduce serum levels of AChR IgG, AChR IgG2b, INF-γ, IL-2, IL-10 and IL-12, the expression of CD28, CTLA4, B7.1 and B7.2 in mononuclear cells of peripheral blood, and enhance muscle contraction function and AChR content in the muscleOverall, the suppression of the CD28/B7 pathway by CTLA4-Ig can have the potential to retard the occurrence and development of MG.

Molecular hydrogen can reduce the oxidative stress of ischemia-reperfusion injury in various organs for transplantation and potentially improve survival rates in recipients. This study aimed to evaluate the protective effects of a hydrogen-rich preservation solution against ischemia-reperfusion injury after cold ischemia in rat lung transplantation.Lewis rats were divided into a nontransplant group (n = 3), minimum-ischemia group (n = 3), cold ischemia group (n = 6), and cold ischemia with hydrogen-rich (more than 1.0 ppm) preservation solution group (n = 6). The rats in the nontransplant group underwent simple thoracotomy, and the rats in the remaining 3 groups underwent orthotopic left lung transplantation. The ischemic time was30 minutes in the minimum-ischemia group and 6 hours in the cold ischemia groups. After 2-hour reperfusion, we evaluated arterial blood gas levels, pulmonary function, lung wet-to-dry weight ratio, and histologic features of the lung tissue. The expression of proinflammatory cytokines was measured using quantitative polymerase chain reaction assays, and 8-hydroxydeoxyguanosine levels were evaluated using enzyme-linked immunosorbent assays.When compared with the nontransplant and minimum-ischemia groups, the cold ischemia group had lower dynamic compliance, lower oxygenation levels, and higher wet-to-dry weight ratios. However, these variables were significantly improved in the cold ischemia with hydrogen-rich preservation solution group. This group also had fewer signs of perivascular edema, lower interleukin-1β messenger RNA expression, and lower 8-hydroxydeoxyguanosine levels than the cold ischemia group.The use of a hydrogen-rich preservation solution attenuates ischemia-reperfusion injury in rat lungs during cold ischemia through antioxidant and anti-inflammatory effects.