Vena Cava Of Rats Research Articles

Abstract 2092: Ultrasound And Histology Protocol For Characterization Of Venous Stents Effect In Deep Veins In Vivo

Background: Deep venous stenting is a common strategy to treat deep vein thrombosis (DVT) decreasing future symptoms and complications. We propose a histopathological characterization of stents deployed in the inferior vena cava of rats and demonstrate how it correlates with ultrasound evaluation. Methods: Sprague Dawley rats (643±62g) were anesthetized (isoflurane). IVC diameter was measured by ultrasound on B mode. Through a midline laparotomy, infrarenal IVC side branches were ligated, posterior venous branches cauterized, micro-clips temporarily placed, and a U-stich is placed in the IVC. Using retrograde canulation of the IVC a sharpened guidewire with a stent sheath backloaded is inserted through the U-stich. The stent sheath is advanced with enough running room to deploy the venous self-expanding stent. After removal, U-stitch was tightened for hemostasis. No anticoagulation was used. Venous flow and patency were measured using a color/pulsed-wave Doppler 10-mHz probe post-procedure (PO) and 24 hours after (POD1). Vein wall thickness (VWT), in-stent-restenosis (ISR), and residual thrombosis percentage were quantified using trichrome staining at 7-, 14-, and 21-days. Results: Complete stent patency was observed in 50% of animals, partial patency in 15%, and obstruction in 35% after insertion. Table I. Recanalization at 1-day improved flow was observed in 41% of obstructed or partially patent stents. ISR was 6.2±3.8% of the luminal stent area (LSA), residual thrombosis in 29.9±23%, VWT mean 80±13.5 μm, no statistical differences found between timepoints. Figure 1. Conclusion: Characterization and follow-up of deep vein stents can be achieved by ultrasound and histology. This viable animal model can identify human pathological stent processes as early thrombosis, resolution, recanalization, and ISR. This model can reduce costs for feasibility studies to test novel stents and simulate real-life patency outcomes at multiple time points.

Silver Ion Decreases Foreign Body Reaction and Venous Neointimal Hyperplasia through the Inhibition of Interleukin-33 Expression

Introduction: Vascular prosthetic grafts are widely used in vascular surgery; however, graft infection remains a major concern. Silver-coated vascular grafts have demonstrated anti-infection properties in clinical settings; however, whether the silver irons influence foreign body reaction or neointimal hyperplasia remains unclear. Methods: Sodium alginate and hyaluronic acid (SA/HA) hydrogel patches loaded with rhodamine, with or without silver, were fabricated. Patches were implanted in the subcutaneous or abdominal cavity and inferior vena cava of rats. Samples were harvested on day 14 and examined via immunohistochemical and immunofluorescence analyses. Results: Silver hydrogel was found to decrease the foreign body reaction; after subcutaneous and abdominal cavity implantation in rats, the capsule was found to be thinner in the silver hydrogel group than in the control hydrogel group. The silver hydrogel group had fewer CD68-positive cells and proliferating cell nuclear antigen and interleukin-33 (IL-33) dual-positive cells than the control hydrogel group. Additionally, the silver hydrogel patch reduced the neointimal thickness after patch venoplasty in rats, and the number of IL-33- and IL-1β-positive cells was lower than that in the control patch. Conclusion: Silver-loaded SA/HA hydrogel patches decreased the foreign body reaction and venous neointimal hyperplasia in rats by the inhibition of IL-33 expression.

THE INVESTIGATION OF THE EFFECTS OF VITAMIN D AND OMEGA 3 ON VENA CAVA

Objective: Vitamin D has a protective role in the cardiovascular system and it affects blood pressure. Omega-3 fatty acids are dietary fats gained from fish and plant oils and involve in coronary heart disease and other cardiovascular complications. The aim of the study was to investigate the effects of Vitamin D and Omega 3 on the vascular structure at the cellular level.
 
 Material and Methods: In the current study, a total of 24 rats were divided into 4 groups. Each group contained 6 animals. The groups are as follows; control, vitamin D, Omega 3; and combined Vitamin D and Omega 3. Vena cava samples from all groups were obtained and stained with hematoxylin and eosin (H&E) for histological alternations. Additionally, endothelial and vascular functions were investigated immunohistochemically.
 
 Results: The H&E staining revealed that the treatment of either Vitamin D or Omega 3 did not cause histomorphological changes in the structure of the vena cava under normal conditions. The immunoexpression of inducible nitric oxide synthase was decreased and vascular endothelial growth factor was increased in the vena cava of rats with the combined treatment of Vitamin D and Omega 3.
 
 Conclusion: In conclusion, combined supplements of Vitamin D and Omega 3 did not have harmful effects on the blood vessel however further studies should be performed to determine the beneficial effects of these supplements.

Antithrombotic Activity of an Indolinone Derivative – a Soluble Guanylate Cyclase Stimulator

Introduction . The article presents the results of studying the antithrombotic activity of a novel drug, a soluble guanylate cyclase stimulator (codename – GRS), in experimental models of arterial and venous thrombosis and thromboembolism. Aim . Study the antithrombotic action of GRS compound in comparison with clopidogrel and rivaroxaban in the following models: arterial thrombosis induced by iron chloride application to carotid artery wall in rats; thromboembolism induced by intravenous administration of thrombin solution in mice; venous thrombosis induced by the ligature of inferior vena cava in rats. Materials and methods . Arterial thrombosis was modelled in rats by applying a pad soaked in iron chloride (FeCl 3 ) to the carotid artery, GRS compound was administered orally in median effective dose of 10 mg/kg once 3 hours before pad application. Blood flow in carotid arteries and blood clot mass were registered. Thromboembolism was induced in mice by intravenous administration of thrombin solution, GRS in dose 10 mg/kg or the reference drug clopidogrel were administered once orally daily for 3 days. Animal mortality, survival time and blood clot size were registered. Venous thrombosis was induced in rats by the ligature of inferior vena cava below renal veins, GRS in dose 10 mg/kg, reference drug rivaroxaban in 5 mg/kg dose or their combination in these doses were administered once orally 1 hour before vein ligature. The mass of dry and wet blood clots was registered. Results and discussion . In arterial thrombosis model GRS compound in 10 mg/kg, administered 3 hours before iron chloride application, increased the time to blood flow cessation in the carotid artery by 35 % and reduced the frequency of complete artery occlusion by 2 times compared to the control group ( р < 0.05). 60 min after arterial thrombosis modelling complete occlusion was observed in 28 % of animals in GRS group and in 75 % of control animals, while after 24 hours the occlusion was observed in 14 % of animals in GRS group and in 50 % of control group animals ( p < 0.05). In thrombin-induced thromboembolism model in mice GRS did not reduce the size of blood clots in pulmonary vessels, while clopidogrel reduced it by 48 %. In GRS group 80 % of animals died of thrombosis, compared to 30 % in clopidogrel group and 90 % in the control group. In ligature-induced venous thrombosis in rats GRS after single oral administration reduced the mass of dry blood clot, being as potent as rivaroxaban. Combined administration of GRS and rivaroxaban did not enhance their antithrombotic action. Conclusion . GRS after single oral administration in 10 mg/kg dose had potent antithrombotic action in models of arterial and venous thrombosis in rats, while not preventing pulmonary vessel thrombosis in mice after thrombin administration. Therapeutic action of GRS in experimental venous thrombosis was as potent as that of rivaroxaban. Antithrombotic action of GRS compound results not only from its antiplatelet action, but also from the alleviation of endothelial dysfunction in arteries and veins.

Downregulation of microRNA-200c-3p alleviates the aggravation of venous thromboembolism by targeting serpin family C member 1

ABSTRACT Venous thromboembolism (VTE) is the third most prevalent cardiovascular complication. Increasing studies have demonstrated that some microRNAs (miRNAs) are aberrantly expressed in VTE and play crucial roles in mediating the development of VTE. Therefore, our study intends to explore the detailed function and molecular mechanism of miR-200c-3p in VTE progression. In our research, VTE rat models were first established via inferior vena cava (IVC) ligation and the time-dependent effects of IVC ligation on thrombus formation were discovered. The results of reverse transcription quantitative polymerase-chain reaction (RT-qPCR) and western blotting showed that serpin family C member 1 (SERPINC1) was downregulated in VTE rat models and showed an inverse correlation with thrombus load. MiRNA target prediction tools and luciferase reporter assay confirmed SERPINC1 as a target for miR-200c-3p. VTE rats were injected with miR-200c-3p inhibitor for 24 h to investigate whether miR-200c-3p knockdown influences thrombus formation in vivo. Histological examination through hematoxylin-eosin staining revealed that miR-200c-3p downregulation markedly inhibited the formation of thrombus in IVC of rats. Additionally, miR-200c-3p was upregulated while SERPINC1 was downregulated in serum and inferior vena cava of VTE rats as well as in plasma of patients with VTE. Linear regression analysis demonstrated that miR-200c-3p expression was negatively correlated to SERPINC1 expression in VTE rats and patients with VTE. Our study determines the previously unelucidated function of miR-200c-3p in VTE, which might provide a potential novel insight for the treatment of VTE.

In vivo magnetic particle imaging: angiography of inferior vena cava and aorta in rats using newly developed multicore particles

Magnetic Particle Imaging (MPI) is a new imaging modality, which maps the distribution of magnetic nanoparticles (MNP) in 3D with high temporal resolution. It thus may be suited for cardiovascular imaging. Its sensitivity and spatial resolution critically depend on the magnetic properties of MNP. Therefore, we used novel multicore nanoparticles (MCP 3) for in-vivo MPI in rats and analyzed dose requirements, sensitivity and detail resolution. 8 rats were examined using a preclinical MPI scanner (Bruker Biospin GmbH, Germany) equipped with a separate receive coil. MCP 3 and Resovist were administered intravenously (i.v.) into the rats’ tail veins at doses of 0.1, 0.05 and 0.025 mmol Fe/kg followed by serial MPI acquisition with a temporal resolution of 46 volumes per second. Based on a qualitative visual scoring system MCP 3–MPI images showed a significantly (P ≤ 0.05) higher image quality than Resovist-MPI images. Morphological features such as vessel lumen diameters (DL) of the inferior vena cava (IVC) and abdominal aorta (AA) could be assessed along a 2-cm segment in mesenteric area only after administration of MCP 3 at dosages of 0.1, 0.05 mmol Fe/kg. The mean DL ± SD estimated was 2.7 ± 0.6 mm for IVC and 2.4 ± 0.7 mm for AA. Evaluation of DL of the IVC and AA was not possible in Resovist-MPI images. Our results show, that MCP 3 provide better image quality at a lower dosage than Resovist. MCP 3-MPI with a clinically acceptable dose of 0.05 mmol Fe/kg increased the visibility of vessel lumens compared to Resovist-based MPI towards possible detection of vascular abnormalities such as stenosis or aneurysms, in vivo.

Neointimal hyperplasia in the inferior vena cava of adenine-induced chronic kidney disease rats with aortocaval fistulas

The failure of autologous arteriovenous fistulas (AVFs) occurs primarily due to stenosis in the anastomotic site, which is mainly related to the development of neointimal hyperplasia (NIH). Therefore, we conducted a study to establish a novel approach to create aortocaval fistulas (ACFs) in adenine-induced (AD) chronic kidney disease (CKD) rats to study the NIH in the inferior vena cava. Ten adult female rats received a 0.75% adenine-rich diet for 4weeks to induce CKD and underwent ACF surgery. Ten healthy rats served as controls. A 5-10-mm segment of a vein immediately adjacent to that the portion of the vein used for creating the fistula was surgically removed at the time of creating the fistula, and reconstruction of the failed fistula from the same patient was used as controls. ACF was assessed using duplex scans and histopathological analyses. At the end of the experiment, AD rats showed higher serum creatinine and urea nitrogen than those of vehicle-treated rats. Remarkable histological changes in kidney tissues demonstrated successful CKD models. Sections of the ACF in AD rats and veins removed at the time of the reconstruction of the failed fistula of the patient demonstrated that the eccentric neointima formation is irregularly thickened, with several small vessels within a more cellular region of the neointima. Immunohistochemistry demonstrated the presence of myofibroblasts, contractile smooth muscle cells and macrophages within the neointima. Our rat models with ACFs showed typical features of NIH in the formation of fistula stenosis, which can resemble clinical findings in uremic patients.

α-Lipoic acid exerts a primary prevention for the cardiac dysfunction in aortocaval fistula-created rat hearts

Aimα-Lipoic acid exerts a powerful antioxidant effect by acting as a free radical scavenger and inducing endogenous antioxidants such as vitamin E and glutathione. In the present study, we examined the effects of α-lipoic acid on cardiac dysfunction in rat hearts with aortocaval fistulae. Main methodsAortocaval fistulae were created between the abdominal aorta and inferior vena cava in male rats. Hemodynamic parameters were measured 14 days after surgery using an intravascular pressure transducer, and then these hearts were harvested for tissue weight measurement, pathological evaluation, and mRNA isolation. ResultsIn vehicle-treated rats, left ventricular end-diastolic pressure and left ventricular weight significantly increased at 14 days after fistula creation. Fistula-creation resulted in expression of 4-hydroxy-2-nonenal, NADPH oxidase subunit p67phox and BNP mRNA in a time-dependent manner in the left ventricle.Long-term treatment (initiated 2 days before surgery, and continued for 14 days after fistula creation; days -2 to 14) with α-lipoic acid (30 mg/kg/day) markedly suppressed the increases in left and right ventricular weight, and left ventricular end-diastolic pressure. α-Lipoic acid treatment from days -2 to 14 prominently prevented the expression of 4-hydroxy-2-nonenal and NADPH oxidase subunit p67phox, and significantly raised BNP mRNA levels. Short-term treatment with α-lipoic acid from day - 2 to 7 was effective in preventing cardiac enlargement and dysfunction, similar to long-term treatment, but treatment from days 7–14 was not effective. ConclusionsTreatment with α-lipoic acid can prevent cardiac hyperplasia and dysfunction, probably by inhibiting superoxide production and enhancing BNP mRNA expression in an early phase after fistula creation.

Significance of malondialdehyde, superoxide dismutase and endotoxin levels in Budd-Chiari syndrome in patients and a rat model.

Budd-Chiari syndrome (BCS) is a rare clinical syndrome caused by the obstruction of hepatic venous outflow. In theory, hepatic congestion and hypoxia induce pathological damage and changes in the liver. However, at present, laboratory evidence supporting this theory is lacking. The aim of the present study was to assess the expression and significance of the hypoxia-associated indicators malondialdehyde (MDA), superoxide dismutase (SOD) and endotoxin (ET) in the liver and serum of subjects with BCS. An animal model of BCS was established by partial ligation of the inferior vena cava (IVC) in rats. The levels of MDA, SOD and ET in the serum of BCS patients, as well as in the liver and serum of rats with BCS, were detected and analyzed. In human patients with BCS, the serum levels of MDA, ET and SOD were significantly different from those in healthy control subjects. In the animal model, similar trends were observed regarding the MDA, ET and SOD levels in liver homogenate and serum (P<0.05), the degree of which was more pronounced in the liver homogenate than in the serum. At 6 weeks after the surgery, these indicators reached peak/valley levels in the experimental group and were at least partially restored by week 12. A negative correlation between MDA and SOD, a positive correlation between MDA and ET, and a negative correlation between SOD and ET was identified. In conclusion, the levels of hypoxia-associated indicators significantly changed with BCS progression, suggesting that hypoxia is a major factor in the pathogenesis of BCS.

Decellularized Carotid Artery Functions as an Arteriovenous Graft

BackgroundProsthetic arteriovenous grafts (AVG) continue to have a high rate of failure in clinical use, yet there is continued clinical demand for them. However, there is no small animal model of AVG to test novel tissue-engineered vascular grafts. We established a new rat arteriovenous graft model to compare the healing of decellularized carotid artery (CA) to autologous CA. Materials and methodsThe infrarenal vena cava and aorta of Wistar rats were exposed and dissected free below renal artery. A longitudinal 1 mm venotomy and arteriotomy were made on the anterior walls. The conduit was either autologous CA or heterologous decellularized CA; a conduit was sewn to the inferior vena cava and aorta in end-to-side fashion. Rats were sacrificed on postoperative day 21 for examination. ResultsAll rats survived without heart failure. Conduits had 100% patency rate (day 21) in both the control and decellularized CA groups (n = 6). Both control and decellularized CA showed similar rates of reendothelialization, inflammatory cell infiltration, and cell turnover. The outflow vein beyond the autologous or decellularized conduits showed similar neointimal thickness and cell turnover. ConclusionsDecellularized CA may be a viable tissue engineering graft for use as an arteriovenous graft for dialysis access. The rat aorta-vena cava graft is a useful model to test new materials including tissue-engineered grafts for use as AVG.

Study on Urinary Metabolic Profile in Rats with Deep Venous Thrombosis Based on Pattern Recognition

To study the urinary metabolic profile in rats with deep venous thrombosis （DVT） based on metabolomics and to screen out small molecular biomarkers for the diagnosis and forensic identification of DVT. Inferior vena cava of rats was ligated to construct DVT models. The rats were randomly divided into three groups： DVT, sham, and control groups, 10 in each group. The urine of DVT and sham rats was collected during 24 hours in the metabolic cage at 48 hours after operating, meanwhile, 24 hours urine was collected in control group. The metabolic profile was analyzed by nuclear magnetic resonance. SIMCA-P 14.1 software was used for pattern recognition. The variable importance in projection （VIP） value from orthogonal PLS-DA （OPLS-DA） model combined with Mann-Whitney U test were used to search the different metabolites in the urine. The metabolic profiles of urine from DVT, sham, and control groups had significant differences. The DVT, sham, and control groups could be distinguished by the partial least squares method-discriminant analysis （PLS-DA） model. Compared with the urine of the rats in control groups, the levels of leucine, glutamine, creatine, creatinine and sucrose in the urine of DVT rats were up-regulated, and the levels of 3-hydroxybutyrate, lactate, acetone, α-oxoglutarate, citrate and hippurate were down-regulated. The different metabolites in the urine of DVT rats are expected to become its candidate biomarkers. The results can provide a research basis for the diagnosis, treatment and forensic identification of DVT.

Natriuretic peptides system in the pulmonary tissue of rats with heart failure: potential involvement in lung edema and inflammation.

Congestive heart failure (CHF) often leads to progressive cardiac hypertrophy and salt/water retention as evident by peripheral and lung edema. Although the pathogenesis of CHF remains largely unclarified, it is widely accepted that neurohormonal changes and inflammatory processes are profoundly involved in structural and functional deterioration of vital organs including, heart, kidney and lungs. Corin, a cardiac serine protease, is responsible for converting pro-ANP and pro-BNP to biologically active natriuretic peptides (NPs). Although the involvement of corin in cardiac hypertrophy and heart failure was extensively studied, the alterations in corin and PCSK6, a key enzyme in the conversion of procorin to corin, have not been studied in the pulmonary tissue. Thus, this study aims at examining the status of PCSK6/Corin in the lung of rats with CHF induced by the creation of aorto-caval fistula (ACF) between the abdominal aorta and vena cava in SD rats. Rats with ACF were divided into 2 subgroups based on the pattern of their daily sodium excretion, compensated and decompensated CHF. Placement of ACF led to cardiac hypertrophy, pulmonary congestion, and renal dysfunction, which were more severe in the decompensated subgroup, despite remarkable elevation of circulatory ANP and BNP levels. Corin mRNA and immunoreactive peptide were detected in pulmonary tissue of all experimental groups. However, the expression and abundance of pulmonary corin significantly increased in the decompensated animals, but not in the compensated ones. Noteworthy, the expression of PCSK6 and ANP/BNP in the pulmonary tissue followed a similar pattern as corin. The upregulation of pulmonary Corin/PCSK6 and NPs were accompanied by local activation of cathepsin L and certain cytokines including IL-6. In light of the anti-inflammatory role of NPs, we postulate that the obtained upregulation of pulmonary PCSK6/Corin along NPs in rats with decompensated CHF may represent a counterbalance response to the inflammatory milieu characterizing CHF especially in severe cases.

MicroRNA-30c suppresses the pro-fibrogenic effects of cardiac fibroblasts induced by TGF-β1 and prevents atrial fibrosis by targeting TGFβRII.

Atrial fibrosis serves as an important contributor to atrial fibrillation (AF). Recent data have suggested that microRNA‐30c (miR‐30c) is involved in fibrotic remodelling and cancer development, but the specific role of miR‐30c in atrial fibrosis remains unclear. The purpose of this study was to investigate the role of miR‐30c in atrial fibrosis and its underlying mechanisms through in vivo and in vitro experiments. Our results indicate that miR‐30c is significantly down‐regulated in the rat abdominal aortic constriction (AAC) model and in the cellular model of fibrosis induced by transforming growth factor‐β1 (TGF‐β1). Overexpression of miR‐30c in cardiac fibroblasts (CFs) markedly inhibits CF proliferation, differentiation, migration and collagen production, whereas decrease in miR‐30c leads to the opposite results. Moreover, we identified TGFβRII as a target of miR‐30c. Finally, transferring adeno‐associated virus 9 (AAV9)‐miR‐30c into the inferior vena cava of rats attenuated fibrosis in the left atrium following AAC. These data indicate that miR‐30c attenuates atrial fibrosis via inhibition of CF proliferation, differentiation, migration and collagen production by targeting TGFβRII, suggesting that miR‐30c might be a novel potential therapeutic target for preventing atrial fibrosis.

Changes in hydrogen sulfide in rats with hepatic cirrhosis in different stages.

This study aimed to observe changes in the hydrogen sulfide (H2S) system in the blood and liver tissue of rats with hepatic cirrhosis at different stages by studying the effect of H2S on the course of hyperdynamic circulation in rats with hepatic cirrhosis. H2S concentration in the blood from the portal vein and inferior vena cava of hepatic cirrhosis rat model induced with carbon tetrachloride was detected on the 15th, 30th, and 52nd day. The expression of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) protein, and CBS and CSE mRNA in the liver was detected by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. The results indicated that H2S concentration in the blood from the portal vein and inferior vena cava of rats with hepatic cirrhosis was significantly lower than that in the control group. H2S was gradually decreased with the development of the disease and significantly lower in the blood from portal vein than in the blood of inferior vena cava at the mid-stage and the late stage groups. The expression levels of CBS and CSE protein, and CBS and CSE mRNA in the livers with hepatic cirrhosis at different stages were all higher than those in the control group, and the expression gradually increased with the development of the disease. The expression of CBS was lower than CSE in the same stages. The results indicated that the CSE mRNA was expressed predominantly in the cirrhosis groups as compared with CBS mRNA. Among experimental rats, the H2S system has an important effect on the occurrence and development of hyperdynamic circulation in rats with hepatic cirrhosis. This finding adds to the literature by demonstrating that H2S protects vascular remodelling in the liver, and that CSE is indispensable in this process.

Integrating microRNA and messenger RNA expression profiles in a rat model of deep vein thrombosis.

Deep vein thrombosis (DVT) is a disease involving multiple genes and systems. MicroRNAs (miRNAs) represent a class of non-coding small RNAs that post-transcriptionally suppress their target genes. The expression patterns of miRNA and messenger RNA (mRNA) in DVT remain poorly characterized. The aim of the present study was to evaluate miRNA and mRNA expression profiles in a stasis-induced DVT rat model. Male SD rats were randomly divided into three groups as follows: DVT, sham and control. The inferior vena cava (IVC) of rats was ligated to construct stasis-induced DVT models. Rats were sacrificed three days after ligation, and morphological changes in the vein tissues were observed by hematoxylin and eosin and Masson staining. The miRNA and mRNA expression profiles were evaluated by microarrays, followed by bioinformatics analysis. The microarray analysis identified 22 miRNAs and 487 mRNAs that were significantly differentially expressed between the experimental and control groups, and between the experimental and sham groups, but not between the control and sham groups (P≤0.05; ≥2.0-fold change). By subsequent bioinformatics analysis, a 19 miRNA-98 mRNAs network was constructed in the stasis-induced DVT rat model. Notably, the majority of these miRNAs and mRNAs are reported to be expressed by endothelial cells (ECs) and are associated with the function of ECs. The results provide evidence indicating that the regulatory association of miRNA and mRNA points to key roles played by ECs in thrombosis. These findings advance our understanding of the molecular regulatory mechanisms underlying the pathophysiology of DVT.

A cross-circulated bicephalic model of head transplantation.

A successful cephalosomatic anastomosis ("head transplant") requires, among others, the ability to control long-term immune rejection and avoidance of ischemic events during the head transference phase. We developed a bicephalic model of head transplantation to study these aspects. The thoracic aorta and superior vena cava of a donor rat were anastomosed with the carotid artery and extracorporeal veins of a recipient rat by vascular grafts. Before thoracotomy in the donor rat, the axillary artery and vein of the donor were connected to the carotid and the extracranial vein of the third rat through a silicone tube. The silicone tube was passed through a peristaltic pump to ensure donor brain tissue blood supply. There is no ischemia reperfusion injury in donor brain tissue analyzed by electroencephalogram. Postoperative donor has pain reflex and corneal reflex. Peristaltic pump application can guarantee the blood supply of donor brain tissue per unit time, while the application of temperature change device to the silicone tube can protect the brain tissue hypothermia, postoperative experimental data show that there is no brain tissue ischemia during the whole operation. The application of vascular grafting can also provide the possibility of long-term survival of the model.

Tubular Cardiac Tissues Derived from Human Induced Pluripotent Stem Cells Generate Pulse Pressure In Vivo

Human induced pluripotent stem (iPS) cell-derived cardiac cells provide the possibility to fabricate cardiac tissues for transplantation. However, it remains unclear human bioengineered cardiac tissues function as a functional pump in vivo. Human iPS cells induced to cardiomyocytes in suspension were cultured on temperature-responsive dishes to fabricate cardiac cell sheets. Two pairs of triple-layered sheets were transplanted to wrap around the inferior vena cava (IVC) of nude rats. At 4 weeks after transplantation, inner pressure changes in the IVC were synchronized with electrical activations of the graft. Under 80 pulses per minute electrical stimulation, the inner pressure changes at 8 weeks increased to 9.1 ± 3.2 mmHg, which were accompanied by increases in the baseline inner pressure of the IVC. Immunohistochemical analysis revealed that 0.5-mm-thick cardiac troponin T-positive cardiac tissues, which contained abundant human mitochondria, were clearly engrafted lamellar around the IVC and surrounded by von Willebrand factor-positive capillary vessels. The mRNA expression of several contractile proteins in cardiac tissues at 8 weeks in vivo was significantly upregulated compared with those at 4 weeks. We succeeded in generating pulse pressure by tubular human cardiac tissues in vivo. This technology might lead to the development of a bioengineered heart assist pump.

Towards a Tissue-Engineered Contractile Fontan-Conduit: The Fate of Cardiac Myocytes in the Subpulmonary Circulation.

The long-term outcome of patients with single ventricles improved over time, but remains poor compared to other congenital heart lesions with biventricular circulation. Main cause for this unfavourable outcome is the unphysiological hemodynamic of the Fontan circulation, such as subnormal systemic cardiac output and increased systemic-venous pressure. To overcome this limitation, we are developing the concept of a contractile extracardiac Fontan-tunnel. In this study, we evaluated the survival and structural development of a tissue-engineered conduit under in vivo conditions. Engineered heart tissue was generated from ventricular heart cells of neonatal Wistar rats, fibrinogen and thrombin. Engineered heart tissues started beating around day 8 in vitro and remained contractile in vivo throughout the experiment. After culture for 14 days constructs were implanted around the right superior vena cava of Wistar rats (n = 12). Animals were euthanized after 7, 14, 28 and 56 days postoperatively. Hematoxylin and eosin staining showed cardiomyocytes arranged in thick bundles within the engineered heart tissue-conduit. Immunostaining of sarcomeric actin, alpha-actin and connexin 43 revealed a well -developed cardiac myocyte structure. Magnetic resonance imaging (d14, n = 3) revealed no constriction or stenosis of the superior vena cava by the constructs. Engineered heart tissues survive and contract for extended periods after implantation around the superior vena cava of rats. Generation of larger constructs is warranted to evaluate functional benefits of a contractile Fontan-conduit.

Abstract 16949: Failing Fontan: Genesis of a Subpulmonary Neo-ventricle From Engineered Heart Tissue

Introduction: Fontan palliation is the treatment of choice for patients with morphological or functional univentricular hearts. The unphysiologic and non-pulsatile pulmonary blood flow results in multiorgan complications and a poor long-term outcome. We evaluated graft survival and histomorphology of a pulsatile Fontan conduit generated from Engineered Heart Tissue (EHT) after implantation in a rat model. Hypothesis: We hypothesized that EHT matures and remains contractile in the setting of venous (Fontan-like) preload. Methods: EHT was generated from ventricular cardiomyocytes of neonatal Wistar rats, fibrinogen, thrombin and DMEM. After culture for 14 days constructs were implanted around the right superior vena cava of Wistar rats (n=12, 300-350 g). Immunosupression was achieved by daily subcutaneous injection of Cyclosporin A (25 mg/kg BW) and Methylprednisolone (2 mg/kg BW). MRI (Bruker) was used to assess condensation of EHTs in vivo. Animals were euthanized after 7, 14, 28 and 56 days postoperatively for histomorphological analysis. Transmission electron microscopy was used to evaluate sarcomeric integrity of cardiomyocytes within the construct. Results: In culture, EHTs started beating around day 8 and remained contractile in vivo throughout the experiment (d7=3/3, d14=2/3, d28=3/3, d56=2/3). All animals survived circumferential implantation of EHTs around the right SVC via a right thoracotomy. MRI (d14, n=3) revealed no constriction or stenosis of the SVC by the constructs. Hematoxylin and Eosin staining showed densely packed bundles of cardiomyocytes within the EHT conduit and intense vascularisation. Immunolabeling of actinin and connexin 43 indicated adequate maturation of cardiomyocytes after grafting around the right SVC in rats. Conclusions: EHTs placed around the superior caval vein of Wistar rats survive and contract for a considerable time after implantation. Histomorphology revealed a matured phenotype of grafted cardiomyocytes and an adequate vascularisation. The functional benefit of a contractile neo-ventricle to propel pulmonary blood flow in Fontan patients remains to be evaluated.

Renal Production, Uptake, and Handling of Circulating αKlotho.

αKlotho is a multifunctional protein highly expressed in the kidney. Soluble αKlotho is released through cleavage of the extracellular domain from membrane αKlotho by secretases to function as an endocrine/paracrine substance. The role of the kidney in circulating αKlotho production and handling is incompletely understood, however. Here, we found higher αKlotho concentration in suprarenal compared with infrarenal inferior vena cava in both rats and humans. In rats, serum αKlotho concentration dropped precipitously after bilateral nephrectomy or upon treatment with inhibitors of αKlotho extracellular domain shedding. Furthermore, the serum half-life of exogenous αKlotho in anephric rats was four- to five-fold longer than that in normal rats, and exogenously injected labeled recombinant αKlotho was detected in the kidney and in urine of rats. Both in vivo (micropuncture) and in vitro (proximal tubule cell line) studies showed that αKlotho traffics from the basal to the apical side of the proximal tubule via transcytosis. Thus, we conclude that the kidney has dual roles in αKlotho homeostasis, producing and releasing αKlotho into the circulation and clearing αKlotho from the blood into the urinary lumen.