Dark Agouti Research Articles

Pharmacological Inhibition of Vanin Activity Attenuates Transplant Vasculopathy in Rat Aortic Allografts.

Development of transplant vasculopathy is a major cause of graft loss and mortality in solid organ transplant recipients. Previous studies in mice have indicated that vanin-1, a member of the vanin protein family with pantetheinase activity, is possibly involved in neointima formation. Here, we investigated if RR6, a recently developed vanin inhibitor, could attenuate development of transplant vasculopathy. Abdominal allogeneic aorta transplantation from Dark Agouti to Brown Norway rats was performed. Surface neointima was quantified 2 and 4 weeks after transplantation. Systemic vanin activity was measured, and allograft leukocyte infiltration, glutathione-synthesizing capacity, matrix metalloproteinase 9 expression and neointimal smooth muscle cell (SMC) proliferation were assessed by immunohistochemistry. In vitro, the effects of RR6 on SMC proliferation (water-soluble tetrazolium-1 assay) and cytokine-induced apoptosis (flow cytometry) were investigated. RR6 treatment significantly reduced systemic pantetheinase activity during the 4-week follow-up period. RR6 attenuated neointima formation 4 weeks after transplantation. Neointimal SMC proliferation and medial SMC matrix metalloproteinase 9 expression were not altered by RR6. However, RR6 significantly reduced neointimal macrophage influx that was accompanied by increased GCLC messenger RNA expression. In vitro, RR6 inhibited platelet-derived growth factor-induced SMC proliferation and protected SMCs from TNF-α-induced apoptosis. Pharmacological inhibition of vanin activity attenuates development of transplant vasculopathy. This was accompanied by reduced macrophage infiltration and increased glutathione-synthesizing capacity. In vitro, RR6 reduced SMC proliferation and apoptosis that was not confirmed in vivo. Further in-depth studies are warranted to reveal the underlying mechanism(s) of RR6-induced attenuation of transplant vasculopathy in vivo.

Novel genes on rat chromosome 10 are linked to body fat mass, preadipocyte number and adipocyte size

The genetic architecture of obesity is multifactorial. We have previously identified a quantitative trait locus (QTL) on rat chromosome 10 in a F2 cross of Wistar Ottawa Karlsburg (WOKW) and Dark Agouti (DA) rats responsible for obesity-related traits. The QTL was confirmed in congenic DA.WOKW10 rats. To pinpoint the region carrying causal genes, we established two new subcongenic lines, L1 and L2, with smaller refined segments of chromosome 10 to identify novel candidate genes. All lines were extensively characterized under different diet conditions. We employed transcriptome analysis in visceral adipose tissue (VAT) by RNA-Seq technology to identify potential underlying genes in the segregating regions. Three candidate genes were measured in human paired samples of VAT and subcutaneous (SC) AT (SAT) (N=304) individuals with a wide range of body weight and glucose homeostasis parameters. DA.WOKW and L1 subcongenic lines were protected against body fat gain under high-fat diet (HFD), whereas L2 and DA had significantly more body fat after high-fat feeding. Interestingly, adipocyte size distribution in SAT and epigonadal AT of L1 subcongenic rats did not undergo typical ballooning under HFD and the number of preadipocytes in AT was significantly elevated in L2 compared with L1 and parental rats. Transcriptome analysis identified three candidate genes in VAT on rat chromosome 10. In humans, these candidate genes were differentially expressed between SAT and VAT. Moreover, HID1 mRNA significantly correlates with parameters of obesity and glucose metabolism. Our data suggest novel candidate genes for obesity that map on rat chromosome 10 in an interval 102.2-104.7 Mb and are strongly associated with body fat mass regulation, preadipocyte number and adipocyte size in rats. Among those genes, AT head involution defective (HID1) mRNA expression may be relevant for human fat distribution and glucose homeostasis.

Strain differences in toxicity of oral cadmium intake in rats

Influence of genetic background on toxicity of oral cadmium (Cd) administration (30 days, in drinking water; 5 ppm and 50 ppm of cadmium) was examined in Albino Oxford (AO) and Dark Agouti (DA) rats. Similar cadmium deposition was noted in gut and draining mesenteric lymph nodes (MLN) of both strains but intensity and/or the pattern of responses to cadmium in these tissues differ. Less intense intestinal damage and leukocyte infiltration was observed in gut of cadmium-exposed AO rats. While gut-associated lymph node cells of DA rats responded to cadmium with an increase of cell proliferation, oxidative activity, IFN-γ, IL-17 production and expression, no changes of these activities of MLN cells of cadmium-treated AO rats were observed. Spleen, which accumulated cadmium comparable to MLN, responded to metal by drop in cell viability and by reduced responsiveness of proliferation and cytokine production to stimulation in DA rats solely, which suggest tissue dependence of cadmium effects. More pronounced cadmium effects on MLN and spleen cells of DA rats (which accumulated similar cadmium doses as AO rats), showed greater susceptibility of this strain to cadmium. The results presented, for the first time, depict the influence of genetic background to effects of oral cadmium administration.

Exogenous oxytocin reduces signs of sickness behavior and modifies heart rate fluctuations of endotoxemic rats

Besides the well-known roles of oxytocin on birth, maternal bonding, and lactation, recent evidence shows that this hypothalamic hormone possesses cardioprotective, anti-inflammatory and parasympathetic neuromodulation properties. In this study, we explore the heart rate fluctuations (HRF) in an endotoxemic rodent model that was accompanied by the administration of exogenous oxytocin. The assessment of HRF has been widely used as an indirect measure of the cardiac autonomic function. In this context, adult male Dark Agouti rats were equipped with a telemetric transmitter to continuously and remotely measure the electrocardiogram, temperature, and locomotion. In a between-subjects experimental design, rats received the following peripheral treatment: saline solution as a vehicle (V); lipopolysaccharide (LPS); oxytocin (Ox); lipopolysaccharide + oxytocin (LPS+Ox). Linear and non-linear parameters of HRF were estimated starting 3h before to 24h after treatments. Our results showed that exogenous oxytocin does not modify by itself the HRF of oxytocin-treated rats in comparison to vehicle-treated rats. However, in animals undergoing endotoxemia it: a) provokes a less anticorrelated pattern in HRF, b) decreased mean heart rate, c) moderated the magnitude and duration of the LPS-induced hyperthermia, and d) increased locomotion, up to 6h after the LPS injection. The less anticorrelated pattern in the HRF and decreased mean heart rate may reflect a cardiac pacemaker coupling with cholinergic influences mediated by oxytocin during LPS-induced endotoxemia. Finally, the anti-lethargic and long-term temperature moderating effects of the administration of oxytocin during endotoxemia could be a consequence of the systemic anti-inflammatory properties of oxytocin.

Allogeneic Liver Transplantation and Subsequent Syngeneic Hepatocyte Transplantation in a Rat Model: Proof of Concept for in vivo Tissue Engineering

Objectives: Stable long-term functioning of liver cells after transplantation in humans is still not achieved successfully. A new approach for successful engraftment of liver cells may be the transplantation of syngeneic cells into an allogeneic liver graft. We therefore developed a new rat model for combined liver and liver cell transplantation (cLCTx) under stable immunosuppression. Materials and Methods: After inducing a mitotic block, liver grafts from female donor rats (Dark Agouti) were transplanted into female recipients (Lewis). In male Lewis rats, liver cell proliferation was induced with subsequent cell isolation and transplantation into female recipients after organ transplantation. Y-chromosome detection of the transplanted male cells was performed by quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FisH) with localization of transplanted cells by immunohistochemistry. Results: Immunohistochemistry demonstrated the engraftment of transplanted cells, as confirmed by FisH, showing repopulation of the liver graft with 15.6% male cells (± 1.8 SEM) at day 90. qPCR revealed 14.15% (± 5.09 SEM) male DNA at day 90. Conclusion: Engraftment of transplanted syngeneic cells after cLCTx was achieved for up to 90 days under immunosuppression. Immunohistochemistry indicated cell proliferation, and the FisH results were partly confirmed by qPCR. This new protocol in rats appears feasible for addressing long-term functioning and eventually the induction of operational tolerance in the future.

Novel genes on rat chromosome 10 are linked to body fat mass, preadipocyte number and adipocyte size

We have previously identified a quantitative trait locus (QTL) on rat chromosome 10 in a F2 cross of WOKW and Dark Agouti rats responsible for obesity and cholesterol metabolism. To pinpoint the region carrying causal genes, we established two new sub-congenic lines, L1 and L2, with refined segments of chromosome10. All lines were extensively characterized under different diet conditions. We employed transcriptome analysis in visceral adipose tissue (AT) by RNA-Seq technology to identify potential underlying genes in the segregating regions. Three candidate genes were measured in paired samples of visceral and subcutaneous AT from 232 individuals with a wide range of body weight and parameters of glucose homeostasis.

Liver microRNA Profile of Induced Allograft Tolerance.

Although the liver is less immunogenic than other solid organs, most liver transplant recipients receive lifelong immunosuppression. In both experimental models and clinical transplantation, total lymphoid irradiation (TLI) has been shown to induce allograft tolerance. Our goal was to identify the microRNAs (miRNAs) expressed in tolerant liver allograft recipients in an experimental model of TLI-induced tolerance. To identify the miRNAs associated with TLI-induced tolerance, we examined syngeneic recipients (Lewis→Lewis) and allogeneic recipients (Dark Agouti→Lewis) of orthotropic liver transplants that received posttransplant TLI, allogeneic recipients that were not treated posttransplantation and experienced acute rejection, and native Dark Agouti livers. Quantitative-polymerase chain reaction miRNA array cards were used to profile liver grafts. We identified 12 miRNAs that were specifically and significantly increased during acute rejection. In early tolerance, 33 miRNAs were altered compared with syngeneic livers, with 80% of the miRNAs increased. In established tolerance, 42 miRNAs were altered. In addition, miR-142-5p and miR-181a demonstrated increased expression in tolerant livers (both early and established tolerance) as compared with syngeneic livers. A principal component analysis of all miRNAs assayed demonstrated a profile in established tolerance that was closely related to that seen in syngeneic livers. The miRNA profile of established tolerant allografts is very similar to syngeneic grafts, suggesting tolerance may be a return to an immunological state of quiescence.

Long-Term Effects of (-)-Epigallocatechin Gallate (EGCG) on Pristane-Induced Arthritis (PIA) in Female Dark Agouti Rats.

Rheumatoid arthritis (RA)—a widespread chronic inflammatory disease in industrialized countries—is characterized by a persistent and progressive joint destruction. The chronic pro-inflammatory state results from a mutual activation of the innate and the adaptive immune system, while the exact pathogenesis mechanism is still under discussion. New data suggest a role of the innate immune system and especially polymorphonuclear granulocytes (PMNs, neutrophils) not only during onset and the destructive phase of RA but also at the chronification of the disease. Thereby the enzymatic activity of myeloperoxidase (MPO), a peroxidase strongly abundant in neutrophils, may be important: While its peroxidase activity is known to contribute to cartilage destruction at later stages of RA the almost MPO-specific oxidant hypochlorous acid (HOCl) is also discussed for certain anti-inflammatory effects. In this study we used pristane-induced arthritis (PIA) in Dark Agouti rats as a model for the chronic course of RA in man. We were able to shown that a specific detection of the HOCl-producing MPO activity provides a sensitive new marker to evaluate the actual systemic inflammatory status which is only partially detectable by the evaluation of clinical symptoms (joint swelling and redness measurements). Moreover, we evaluated the long-term pharmacological effect of the well-known anti-inflammatory flavonoid epigallocatechin gallate (EGCG). Thereby only upon early and continuous oral application of this polyphenol the arthritic symptoms were considerably diminished both in the acute and in the chronic phase of the disease. The obtained results were comparable to the treatment control (application of methotrexate, MTX). As revealed by stopped-flow kinetic measurements, EGCG may regenerate the HOCl-production of MPO which is known to be impaired at chronic inflammatory diseases like RA. It can be speculated that this MPO activity-promoting effect of EGCG may contribute to the pharmacological mode of action of this polyphenol.

Re-evaluation of hepatocyte replacement by recipient-derived cells after allogenic liver transplantation: Discrepancy between clinical observations and a rat model.

Reports suggest that hepatocyte replacement by recipient-derived cells is an active phenomenon after allogenic liver transplantation in rats. However, this phenomenon is rarely observed in humans, and further evaluation is necessary to bridge the gap between clinical practice and animal experiment. Fifty percent volume of the liver from green fluorescent protein (GFP) transgenic Lewis rats were transplanted into wild-type Dark Agouti (DA) rats, in which GFP negative hepatocytes were considered as host (DA rat)-derived cells. The transplanted liver was observed on whole imaging system and fluorescent microscope 7-10days after transplantation. As a different method from previous reports, hepatocytes isolated from transplanted livers were cultured, and the expression of GFP was examined. The sliced liver (2mm) after allogenic transplantation demonstrated decreased intensity of GFP signals compared with the positive control. The hematoxylin-eosin staining of the section revealed abundant infiltration of inflammatory cells, suggesting an immunological rejection reaction. Large polygonal cells with significantly decreased or negative GFP signals were also demonstrated, which was consistent with the results of previous studies. However, cell culturing demonstrated that none of the examined albumin positive large polygonal cells were host-derived cells. The same results were obtained irrespective of reconstruction of hepatic artery. Our result implies that rejection reaction does not promote parenchymal replacement by recipient-derived cells, in contrast to previous reports. If so, the phenomena occurring in rats are consistent with clinical observation of liver transplantation in humans.

Chronic central serotonin depletion attenuates ventilation and body temperature in young but not adult Tph2 knockout rats.

Genetic deletion of brain serotonin (5-HT) neurons in mice leads to ventilatory deficits and increased neonatal mortality during development. However, it is unclear if the loss of the 5-HT neurons or the loss of the neurochemical 5-HT led to the observed physiologic deficits. Herein, we generated a mutant rat model with constitutive central nervous system (CNS) 5-HT depletion by mutation of the tryptophan hydroxylase 2 (Tph2) gene in dark agouti (DA(Tph2-/-)) rats. DA(Tph2-/-) rats lacked TPH immunoreactivity and brain 5-HT but retain dopa decarboxylase-expressing raphe neurons. Mutant rats were also smaller, had relatively high mortality (∼50%), and compared with controls had reduced room air ventilation and body temperatures at specific postnatal ages. In adult rats, breathing at rest and hypoxic and hypercapnic chemoreflexes were unaltered in adult male and female DA(Tph2-/-) rats. Body temperature was also maintained in adult DA(Tph2-/-) rats exposed to 4°C, indicating unaltered ventilatory and/or thermoregulatory control mechanisms. Finally, DA(Tph2-/-) rats treated with the 5-HT precursor 5-hydroxytryptophan (5-HTP) partially restored CNS 5-HT and showed increased ventilation (P < 0.05) at a developmental age when it was otherwise attenuated in the mutants. We conclude that constitutive CNS production of 5-HT is critically important to fundamental homeostatic control systems for breathing and temperature during postnatal development in the rat.

Platelet-derived Growth Factor-B Protects Rat Cardiac Allografts From Ischemia-reperfusion Injury.

Microvascular dysfunction and cardiomyocyte injury are hallmarks of ischemia-reperfusion injury (IRI) after heart transplantation. Platelet-derived growth factors (PDGF) have an ambiguous role in this deleterious cascade. On one hand, PDGF may exert vascular stabilizing and antiapoptotic actions through endothelial-pericyte and endothelial-cardiomyocyte crosstalk in the heart; and on the other hand, PDGF signaling mediates neointimal formation and exacerbates chronic rejection in cardiac allografts. The balance between these potentially harmful and beneficial actions determines the final outcome of cardiac allografts. We transplanted cardiac allografts from Dark Agouti rat and Balb mouse donors to fully major histocompatibility complex-mismatched Wistar Furth rat or C57 mouse recipients with a clinically relevant 2-hour cold ischemia and 1-hour warm ischemia. Ex vivo intracoronary delivery of adenovirus-mediated gene transfer of recombinant human PDGF-BB upregulated messenger RNA expression of anti-mesenchymal transition and survival factors BMP-7 and Bcl-2 and preserved capillary density in rat cardiac allografts at day 10. In mouse cardiac allografts PDGF receptor-β, but not -α intragraft messenger RNA levels were reduced and capillary protein localization was lost during IRI. The PDGF receptor tyrosine kinase inhibitor imatinib mesylate and a monoclonal antibody against PDGF receptor-α enhanced myocardial damage evidenced by serum cardiac troponin T release in the rat and mouse cardiac allografts 6 hours after reperfusion, respectively. Moreover, imatinib mesylate enhanced rat cardiac allograft vasculopathy, cardiac fibrosis, and late allograft loss at day 56. Our results suggest that PDGF-B signaling may play a role in endothelial and cardiomyocyte recovery from IRI after heart transplantation.

The local effect of octreotide on mechanical pain sensitivity is more sensitive in DA rats than DA.1U rats.

A recent study by the authors indicated that major histocompatibility complex (MHC) genes are associated with the differences in basal pain sensitivity and in formalin model between Dark-Agouti (DA) and novel congenic DA.1U rats, which have the same genetic background as DA rats except for the u alleles of MHC. The objective of the present study is to investigate whether there is a difference in the pristane-induced arthritis (PIA) model and local analgesic effect of octreotide (OCT) between DA and DA.1U rats. The hindpaw mechanical withdrawal threshold (MWT) and heat withdrawal latency (HWL) were observed. The C unit firings of the tibial nerve evoked by non-noxious and noxious toe movements were recorded by electrophysiological methods in normal and PIA models in DA and DA.1U rats before and after local OCT administration. The expression of somatostatin receptor 2A (SSTR2A) was observed by immunohistochemistry. The results demonstrate that DA rats have a higher mechanical sensitivity than DA.1U rats after PIA. Local OCT administration significantly elevated MWT in DA rats under normal and PIA sate, but not in DA.1U rats. The electrophysiological experiments showed OCT significantly attenuated the firings of C units evoked by non-noxious and noxious stimulation in DA rats more than those in DA.1U rats both in normal and PIA states. In addition, the expression of SSTR2A in the dorsal horn of the spinal cord was significantly higher in DA than in DA.1U rats. All of the findings suggest a higher local analgesic effect of OCT in DA rats than DA.1U rats, which might be associated with the MHC genes.

Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

Oral Platelet-Derived Growth Factor and Vascular Endothelial Growth Factor Inhibitor Sunitinib Prevents Chronic Allograft Injury in Experimental Kidney Transplantation Model.

Expression of both platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) is increased during the development of chronic rejection which remains the major reason for late allograft loss in clinical kidney transplantation. Sunitinib is a tyrosine kinase inhibitor which inhibits both VEGF and PDGF receptors. Here we investigated its effect on the development of chronic rejection. Rat aortic denudation model was used to define sunitinib dose. In vitro studies were done to investigate the effect of sunitinib on smooth muscle cell proliferation and migration. Kidney transplantations were performed from dark agouti rat strain (DA) to Wistar furth rat strain rats and syngenic DA-DA grafts were used as controls. Allografts were immunosuppressed either with cyclosporine or with cyclosporine and sunitinib. Grafts were harvested at 5 and 90 days for histology and immunohistochemistry. Serum creatinine levels were measured weekly to monitor graft function. Sunitinib decreased neointimal formation and smooth muscle cell proliferation and migration in a dose-dependent manner. Sunitinib was well tolerated and almost completely prevented chronic rejection changes and preserved significantly better renal graft function after transplantation. Sunitinib also inhibited chronic PDGF-A and -B and VEGF-A and -B expressions. These results demonstrate that combined inhibition of PGDF and VEGF with sunitinib prevents chronic rejection changes in experimental kidney transplantation which indicates that sunitinib could be a potential intervention also in clinical kidney transplantation.

N-octanoyl Dopamine Attenuates the Development of Transplant Vasculopathy in Rat Aortic Allografts Via Smooth Muscle Cell Protective Mechanisms.

Transplant vasculopathy (TV) is a major cause for late graft loss after cardiac transplantation. Endothelial damage and T cell infiltration play a pivotal role in the development of TV. Because N-octanoyl dopamine (NOD) inhibits vascular inflammation and suppresses T cell activation in vitro, we here tested the hypothesis that NOD treatment ameliorates TV. Aortic grafts were orthotopically transplanted in the Dark Agouti to Brown Norway strain combination. Recipient rats were treated with NOD or vehicle administered via osmotic minipumps. Histology and quantitative polymerase chain reaction (qPCR) were performed on nontransplanted aortas and grafts explanted 2 and 4 weeks after transplantation to assess the degree of TV, inflammation, apoptosis, and number of (proliferating) α smooth muscle actin (αSMA) neointimal cells. In vitro analyses of human aortic smooth muscle cells were performed to test the effect of NOD on proliferation (WST-1 assay), cell cycle (flow cytometry and qPCR), and cytokine-induced apoptosis (flow cytometry). Allografts from vehicle-treated recipients developed neointimal lesions predominantly consisting of αSMA-expressing cells. NOD treatment significantly reduced neointima formation and neointimal αSMA cells. In situ, smooth muscle cell proliferation (Ki67) was not influenced by NOD. Macrophage (CD68), T (CD3), and Natural Killer (ANK61) cell infiltration as well as intragraft TNFα and IFNγ mRNA expression were similar in both groups. Medial apoptosis (cleaved caspase-3) was significantly reduced by NOD. In vitro, NOD inhibited proliferation of human aortic smooth muscle cells by causing a G1-arrest and protected from TNFα-induced apoptosis. This study identified NOD as potential treatment modality to attenuate TV. Our data clearly support a vasculoprotective effect of NOD by reducing smooth muscle cell proliferation and inflammation-induced apoptosis.

Proliferation and Migration of Peripheral Retinal Pigment Epithelial Cells Are Associated with the Upregulation of Wingless-Related Integration and Bone Morphogenetic Protein Signaling in Dark Agouti Rats

Objective: The aim of this study was to investigate the possible migration of proliferating peripheral retinal pigment epithelial (RPE) cells and their association with differential gene expressions. Materials and Methods: The RPE layer was obtained from the inner aspect of the eyeball of dark agouti rats (12-13 weeks old) and was mounted on glass slides. The peripheral RPE cell proliferation was evaluated using bromodeoxyuridine immunohistochemistry (n = 10). The cell migration was examined using the Dil tracer technique (n = 40) at the end of weeks 6, 10, 14 and 18. Affymetrix microarray analysis was used to investigate differential gene expressions in peripheral and central RPE cells, which was authenticated by RT-PCR using 4 RPE-specific genes (n = 10). Results: In this study, peripheral RPE cells divided and appeared in clusters, but equatorial and central RPE cells rarely divided. The peripheral RPE cells migrated to the central RPE region in a time-dependent manner up to the end of week 14, but not later. The microarray analysis showed the expression of 9,645 out of a total of 35,220 genes studied. Among the 9,645 genes, 573 were differentially expressed (438 were upregulated and 135 were downregulated) in peripheral RPE cells as compared to central RPE cells. Of these 573 genes, 56 were associated with signaling pathways related to the regulation of cell proliferation, including Pax6, TGFβ, BMP and Wnt, and 404 were associated with pathways of cell migration. Conclusions: In this study, peripheral RPE cells divided and migrated to the central region. This process was associated with differential gene expressions in these cells.

Rhubarb extract partially improves mucosal integrity in chemotherapy-induced intestinal mucositis.

AIMTo investigate the effects of orally gavaged aqueous rhubarb extract (RE) on 5-fluorouracil (5-FU)-induced intestinal mucositis in rats.METHODSFemale Dark Agouti rats (n = 8/group) were gavaged daily (1 mL) with water, high-dose RE (HDR; 200 mg/kg) or low-dose RE (LDR; 20mg/kg) for eight days. Intestinal mucositis was induced (day 5) with 5-FU (150 mg/kg) via intraperitoneal injection. Intestinal tissue samples were collected for myeloperoxidase (MPO) activity and histological examination. Xenopus oocytes expressing aquaporin 4 water channels were prepared to examine the effect of aqueous RE on cell volume, indicating a potential mechanism responsible for modulating net fluid absorption and secretion in the gastrointestinal tract. Statistical significance was assumed at P < 0.05 by one-way ANOVA.RESULTSBodyweight was significantly reduced in rats administered 5-FU compared to healthy controls (P < 0.01). Rats administered 5-FU significantly increased intestinal MPO levels (≥ 307%; P < 0.001), compared to healthy controls. However, LDR attenuated this effect in 5-FU treated rats, significantly decreasing ileal MPO activity (by 45%; P < 0.05), as compared to 5-FU controls. 5-FU significantly reduced intestinal mucosal thickness (by ≥ 29% P < 0.001) as compared to healthy controls. LDR significantly increased ileal mucosal thickness in 5-FU treated rats (19%; P < 0.05) relative to 5-FU controls. In xenopus oocytes expressing AQP4 water channels, RE selectively blocked water influx into the cell, induced by a decrease in external osmotic pressure. As water efflux was unaltered by the presence of extracellular RE, the directional flow of water across the epithelial barrier, in the presence of extracellular RE, indicated that RE may alleviate water loss across the epithelial barrier and promote intestinal health in chemotherapy-induced intestinal mucositis.CONCLUSIONIn summary, low dose RE improves selected parameters of mucosal integrity and reduces ileal inflammation, manifesting from 5-FU-induced intestinal mucositis.

Towards an elastin-based hydrogel platform for the repair of myocardial infarction

Event Abstract Back to Event Towards an elastin-based hydrogel platform for the repair of myocardial infarction Paolo Contessotto1, Gesmi Milcovich1*, Renza Spelat1*, Grazia Marsico1*, Ankit Chaturvedi2*, Doriana Orbanić3*, Tatjana Flora3*, Mark Da Costa1, 4, Mindaugas Rackauskas5*, Heinz Amenitsch6*, Mike Mccartin2*, José Carlos Rodriguez-Cabello3*, Lokesh Joshi7*, Peter Dockery8*, Michelle Kilcoyne7, 9* and Abhay Pandit1 1 National University of Ireland, CURÁM, Centre for Research in Medical Devices, Ireland 2 Vivasure Medical Limited, Ireland 3 Universidad de Valladolid, Ciber-BBN, G.I.R. Bioforge, Spain 4 University College Hospital Galway, Ireland 5 Mater Misericordiae University Hospital, Ireland 6 Institut of Inorganic Chemistry, Graz University of Technology, Austria 7 National University of Ireland, Glycoscience Group, Ireland 8 National University of Ireland, Anatomy, School of Medicine, Ireland 9 National University of Ireland, Microbiology, School of Natural Sciences, Ireland Introduction Elastin is one of the main components of the extracellular matrix (ECM) and has been investigated as a potential therapeutic biomaterial in the treatment of several diseases, such as myocardial infarction [1]. The intrinsic physical elastic properties of elastin-like recombinamers and their interaction with relevant glycosylation in the ECM can be exploited by developing a tailored biomaterial. The objectives of this study are to fabricate an injectable elastin-based hydrogel based on the glycoprofile in normal and ischemic cardiac muscle. We hypothesize that a naturally-inspired glyco-functionalised injectable elastin-like hydrogel will preserve cardiac functionality by the enhancement of cardiomyocyte proliferation. Materials and methods Hydrogel fabrication: Elastin-like recombinamers (ELRs) were functionalised with azide and cyclooctyne groups for crosslinking. The hydrogel construct was characterized by rheology, NMR, and Synchrotron Radiation SAXS (SRSAXS). Moreover, further degradation studies were conducted using elastase (Sigma). The hydrogel was stained with 1% Alcian Blue solution before epicardial injection in the myocardium of a healthy lamb heart. Glycoprofile analysis: Lectin histochemistry and lectin microarray analyses were performed on left ventricle cryosections from healthy male Lewis and Dark Agouti rats (n=5). Myocardial infarction in vivo models: Induction of myocardial infarction is being performed on Romanov sheep (n=7) to better mimic human conditions [2], while cryoinjury method is adopted as a method to reproduce the ischemic event in Danio rerio [3]. All animals were sacrificed after approval by the respective Institutional Ethics Committee. Statistical analysis was performed using Minitab Software. Results and Discussion ELRs-based hydrogel was fabricated and optimized; the optimal gelling time before injection was found between 10 and 12 minutes at room temperature and its successful integration across the cardiomyocytes confirmed by hematoxylin and eosin (H&E) and Wheat Germ Agglutinin (WGA) lectin staining, confirming its potential application for in vivo studies [4]. Glycoprofile analysis was carried out in healthy myocardial left ventricle rat tissue, using a panel of lectins which bind to specific glycan residues that are involved in cardiac signalling and functionality, such as sialic acids (Neu) and N-acetylglucosamine (GlcNAc) [5]. Distinct patterns of binding due to the differential glycosylation in capillaries and cardiomyocytes membranes were found by lectin histochemistry analysis and their presence further validated by lectin microarray technology. Conclusion An ELRs-based hydrogel was optimised for injection through the myocardial walls. The assessment of the glyco-profiles in healthy myocardial tissue was completed to have a baseline before further analysis in the ischemic samples. Glycoprofile comparative analysis between a non-regenerative and a self-regenerative model in Danio rerio can further enable us to tailor our system towards a regenerative system. Seventh Framework Programme Grant Agreement no.: 317304; Centre for Microscopy & Imaging at the National University of Ireland Galway funded by Irish Government’s Programme for Research in Third Level Institutions, Cycles 4 and 5, National Development Plan 2007-2013

Multiple rodent models and behavioral measures reveal unexpected responses to FTY720 and DMF in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a widely-used rodent model for multiple sclerosis (MS), but a single model can hardly capture all features of MS. We investigated whether behavioral parameters in addition to clinical motor function scores could be used to assess treatment efficacy during score-free intervals in the relapsing-remitting EAE model in SJL/J mice. We studied the effects of the clinical reference compounds FTY720 (fingolimod, 0.5mg/kg/day) and dimethyl fumarate (DMF, 20–30mg/kg/day) on clinical scores in several rodent EAE models in order to generate efficacy profiles. SJL/J mice with relapsing-remitting EAE were studied using behavioral tests, including rotarod, gait analysis, locomotor activity and grip strength. SJL/J mice were also examined according to Crawley’s sociability and preference for social novelty test. Prophylactic treatment with FTY720 prevented clinical scores in three of the four EAE rodent models: Dark Agouti (DA) and Lewis rats and C57BL/6J mice. Neither prophylactic nor late-therapeutic treatment with FTY720 reduced clinical scores or reversed deficits in the rotarod test in SJL/J mice, but we observed effects on motor functions and sociability in the absence of clinical scores. Prophylactic treatment with FTY720 improved the gait of SJL/J mice whereas late-therapeutic treatment improved manifestations of reduced social (re)cognition or preference for social novelty. DMF was tested in three EAE models and did not improve clinical scores at the dose used. These data indicate that improvements in behavioral deficits can occur in absence of clinical scores, which indicate subtle drug effects and may have translational value for human MS.

Electrical signal propagated across acupoints along Foot Taiyang Bladder Meridian in rats.

To investigate the electrical signals propagated along Foot Taiyang Bladder Meridian (BL) in a rat model. The experiments were performed on Dark-Agouti (DA), DA.1U and Sprague Dawley (SD) rats. The antidromic electrical stimulation was applied on the nerve innervating "Pishu" (BL 20) to mimic the acupoint electro-acupuncture (EA). The activities recording from adjacent nerve innervating acupoint "Danshu" (BL 19) or "Weishu" (BL 21) were recorded as indics for acupoint, including the mechanical threshold and discharge rate. After mimic EA on BL 20, C and Aδ units from adjacent BL 19 or BL 21 were sensitized including the decrease in mechanical threshold and increase in discharge rates in DA, DA.1U and SD rats, especially in DA rats. The average discharge rate increased from 2.40±0.26 to 6.06±0.55 and from 1.92±0.42 to 6.17±1.10 impulse/min (P<0.01), and the mechanical threshold decreased from 0.52±0.12 to 0.24±0.05 and from 0.27±0.02 to 0.16±0.01 mmol/L (P<0.01) in C (n=15) and Aδ (n=18) units in DA rats. The net change in discharge rates from C units were 152.5%, 144.7% and 42.4% in DA, DA.1U and SD rats, respectively, among which DA rat's was the highest (P<0.05). In Aδ units, the net change in DA rats were also the highest (221.5%, 139.2% and 49.2% in DA, DA.1U and SD rats). These results showed that mimic acupoint EA activated adjacent acupoints along BL in three rat strains, which might be related to propagated sensation along meridians (PSM). In addition, DA rats were more sensitive and might be a good model animal for PSM research.