Recipient Rats Research Articles

Remote ischemic conditioning in Ossabaw minipigs induces the release of humoral cardioprotective triggers, but the myocardium does not respond with reduced infarct size.

Ischemic preconditioning (IPC; brief cycles of coronary occlusion/reperfusion) is operative in all species tested so far and reduces infarct size through the release of trigger molecules and activation of signal transducer and activator of transcription (STAT)3 in pigs. We have recently demonstrated that IPC failed to protect Ossabaw minipigs, which had a genetic predisposition to, but not yet established a metabolic syndrome, from infarction and did not activate STAT3. We now subjected Ossabaw minipigs to remote ischemic conditioning (RIC; 4 × 5 min/5 min bilateral hindlimb ischemia-reperfusion) and analyzed the release of cardioprotective triggers into the circulation with the aim to distinguish whether IPC failed to stimulate trigger release or to activate intracellular signaling cascades upstream of STAT3. RIC or a placebo protocol, respectively, was induced in anesthetized pigs before 60 min/180 min coronary occlusion/reperfusion. Plasma, prepared from Ossabaw minipigs after RIC or placebo, was infused into isolated rat hearts subjected to 30 min/120 min global ischemia-reperfusion. In the Ossabaw minipigs, RIC did not reduce infarct size (49.5 ± 12.1 vs. 56.0 ± 11.8% of area at risk with placebo), and STAT3 was not activated. In isolated rat hearts, infusion of RIC plasma reduced infarct size (19.7 ± 6.7 vs. 33.2 ± 5.5% of ventricular mass with placebo) and activated STAT3. Pretreatment of rat hearts with the STAT3 inhibitor stattic abrogated such infarct size reduction and STAT3 activation. In conclusion, Ossabaw minipigs release cardioprotective triggers in response to RIC into the circulation, and lack of cardioprotection is attributed to myocardial nonresponsiveness.NEW & NOTEWORTHY Ischemic conditioning reduces myocardial infarct size in all species tested so far. In the present study, we used Ossabaw minipigs that had a genetic predisposition to, but not yet established a metabolic syndrome. In these pigs, remote ischemic conditioning (RIC) induced the release of cardioprotective triggers but did not reduce infarct size. Transfer of their plasma, however, reduced infarct size in isolated recipient rat hearts, along with signal transducer and activator of transcription (STAT)3 activation.

Abstract 12689: Electron Transport Chain Inhibition to Decrease Reperfusion Injury in Transplanted Donation After Circulatory Death Rat Hearts

Introduction: Intrinsic to the donation after circulatory death (DCD) donation is ischemia and reperfusion injury, primarily mediated through release of reactive oxygen species (ROS) from the damaged mitochondria. Amobarbital (AMO) is a transient, reversible inhibitor of complex I known to decrease ROS generation. We studied whether AMO treatment during reperfusion can improve function in transplanted DCD hearts. Methods: Sprague Dawley rats, were assigned to four heart donor groups- DCD or DCD+AMO and control beating-heart donor (CBD) or CBD+AMO (n=6-8 each). Donor rats were anesthetized using ketamine/xylazine, intubated and connected to a ventilator. The carotid access was used to deliver heparin (1000 U/kg) and vecuronium bromide (4 mg/kg) to paralyze diaphragmatic muscles. The DCD process was initiated by disconnecting the ventilator. Donor hearts were procured following 25 minutes of in-vivo ischemia in the DCD groups, while CBD hearts were procured without terminating the ventilation. At procurement, after clamping the aortic arch, all donor hearts received 10 ml of University of Wisconsin cardioplegia solution delivered through the carotid cannula. The CBD+AMO and DCD+AMO groups received AMO (2 mM), dissolved in the cardioplegia. Heterotopic heart transplantation was performed in recipient rat’s abdomen. After 14 days the transplanted heart function was measured with a balloon tip catheter. Results: Compared to CBD hearts, DCD hearts had significantly lower developed pressure, the rate pressure product (developed pressure times heart rate), and negative dP/dt indicating decreased contractile and impaired diastolic function (Figure). AMO treatment of the DCD hearts resulted in significantly improved cardiac function compared to the untreated DCD hearts. Conclusions: A single treatment with AMO during the heart procurement improves the transplanted DCD heart function which was comparable to the control CBD hearts.

Age- and sex-specific effects of maternal separation on the acoustic startle reflex in rats: early baseline enhancement in females and blunted response to ambiguous threat.

Early life adversity (ELA) increases the incidence of later-life anxiety disorders. Dysregulated threat processing, including responsivity to ambiguous threats, is an indicator of anxiety disorders and can be influenced by childhood experiences. The acoustic startle response is a defensive reflex displayed by mammals when exposed to sudden intense stimuli reflecting individual variations in vigilance. These measures can be altered by previous experience and experimental modifications, including the introduction of unconditioned aversive stimuli. Rats emit ultrasonic vocalizations (USVs) in the 22 KHz range in negative contexts. As such, 22 KHz USVs are an ethologically relevant social cue of environmental threat shown to induce anxiety-like behavior in recipient rats. Because the timing of symptom manifestation after early life adversity can differ between sexes, the current study sought to identify the age- and sex-specific effects of daily maternal separation (MS) on responsivity to ambiguous threat in rats. In Experiment 1, rat pups underwent MS or control rearing from postnatal day (P) 2-20, then underwent behavioral testing beginning on P24, 34, or 54 to determine whether MS modified the baseline startle response or the modulation of startle by 22 KHz USVs. In Experiment 2, rats were tested in a light-enhanced startle paradigm at P54 after MS or control rearing to determine whether MS influenced light-enhanced startle. Results show an enhancement of the baseline startle magnitude by MS in females at P34. At P54, MS reduced the modulation of the startle response by 22 KHz USVs and prevented light-enhanced startle, indicating an MS-induced deficit in defensive responsivity when exposed to potential threat.

Bio-engineering of 3-D cell sheets for diabetic rats: Interaction between mesenchymal stem cells and beta cells in functional islet regeneration system

Type 1 diabetes is an autoimmune disease that emerges with the destruction of beta cells of pancreatic Langerhans islets. Three different therapeutical approaches have been developed so far; pancreas transplantation, islet transplantation, and cell-based therapies. Bioengineering cell sheets for tissue generating is one of the latest approaches that have been used to construct cell-sheets instead of single cells so that it mimics the in vivo environments more. In this study, extra-hepatic functional islet tissue was constructed by transferring the 3-D beta cells and GFP labelled MSCs MSC sheets to the subcutaneous site of rats with STZ-induced diabetes. rBM-MSCs and beta cells were cultured on the 6-well PIPAAm culture dishes. Obtained rBM-MSCs as two-cell sheets and beta cells cultured in droplets with matrigel has transplanted into the dorsal subcutaneous area of diabetic rats. Fasting blood glucose levels and body weights were evaluated for 30 days after transplantation. Immunocytochemistry analysis for the anti-apoptotic, anti-inflammatory, and angiogenetic effects of MSCs on the 30th day of subcutaneous cell transplantation. All recipient rats transplanted with beta-cells with MSCs returned toward normoglycemia by day 5 and remained at this level for 30 days. Immunocytochemical analyses supported that the MSCs and beta cells preserved their viability and function. MSCs secrete cytokines and growth factors TGF-β and IL-6; MSCs of the important features of the anti-apoptotic and anti-inflammatory properties, thanks to apoptosis of beta cells preserve graft explained by inhibition. In transplantation of MSCs induced angiogenesis and neovascularization, PECAM-1 and GFP positive simultaneously determining endothelial cells was observed indicating. Subcutaneous 3D beta-cell transplantation would be possible with the MSC-sheets as a feeder layer of beta cells. The beta-cell line is glucose-sensitive and has a high insulin release potential, and can be used as an alternative to islets in in vivo transplant studies.

333.9: The Effect of Short-term Inhalation of Isoflurane on the Outcomes of Intraportal Hepatocyte Transplantation

Introduction: Liver transplantation is currently established as a treatment for endo-stage liver disease. However, this treatment is considered too invasive for those patients. Hepatocyte transplantation (HTx), which is less invasive in comparison to liver transplantation, has been expected to serve as an alternative therapy to liver transplantation. However, the outcomes of HTx are still far from satisfactory, and it is well known that there is a large discrepancy between the experimental results of animal models and clinical outcomes of HTx. Of particular note, clinical HTx is only performed without general anesthesia, while inhalation anesthetics are usually used in animal experiments. Isoflurane, which is one of the most widely used inhalation anesthetics in animal experiments, is known to have a strong vasodilatory effect, portal pressure inhibitory effect, and cytoprotective effect against cytokine-induced injury. Therefore, we hypothesized that isoflurane may be a possible reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx. Methods: F344/NSLc rat hepatocytes were isolated. Isolated hepatocytes (1.0×107 cells) were transplanted to analbuminemic rats with (ISO group) and without (AW group) isoflurane. These analbuminemic rats had a syngeneic background to the donor rats. The serum albumin, AST, ALT, LDH levels and several inflammatory mediators in the recipient rats were analyzed. Immunohistochemical staining and ex vivo imaging of transplanted hepatocytes were also performed. Results: The serum albumin levels of the ISO group were significantly higher in comparison to the AW group (p<0.05) (Figure 1). The serum AST, ALT, LDH levels of the ISO group were significantly suppressed in comparison to the AW group (p<0.0001, respectively). The serum levels of IL-1β (p<0.01), IL-10 (p<0.01), IL-18 (p<0.01), MCP-1 (p<0.05), RANTES (p<0.01), Fractalkine (p<0.01), and LIX (p<0.05) in the ISO group were significantly downregulated in comparison to the AW group. The ischemic regions of the recipient livers, which were easily detected in the livers of both groups, tended to be more evident in the AW group (3.69±1.05%) in comparison to the ISO group (2.42±1.76%) (Figure 2). Conclusions: This study showed that the anti-inflammatory effects of isoflurane could efficiently contribute to hepatocyte engraftment. According to the detailed analyses in this study, the anti-inflammatory effects, rather than vasodilatory effects, of isoflurane appeared to be the main mechanism of the abovementioned benefits. Isoflurane may at least in part be a reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx.

337.2: Comparison of Hypothermic Static and Normothermic Ex-situ Donor Heart Preservation in Heterotopic Heart Transplantation With the Murine Model

Introduction and Background: Hypothermic static and Normothermic ex-situ preservation methods are the most widely used preservation technique worldwide. While cold static storage is associated with subsequent ischemic injury, Normothermic perfusion preservation has the potential to elongate preservation time with less ischemia. The current study compares the hypothermic and normothermic preservation methods in terms of graft performance, morphologic changes, and acute immune response in an experimental model. Materials and Method: Twenty-five rats underwent heterotopic abdominal heart transplantation after two hours of donor heart hypothermic storage (HT group; n=10) or normothermic ex-situ preservation (NT group; n=15). Blood samples were obtained just before and after 4-hours of implantation to analyze surface markers of immune cells (CD4, CD8, CD161, CD45R). ECG and Echocardiography were performed before harvesting and after implantation. Hearts were extracted after four hours of implantation for Hematoxylin-Eosin (HE) and TUNEL-staining, and recipient rats were euthanized. Results: 19 (76%) rats successfully survived after implantation (HT group: 100%, NT group: 60%). The mean ischemic time of the donor heart was 162.7±8.34 minutes in the HT group and 45±3.7 minutes in the NT group. The NT group showed no significant change in the heart rate before and after implantation but significantly decreased in the first hour of implantation in the HT group (259.5±24.3rpm) and normalized at 3 hours (338.3±34.1rpm). Ejection fraction and fractional shortening significantly decreased after implantation in both groups but were less significant in the NT group (P=0.001). Granulocyte was less significantly increased in the NT group compared to the HT group after the experiment (p=0.037). Although the gross structure was well preserved in both groups in HE-staining, the number of TUNEL positive cells was significantly higher in the HT group. Conclusions: Our findings suggest that normothermic ex-situ preservation is associated with well-preserved donor hearts but similar recipient immune response in comparison with hypothermic static preservation.

Symposium 1-3 - Study for the male immunocontraception of wild animals using sperm antigens.

Testicular immune tolerance is essential for establishment of spermatogenesis, because germ cells have antigenicity. Once the tolerogenic conditions are disturbed by any factors, activated immune cells begin to attack the spermatogenic cells. Previously, we investigated early regression of spermatogenesis in the pig. The males showed aspermia due to prevalence of autoimmune orchitis, but their libido was normal. This suggested that induced autoimmune orchitis might be preferable to immunocontraception with GnRH, which eliminates both spermatogenesis and libido through abolition of testicular endocrine function. Based on this idea, we investigated the concept of immunocontraception using sperm antigens and obtained the following results. Autoimmune orchitis was induced in rats by injection of sperm emulsified with adjuvant. Although the males successfully copulated with females, the number of resulting fetuses was reduced. The condition was also similarly induced in goats. We found that lipopolysaccharide was effective for activating the immune reaction in rats. This type of orchitis is related to cellular immunity. In order to identify the highly antigenic sperm-derived molecules involved, we developed a culture system for rat T lymphocytes that could be activated using sperm antigens. The cells became highly proliferative after culture, and flow cytometry showed that they were CD4 + and TNFα + . However, transfer of the cells did not induce autoimmune orchitis in recipient rats.

404.7: Study on the Regulatory Effect of iPS-Derived Exosomes on the Local Immune Microenvironment in DCD Kidney Transplanted Rats

Introduction: Ischemia reperfusion injury (IRI) of donation After Cardiac death (DCD) transplanted kidney is the key factor of acute renal injury, acute and chronic rejection of renal transplantation and transplanted kidney disease.The latest researches show that mesenchymal stem cells (MSCs) have the function of protecting damaged tissues and organs and strong immune regulation. Induced pluripotent stem (iPS) cells have higher proliferative capacity and exosome secretion potential than MSCs. In addition to the functions of stem cells, exosomes have the unique ability to cross the blood-brain barrier and deliver remotely, which means that exosomes have better advantages in clinical disease treatment compared with the therapeutic potential of stem cells. Therefore, the main purpose of this study is to clarify the role of IPS derived exosomes in protecting the IRI of DCD transplanted kidney and regulating its local immune microenvironment. Methods: The iPS cells were passaged to the third passage and massively expanded. After 48 hours of culture, 100 mL of cell culture supernatant was collected and exosomes were extracted from the supernatant by the kit method, and the morphology and surface membrane marker proteins of the exosomes were identified. The SD rats were established with DCD renal transplantation model and 10, 50, and 100 μg of exosomes were infused into that SD rats respectively, and a PBS control group was set at the same time. On the 3rd, 5th, 7th, 14th and 28th days after transplantation, the content of cytokines in the serum of the recipient rats, the proportion of lymphocyte subsets CD4+CD25+Foxp3+Tregs in the spleen and the relative expression of Foxp3 mRNA were detected. The proliferation of renal tubular cells was detected by PCNA immunofluorescence. The pathological changes of renal tissue were determined by HE staining, and the apoptosis of renal tubular epithelial cells was detected by Tunel staining. The infiltration of inflammatory cells was detected by immunohistochemistry. The expression changes of regulatory T cells in renal tissue and macrophage phenotypic characteristics were analyzed by flow cytometry. Results: The experimental amount of exosomes was successfully obtained, and the morphology was observed by transmission electron microscope. The exosomes were round or oval membranous vesicles with uniform size, and the edges were clearly visible. The TSG101, CD63, and CD9 proteins were highly expressed on the exosome membrane surface by western-blot. A DCD rat kidney transplantation model was successfully established. The expression levels of IL-12, IL-2, IFN-γ, and TNF-α in the serum of each group were increased to varying degrees from the 3rd day after operation. On the 3rd and 7th days, the levels of IL-12, IL-2, IFN-γ, and TNF-α in the 100 μg exosome infusion group were significantly lower than those in the 10 μg, 50 μg and PBS groups (P<0.05), and the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than 10μg, 50μg and PBS groups (P<0.05). On the 28th day, the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than other groups (P<0.01). Compared with other groups, the CD4+CD25+Tregs and CD4+CD25+Foxp3+Tregs accounted for the highest proportion of CD4+T cells in the spleen of the 100μg infusion group (P<0.05). On the 28th day, the expression level of Foxp3 mRNA in the 100 μg infusion group was significantly higher than that in the 50 μg group (P<0.05). The stainings of PCNA, HE and Tunel showed that SD rats infused with 100μg exosomes could significantly reduce the pathological damage, inflammatory response and apoptosis of renal tubular epithelial cells in the transplanted kidney, promote the proliferation of renal tubular epithelial cells, and have a protective effect on IRI kidney. On the 28th day, the expression of CD4+CD25+Foxp3+Tregs cells in the transplanted kidney tissue was significantly increased, which further promoted the differentiation of macrophages into the immunomodulatory M2 subset. Conclusions: The infusion of iPS-derived high-dose exosomes is involved in the protection of the DCD kidney transplant during IRI process, and alleviates the acute and chronic injury of the DCD kidney transplant, and promotes the formation of the local immunosuppressive microenvironment in the DCD kidney transplantation. The National Natural Science Foundation of China (No.81900686).

Efficient engraftment and viral transduction of human hepatocytes in an FRG rat liver humanization model

Humanized liver rodent models, in which the host liver parenchyma is repopulated by human hepatocytes, have been increasingly used for drug development and disease research. Unlike the leading humanized liver mouse model in which Fumarylacetoacetate Hydrolase (Fah), Recombination Activating Gene (Rag)-2 and Interleukin-2 Receptor Gamma (Il2rg) genes were inactivated simultaneously, generation of similar recipient rats has been challenging. Here, using Velocigene and 1-cell-embryo-targeting technologies, we generated a rat model deficient in Fah, Rag1/2 and Il2rg genes, similar to humanized liver mice. These rats were efficiently engrafted with Fah-expressing hepatocytes from rat, mouse and human. Humanized liver rats expressed human albumin and complement proteins in serum and showed a normal liver zonation pattern. Further, approaches were developed for gene delivery through viral transduction of human hepatocytes either in vivo, or in vitro prior to engraftment, providing a novel platform to study liver disease and hepatocyte-targeted therapies.

Preclinical Pharmacokinetics, Biodistribution, and Acute Toxicity Evaluation of Caerin 1.9 Peptide in Sprague Dawley Rats.

Caerin 1.9 is a natural peptide derived from the skin secretions of the Australian tree frog (Litoria) with broad-spectrum antimicrobial and anticancer bioactivity. It improves the efficacy of a therapeutic vaccine and immune checkpoint inhibitor therapy when injected intratumorally and inhibits TC-1 tumor growth when applied topically through intact skin in a TC-1 murine tumor model. This paper investigated the pharmaceutical kinetic profile, the tissue distribution, and the acute safety investigation of Caerin 1.9 peptide in Sprague Dawley (SD) rats. The results showed that subcutaneous injection of Caerin 1.9 at 100 mg/kg is safe and does not cause mortality or organ malfunction in the recipient rats. For the consecutive injection of F3 at 10 mg/kg, the peak concentration (Cmax) of F3 displayed at 1 hr after injection in male rats was 591 ng/mL, the average drug retention time was 0.807 hr, T1/2 was 4.58 hr, and AUC0-last was 1890 h × ng/mL. In female rats, Cmax was 256 ng/mL, with an average drug retention time of 2.96 hr, T1/2 of 1.33 hr, and AUC0-last of 740 h × ng/mL. The results showed that the concentration of Caerin 1.9 in the peripheral blood peaked at 1 hour. As injected concentration increased, T1/2 extended, and Cmax, AUC0-last, and volume of distribution at a steady state all increased. After 14 days of repeated subcutaneous injection at 10.0 mg/kg, no accumulation of Caerin 1.9 in plasma was observed. The results of tissue distribution showed that the Caerin 1.9 is below the LC-MS/MS detection threshold at a minimum concentration of 40 ng/g. In conclusion, Caerin 1.9 is well tolerated in rats and could be used with current immunotherapies for better management of solid tumors and genital warts.

Wasabi Component 6-(Methylsulfinyl)hexly Isothiocyanate and Derivatives Improve the Survival of Skin Allografts.

We tested the effect of 6-(Methylsulfinyl)hexyl Isothiocyanate (6-MITC) and derivatives (I7447 and I7557) on the differentiation and maturation of human myeloid dendritic cells (DCs) in vitro, and skin transplantation in vivo. Triggering of CD14+ myeloid monocyte development toward myeloid DCs with and without 6-MITC and derivatives to examine the morphology, viability, surface marker expression, and cytokine production. Stimulatory activity on allogeneic naive T cells was measured by proliferation and interferon-γ production. The skin allograft survival area model was used to translate the 6-MITC and derivatives’ antirejection effect. All of the compounds had no significant effects on DC viability and reduced the formation of dendrites at concentrations higher than 10 μM. At this concentration, 6-MITC and I7557, but not I7447, inhibited the expression of CD1a and CD83. Both 6-MITC and I7557 exhibited T-cells and interferon-γ augmentation at lower concentrations and suppression at higher concentration. The 6-MITC and I7557 prolonged skin graft survival. Both the 6-MITC and I7557 treatment resulted in the accumulation of regulatory T cells in recipient rat spleens. No toxicity was evident in 6-MITC and I7557 treatment. The 6-MITC and I7557 induced human DC differentiation toward a tolerogenic phenotype and prolonged rat skin allograft survival. These compounds may be effective as immunosuppressants against transplant rejection.

Integrated Analyses of Microbiomics and Metabolomics Explore the Effect of Gut Microbiota Transplantation on Diabetes-Associated Cognitive Decline in Zucker Diabetic Fatty Rats.

Diabetes-associated cognitive decline (DACD), one of the complications of type 2 diabetes (T2DM), correlates significantly with the disorder in glycolipid metabolism, insulin/leptin resistance, and accumulation of β-amyloid (Aβ). Although gut microbiota transplantation (GMT), a novel non-invasive physiotherapy strategy, has been a promising intervention to alleviate the symptoms of T2DM, its protective effect on progressive cognitive decline remains elusive. Here, we transplanted the gut microbiota of healthy or cognitive decline donor rats into ZDF or LZ rats, and integrated microbiomics and metabolomics to evaluate the directional effect of the gut microbiota on the recipient rats. The basal metabolism phenotype changed in ZDF rats instead of in LZ rats. One possible mechanism is that the microbiota and metabolites alter the structure of the intestinal tract, stimulate the brain insulin and leptin signaling pathways, and regulate the deposition of Aβ in the brain. It is worth noting that 10 species of genera, such as Parabacteroides, Blautia, and Lactobacillus, can regulate 20 kinds of metabolites, such as propanoic acid, acetic acid, and citramalic acid, and having a significant improvement on the cognitive behavior of ZDF rats. In addition, the correlation analysis indicated the gut microbiota and metabolites are highly associated with host phenotypes affected by GMT. In summary, our study indicates that altering the microbiota-gut-brain axis by reshaping the composition of gut microbiota is a viable strategy that has great potential for improving cognitive function and combatting DACD.

Mitochondrial therapy: direct visual assessment of the possibility of preventing myocardial infarction under chronic neurogenic pain and B16 melanoma growth in the experiment

On models of chronic neurogenic pain (CNP) and the growth of a malignant tumor (metastasizing B16 melanoma) in male mice, we studied an effect produced by mitochondrial therapy (MCT) on the state of the myocardium. Some structural correlates of the compensatory-restorative effect by mitochondria transplanted from healthy recipient rats were revealed. It has been identified that MCT contributes to the preservation of the structural integrity of the myocardial tissue, the inclusion of an auxiliary link in the cellular mechanisms of tissue restoration: fibroblasts, histiocytes, lymphocytes, eosinophils and other connective tissue elements, which implement the intercellular mechanism of information transfer that provides the external regulatory function of MCT. The ability of mitochondria to prevent the DNA decay determines the possibility of initiation of the operation of the nuclear mechanisms of the cardiomyocyte division, which is characteristic of a population of young cells and which indicates the determining position of exogenous mitochondria.

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Generation of 3D lacrimal gland organoids from human pluripotent stem cells.

Lacrimal glands are the main exocrine glands of the eyes. Situated within the orbit, behind the upper eyelid and towards the temporal side of each eye, they secrete lacrimal fluid as a major component of the tear film. Here we identify cells with characteristics of lacrimal gland primordia that emerge in two-dimensional eye-like organoids cultured from human pluripotent stem cells1. When isolated by cell sorting and grown under defined conditions, the cells form a three-dimensional lacrimal-gland-like tissue organoid with ducts and acini, enabled by budding and branching. Clonal colony analyses indicate that the organoids originate from multipotent ocular surface epithelial stem cells. The organoids exhibit notable similarities to native lacrimal glands on the basis of their morphology, immunolabelling characteristics and gene expression patterns, and undergo functional maturation when transplanted adjacent to the eyes of recipient rats, developing lumina and producing tear-film proteins.

Spatial Transcriptomic Analysis of Acute Heart Rejection Model

<h3>Purpose</h3> Heart transplantation (HTx) is sometimes the only treatment for end-stage cardiac disease, but survival after HTx is limited. Understanding the molecular mechanisms behind allograft injury, dysfunction, and failure, are critical in improving the patients' outcome. Rapidly evolving single cell and spatial transcriptomic technologies have opened a new field of precision to diagnostics and therapeutics of cardiovascular diseases. Here we studied the cellular mechanisms of acute rejection in a rat heart transplant model with spatial transcriptomics methods. <h3>Methods</h3> We investigated the effects of acute rejection in heterotopic rat heart transplantation model with syngeneic (DA-DA) and major MHC-mismatched allogeneic (DA-WF) recipients. The recipient rats were sacrificed 5 days after transplantation, and graft cross-sections were analyzed with the Visium Spatial Gene Expression platform (10xGenomics). Raw sequencing reads were processed using SpaceRanger. All main data analysis steps were performed using Seurat. Clusters were identified based on the expression of several known cell markers. <h3>Results</h3> From all samples together, a total of 17 clusters of spots were identified. Our analysis suggests that two of the clusters presented mainly cardiomyocytes, one cluster of smooth muscle cells, two clusters of fibroblasts, 11 immune cells and one cluster of erythrocytes. Endothelial cells did not form a unique cluster. Syngeneic HTx alone induced marked transcriptomic changes in 5 clusters based on differential gene expression analysis, mainly increasing the proportion of clusters with certain types of macrophages. Few clusters remained unchanged between the allogenic and the syngeneic groups. Allogenic group had four unique clusters representing mainly different immune cell populations: granulocytes, lymphocytes and a type of macrophages that were not present in other samples. One cluster consisted of erythrocytes from hemorrhagic area. <h3>Conclusion</h3> Acute rejection changes the cellular composition of the heart muscle dramatically and induces transcriptomic changes in the structural cells of the heart. Our spatial transcriptomic analysis provides novel transcriptomic data on cellular changes occurring following acute rejection. This method allows us to study the cells and mechanisms involved in rejection and look for potential targets for drug development and therapeutics.

Metformin Attenuates Ischemia-Reperfusion Injury in a Rat Lung Transplantation Model

<h3>Purpose</h3> Ischemia-reperfusion injury (IRI), an inevitable acute lung injury during lung transplantation (LTx), contributes to the development of severe complications and limits the success of LTx. Metformin exerts beneficial actions to alleviate acute lung injury induced by a diversity of factors. However, the effect of metformin on IRI has not yet been elucidated in LTx. In this study, we conducted an animal experiment to investigate the effects of metformin on IRI and explore the possible therapeutic mechanism. <h3>Methods</h3> LTx was performed on five groups of rats: sham operation (SM), ischemia-reperfusion (IR), metformin-IR (MI), metformin-anisomycin (MAPK activator)-IR (MAI), metformin-phorbol 12-myristate 13-acetate (PMA, NETosis activator)-IR (MPI). In MI, MAI, and MPI groups, metformin (400 mg/kg) was administered by gavage 72h, 48h, 24h, and 30min before cold ischemia. Anisomycin (15 mg/kg) and PMA (3 ug/kg) were intraperitoneally injected 30min before cold ischemia in MAI and MPI groups, respectively. Each group (n = 5) received a cold ischemic time of 1.5h followed by LTx except the SM group (n = 5). After reperfusion for two hours, recipient rats were sacrificed for further examination of arterial blood gas analysis, histopathology examination, and lung wet-to-dry weight (W/D) ratio. <h3>Results</h3> Compared to the IR group, the MI group showed significantly better PaO₂/FiO₂ ratio and SaO₂, lower W/D ratio, perivascular cuff area/vessel area (P/V) ratio, lung injury score, and red blood cell (RBC) infiltration (all <i>P</i> < 0.05). After the administration of MAPK signaling pathway and NETosis activators, significant deterioration was found in the MAI group with higher lung injury score, RBC infiltration, and W/D ratio (all <i>P</i> < 0.05) and the MPI group with lower SaO₂ and higher lung injury score and P/V ratio (all <i>P</i> < 0.05) (Figure 1). <h3>Conclusion</h3> Metformin attenuates IRI and may be attributed to inhibition of MAPK signaling pathway and NETosis in rat LTx.

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding root replacement, the possible dilation of the pulmonary autograft and subsequent aortic regurgitation is described. Several animal models have been proposed. However, these are usually limited to ex-vivo models or in-vivo experiments with relatively expensive large animal models. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position. A total of 39 adult Lewis rats were included. Immediately after euthanasia, the pulmonary root was harvested from a donor animal (n=17). Syngeneic recipient (n=17) and sham-operated (n=5) rats were sedated and ventilated. In the recipient group, the PAG was implanted with an end-to-end anastomosis in infra-renal abdominal aortic position. Sham-operated rats underwent only transection and re-anastomosis of the aorta. Animals were followed with serial ultrasound studies for two months and post-mortem histological analysis. The median PAG diameter in the native position was 3.20 mm (IQR=3.18-3.23). At follow-up, the median diameter of the PAG was 4.03 mm (IQR=3.74-4.13) at 1 week, 4.07 mm (IQR=3.80-4.28) at 1 month, and 4.27 mm (IQR=3.90-4.35) at 2 months (p<0.01). Peak systolic velocity was 220.07 mm/s (IQR=210.43-246.41) at 1 week, 430.88 mm/s (IQR=375.28-495.56) at 1 month, and 373.68 mm/s (IQR=305.78-429.81) at 2 months (p=0.02) and did not differ from the sham-operated group at the end of the experiment (p=0.5). Histological analysis did not show any sign of endothelial thrombosis. This study showed that rodent models may allow for the evaluation of the long-term adaptation of the pulmonary root to a high-pressure system. A systemically placed syngeneic PAG implantation represents a simple and feasible platform for the development and evaluation of novel surgical techniques and drug therapies to further improve the outcomes of the Ross operation.

The Calcified Vasculature in Chronic Kidney Disease Secretes Factors that Inhibit Bone Mineralization.

ABSTRACTVascular calcification and bone disorder progress simultaneously in chronic kidney disease (CKD). Still, how the complex pathological mechanisms are linked is only sparsely understood. Up to now, the focus has been on the disturbed bone metabolism in developing vascular calcification. However, our group has recently demonstrated that vascular calcification has negative effects on bone formation and mineralization as shown in the bone of normal recipient rats transplanted with the calcified aorta from CKD rats. In the present in vitro study, the hypothesis of a direct crosstalk between the vasculature and bone was examined. Calcified aortas from 5/6 nephrectomized rats and normal aortas from control rats were excised and incubated ex vivo. The calcified aorta secreted large amounts of sclerostin, dickkopf‐1 (Dkk1), and activin A. Both normal and calcified aortas secreted frizzle‐related protein 4 (SFRP4). Aorta rings were co‐incubated with the osteoblast‐like cell line UMR‐106. The calcified aorta strongly inhibited calcium crystal formation in UMR‐106 cells, together with a significant upregulation of the mineralization inhibitors osteopontin and progressive ankylosis protein homolog (ANKH). The strong stimulation of osteopontin was blocked by lithium chloride, indicating involvement of Wnt/β‐catenin signaling. The present in vitro study shows detrimental effects of the calcified aorta on bone cell mineralization. These findings support the hypothesis of an active role of the calcified vasculature in the systemic CKD–mineral and bone disorder (CKD‐MBD), resulting in a pathological vascular–bone tissue crosstalk. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Cytomegalovirus Latency Exacerbated Small-for-size Liver Graft Injury Through Activation of CCL19/CCR7 in Hepatic Stellate Cells.

The interplay between cytomegalovirus (CMV) latency and graft malfunction after living donor liver transplantation remains poorly defined because of the complexity of clinical confounding factors. Here, we aimed to investigate the effects of CMV latency on small-for-size graft injury and to get further insight into the pathogenic role of hepatic stellate cells (HSCs) in this process. Rat orthotopic liver transplantation with small-for-size grafts was performed in a CMV latent model developed in immunocompetent Sprague Dawley rats using Priscott strain. Posttransplant graft injury including hepatocyte damage, stellate cell activation, and fibrogenesis was evaluated. Differential gene expression of HSCs in response to CMV latency was screened by cDNA microarray. Clinical validation was further conducted in human biopsies. CMV latency aggravated hepatocyte apoptosis/necrosis in the early phase and enhanced HSC expansion and graft fibrosis during the middle-late phase in small-for-size liver grafts of the rat model. cDNA microarray mining revealed CCL19/CCR7 as one of the most noteworthy pathways bridging HSC activation and liver graft injury in the presence of CMV latency. Together with CCL19 upregulation, coherent overexpression of CCR7 in accumulated HSCs was confirmed in both rat and human CMV latent recipients. Moreover, addition of CCL19 in vitro promoted HSC migration by increasing the level of matrix metalloproteinase-2. Our data demonstrated that CMV latency aggravated early/late phase liver graft damage and fibrogenesis via CCL19/CCR7/HSCs axis. Blockade of CMV latency-related stellate cell activation may shed light on the strategy of graft protection clinically.