Gene Expression of Inflammatory Cytokines in Major Organs by Extracorporeal Circulation.

(1) Background: Extracorporeal circulation (ECC) is indispensable for cardiac surgery. Despite the fact that ECC causes non-physiological damage to blood components, its pathophysiology has not been fully elucidated. In our previous study, we constructed a rat ECC system and observed a systemic inflammatory response during and after blood tests assessing ECC, while the damage per organ localization caused by ECC was not examined. In this study, we used a rat model to assess the gene expression of inflammatory cytokines in major organs during ECC. (2) Methods: The ECC system consisted of a membranous oxygenator, tubing line, and a small roller pump. Rats were divided into a SHAM (which received surgical preparation only, without ECC) group and an ECC group. Proinflammatory cytokines were measured using real-time PCR in major organs after ECC to evaluate local inflammatory responses in the organs. (3) Results: Interleukin (IL)-6 levels were significantly elevated in the ECC group compared to the SHAM group, especially in the heart and lungs. (4) Conclusions: This study suggests that ECC promotes organ damage and the inflammatory response, but the degree of gene expression of proinflammatory cytokines varies from organ to organ, suggesting that it does not uniformly cause organ damage.

Diabetic Pathophysiology Enhances Inflammation during Extracorporeal Membrane Oxygenation in a Rat Model.

Systemic inflammatory responses in patients undergoing extracorporeal membrane oxygenation (ECMO) contribute significantly to ECMO-associated morbidity and mortality. In recent years, the number of type 2 diabetes mellitus patients has increased, and the number of these patients undergoing ECMO has also increased. Type 2 diabetes mellitus is a high-risk factor for complications during ECMO. We studied the effects of ECMO on inflammatory response in a diabetic rat ECMO model. Twenty-eight rats were divided into 4 groups: normal SHAM group (normal rats: n = 7), diabetic SHAM group (diabetic rats: n = 7), normal ECMO group (normal rats: n = 7), and diabetic ECMO group (diabetic rats: n = 7). We measured the plasma levels of cytokines, tumor necrosis factor-α, and interleukin-6. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine (Cr), and liver-type fatty acid binding protein (L-FABP) were examined in the rat cardiopulmonary bypass model to ascertain organ damage. In addition, the lung wet-to-dry weight (W/D) ratio was measured as an index of pulmonary tissue edema. A pathologic evaluation of kidneys was conducted by hematoxylin-eosin (HE) and periodic-acid-methenamine-silver (PAM) staining. In the diabetic ECMO group, levels of cytokines, AST, ALT, LDH, and L-FABP increased significantly, reaching a maximum at the end of ECMO in comparison with other groups (p < 0.05). In addition, hematoxylin-eosin and periodic acid-methenamine-silver staining of renal tissues showed marked injury in the ECMO group (normal ECMO and diabetic ECMO groups). Furthermore, when the normal ECMO and diabetic ECMO groups were compared, severe organ injury was seen in the diabetic ECMO group. There was remarkable organ injury in the diabetic ECMO group. These data demonstrate that diabetes enhances proinflammatory cytokine release, renal damage, and pulmonary edema during ECMO in an animal model.

Establishment of a novel miniature veno-venous extracorporeal membrane oxygenation model in the rat.

Recently, veno-venous extracorporeal membrane oxygenation (V-V ECMO) has been commonly used in the world to support patients with severe respiratory failure. However, V-V ECMO is a new technology compared to veno-arterial extracorporeal membrane oxygenation and cardiopulmonary bypass, and there are few reports of basic research. Although continuing research is desired, clinical research that standardizes conditions such as patients' background characteristics is difficult. The purpose of this study was to establish a simple and stably maintainable miniature V-V ECMO model to study the mechanisms of the biological reactions in circulation during V-V ECMO. The V-V ECMO system consisted of an original miniature membrane oxygenator, polyvinyl chloride tubing line, and roller pump. The priming volume of this system was only 8 mL. Polyethylene tubing was used to cannulate the right femoral vein as the venous return cannula for the V-V ECMO system. A 16-G cannula was passed through the right internal jugular vein and advanced into the right atrium as the conduit for venous uptake. The animals were divided into 2 groups: SHAM group and V-V ECMO group. V-V ECMO was initiated and maintained at 50-60 mL/kg/min, and oxygen was added into the oxygenator during V-V ECMO at a concentration of 100% (pump flow:oxygen = 1:10). Blood pressure was measured continuously, and blood cells were measured by blood collection. During V-V ECMO, the blood pressure and hemodilution rate were maintained around 80 mm Hg and 20%, respectively. Hb was kept at >10 g/dL, and V-V ECMO could be maintained without blood transfusion. It was possible to confirm oxygenation of and carbon dioxide removal from the blood. Likewise, the pH was adequately maintained. There were no problems with this miniature V-V ECMO system, and extracorporeal circulation progressed safely. In this study, a novel miniature V-V ECMO model was established in the rat. A miniature V-V ECMO model appears to be very useful for studying the mechanisms of the biological reactions during V-V ECMO and to perform basic studies of circulation assist devices.

The apparition macrophage and Döderlein bacillus is negatively correlated in class I Papanicolaou smear: A morphological examination.

BACKGROUND: Nonspecific vaginitis, also known as Bacterial vaginosis, unlike genital candidiasis and trichomoniasis, is caused by microbiome breakdown. Döderlein's bacillus are gram-positive bacillus that form a microbiome, reproduce in the female vagina after gaining sexual maturity, secrete lactic acid, and prevent the growth of other vaginitis-causing bacteria. Clue cell are squamous epithelial cells with Gardnerella sp. attached to their cell surface. The presence of clue cell is one of the diagnostic criteria for nonspecific vaginitis. Additionally, although macrophages are reported to protect against candidal vaginitis, there are no reports of studies examining the association between macrophages and clue cell. MATERIALS AND METHODS: After re-staining 300 class I specimens by cervical cancer screening with Papanicolaou staining, the appearance of Döderlein's bacillus, macrophages, and clue cell was observed. RESULT: Age group and appearance of Döderlein's bacillus were negatively correlated. The rate of appearance of macrophages was positively correlated with the age group. In people aged 50 years or more, the appearance rate of clue cells was significantly lower in the macrophage appearance group than that in the non-appearance group. CONCLUSION: This study suggested that macrophages, and not Döderlein's bacillus, may play an important role in defense against nonspecific vaginitis.

PaO2 greater than 300 mmHg promotes an inflammatory response during extracorporeal circulation in a rat extracorporeal membrane oxygenation model.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is being increasingly used for mechanical support of respiratory and cardio-circulatory failure. An excessive systemic inflammatory response is observed during sepsis and after cardiopulmonary bypass (CPB) with similar clinical features. We hypothesized that hyperoxia condition encourages the systemic inflammatory response and organ disorder during ECMO. To prove this hypothesis correct, we investigated the systemic inflammatory responses at normal and high levels of arterial oxygen pressure (PaO2) in the rat ECMO model. METHODS: Rats were randomly assigned to one of the following groups depending on the value of PaO2 during ECMO: A group (n=11, PaO2 100-199 mmHg), B group (n=10, PaO2 200-299 mmHg), C group (n=8, PaO2 300-399 mmHg), and D group (n=11, PaO2 >400 mmHg). Serum cytokine levels [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10)] were measured before, 60, and 120 min after the initiation of ECMO. The wet-to-dry weight (W/D) ratio of the left lung was also measured, and dihydroethidium (DHE) staining, reflecting superoxide generation, of lung and liver tissues was performed 120 min after ECMO initiation. RESULTS: In the C and D groups, the pro-inflammatory cytokines (TNF-α and IL-6) significantly increased during ECMO compared with the other groups. On the other hand, the increase in anti-inflammatory cytokines (IL-10) was more suppressed in the C and D groups than in the other groups. The W/D ratio increased significantly more in the C and D groups than in the other groups. In addition, DHE fluorescence had a tendency to increase as the PaO2 rose. CONCLUSIONS: These data demonstrate that it is better to avoid administration of too much oxygen during ECMO to attenuate lung injury linked to generation of superoxide and the systemic inflammatory response.

Evaluation of Inflammation Caused by Cardiopulmonary Bypass in a Small Animal Model.

Extracorporeal circulation (ECC) methods are being increasingly used for mechanical support of respiratory and cardio-circulatory failure. Especially, cardiopulmonary bypass (CPB) during cardiovascular surgery, sustenance of the patient's life by providing an appropriate blood flow and oxygen supply to principal organs. On the other hand, systemic inflammatory responses in patients undergoing cardiovascular surgery supported by CPB contribute significantly to CPB-associated mortality and morbidity. Our previous research showed that CPB causes a systemic inflammatory response and organ damage in a small animal CPB model. We have been studying the effects of hyperoxia and blood plasma substitute on CPB. In this review, we present a study focusing on the systemic inflammatory response during CPB, along with our findings.

Ghrelin Pre-treatment Attenuates Local Oxidative Stress and End Organ Damage During Cardiopulmonary Bypass in Anesthetized Rats.

Cardiopulmonary bypass (CPB) induced systemic inflammation significantly contributes to the development of postoperative complications, including respiratory failure, myocardial, renal and neurological dysfunction and ultimately can lead to failure of multiple organs. Ghrelin is a small endogenous peptide with wide ranging physiological effects on metabolism and cardiovascular regulation. Herein, we investigated the protective effects of ghrelin against CPB-induced inflammatory reactions, oxidative stress and acute organ damage. Adult male Sprague Dawley rats randomly received vehicle (n = 5) or a bolus of ghrelin (150 µg/kg, sc, n = 5) and were subjected to CPB for 4 h (protocol 1). In separate rats, ghrelin pre-treatment (protocol 2) was compared to two doses of ghrelin (protocol 3) before and after CPB for 2 h followed by recovery for 2 h. Blood samples were taken prior to CPB, and following CPB at 2 h and 4 h. Organ nitrosative stress (3-nitrotyrosine) was measured by Western blotting. CPB induced leukocytosis with increased plasma levels of tumor necrosis factor-α and interleukin-6 indicating a potent inflammatory response. Ghrelin treatment significantly reduced plasma organ damage markers (lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase) and protein levels of 3-nitrotyrosine, particularly in the brain, lung and liver, but only partly suppressed inflammatory cell invasion and did not reduce proinflammatory cytokine production. Ghrelin partially attenuated the CPB-induced elevation of epinephrine and to a lesser extent norepinephrine when compared to the CPB saline group, while dopamine levels were completely suppressed. Ghrelin treatment sustained plasma levels of reduced glutathione and decreased glutathione disulphide when compared to CPB saline rats. These results suggest that even though ghrelin only partially inhibited the large CPB induced increase in catecholamines and organ macrophage infiltration, it reduced oxidative stress and subsequent cell damage. Pre-treatment with ghrelin might provide an effective adjunct therapy for preventing widespread CPB induced organ injury.

Novel temporary left ventricular assist system with hydrodynamically levitated bearing pump for bridge to decision: initial preclinical assessment in a goat model.

The management of heart failure patients presenting in a moribund state remains challenging, despite significant advances in the field of ventricular assist systems. Bridge to decision involves using temporary devices to stabilize the hemodynamic state of such patients while further assessment is performed and a decision can be made regarding patient management. We developed a new temporary left ventricular assist system employing a disposable centrifugal pump with a hydrodynamically levitated bearing. We used three adult goats (body weight, 58-68 kg) to investigate the 30-day performance and hemocompatibility of the newly developed left ventricular assist system, which included the pump, inflow and outflow cannulas, the extracorporeal circuit, and connectors. Hemodynamic, hematologic, and blood chemistry measurements were investigated as well as end-organ effect on necropsy. All goats survived for 30 days in good general condition. The blood pump was operated at a rotational speed of 3000-4500 rpm and a mean pump flow of 3.2 ± 0.6 L min. Excess hemolysis, observed in one goat, was due to the inadequate increase in pump rotational speed in response to drainage insufficiency caused by continuous contact of the inflow cannula tip with the left ventricular septal wall in the early days after surgery. At necropsy, no thrombus was noted in the pump, and no damage caused by mechanical contact was found on the bearing. The newly developed temporary left ventricular assist system using a disposable centrifugal pump with hydrodynamic bearing demonstrated consistent and satisfactory hemodynamic performance and hemocompatibility in the goat model.

Novel Rotational Speed Modulation System Used With Venoarterial Extracorporeal Membrane Oxygenation.

BACKGROUND: Femoral venoarterial extracorporeal membrane oxygenation (VA-ECMO) is widely used to maintain blood flow in patients with cardiogenic shock. However, retrograde blood flow increases left ventricular (LV) afterload during femoral VA-ECMO. Additional support by means of an intraaortic balloon pump (IABP) alleviates LV afterload but is associated with significant adverse events. We previously developed a system for rotational speed modulation in synchrony with the native cardiac cycle, for use with implantable continuous-flow LV assist devices. Here, we aimed to evaluate whether our novel rotation speed modulation system can improve coronary artery flow and reduce LV during femoral VA-ECMO. METHODS: VA-ECMO was installed by means of right atrial drainage and distal abdominal aortic perfusion in six adult goats. Cardiogenic shock was induced with ß-adrenergic antagonist infusion. An IABP was placed in the descending aorta. LV stroke work, LV end-systolic pressure, and coronary arterial flow were evaluated. Data were collected under five conditions (modes): baseline, circuit-clamp (cardiogenic shock), continuous mode (constant rotational speed), counterpulse mode (increasing rotational speed during diastole), and continuous mode with IABP support. RESULTS: LV stroke work and LV end-systolic pressure tended to be lower in the counterpulse mode, indicating decreased LV work load and afterload in this mode. Furthermore, coronary arterial flow tended to be higher in the counterpulse mode. CONCLUSIONS: Our system enabled an increase in coronary arterial flow and a decrease in LV work load and afterload during VA-ECMO. The system offers the effects of VA-ECMO and an IABP in a single device.

Effect of Hydroxyethyl Starch Priming on the Systemic Inflammatory Response and Lung Edema after Cardiopulmonary Bypass in a Rat Model.

Cardiopulmonary bypass (CPB) preserves patients' lives during open heart surgery by providing sufficient oxygen delivery and blood supply to vital organs. However, previous studies have suggested that the interaction of hemodilution and vascular hyperpermeability induces tissue edema and an inflammatory response during CPB. In this study, we hypothesized the suppression of the systemic inflammatory response and tissue edema during CPB by a plasma substitute (hydroxyethyl starch [HES]). Rats (450-500 g) were divided into a SHAM group (n = 5), a Ringer's acetate CPB group (n = 7), and an HES CPB group (n = 7). In the Ringer's acetate group, the CPB circuit was primed with Ringer's acetate solution, and in the HES CPB group, it was primed with HES formulation (6% HES 130/0.4). Blood samples were collected before (baseline) and 30, 60, 90 and 120 min after initiation of CPB. Plasma cytokine levels of tumor necrosis factor-α, interleukin (IL)-6, and IL-10, and biochemical markers (lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, creatinine, liver-type fatty acid-binding protein, and colloid osmotic pressure [COP]) were measured before and 30, 60, 90, and 120 min after the initiation of CPB. In the Ringer's acetate CPB group, the inflammatory cytokines and biochemical markers increased significantly during CPB compared with the SHAM group, but such increases were significantly suppressed in the HES CPB group. In addition, during CPB, it was possible to preserve normal plasma COP in the HES CPB group. The data suggest that 6% HES 130/0.4 is effective for suppressing the inflammatory response during CPB.

Ghrelin Promotes Functional Angiogenesis in a Mouse Model of Critical Limb Ischemia Through Activation of Proangiogenic MicroRNAs.

Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limited success. Recent in vitro evidence in the literature, using cell lines, proposes that the peptide hormone ghrelin may have angiogenic properties. In this study, we aim to investigate if ghrelin could promote postischemic angiogenesis in a mouse model of CLI and, further, identify the mechanistic pathway(s) that underpin ghrelin's proangiogenic properties. CLI was induced in male CD1 mice by femoral artery ligation. Animals were then randomized to receive either vehicle or acylated ghrelin (150 µg/kg sc) for 14 consecutive days. Subsequently, synchrotron radiation microangiography was used to assess hindlimb perfusion. Subsequent tissue samples were collected for molecular and histological analysis. Ghrelin treatment markedly improved limb perfusion by promoting the generation of new capillaries and arterioles (internal diameter less than 50 µm) within the ischemic hindlimb that were both structurally and functionally normal; evident by robust endothelium-dependent vasodilatory responses to acetylcholine. Molecular analysis revealed that ghrelin's angiogenic properties were linked to activation of prosurvival Akt/vascular endothelial growth factor/Bcl-2 signaling cascade, thus reducing the apoptotic cell death and subsequent fibrosis. Further, ghrelin treatment activated proangiogenic (miR-126 and miR-132) and antifibrotic (miR-30a) microRNAs (miRs) while inhibiting antiangiogenic (miR-92a and miR-206) miRs. Importantly, in vitro knockdown of key proangiogenic miRs (miR-126 and miR-132) inhibited the angiogenic potential of ghrelin. These results therefore suggest that clinical use of ghrelin for the early treatment of CLI may be a promising and potent inducer of reparative vascularization through modulation of key molecular factors.

The potential of the novel leukocyte removal filter in cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) is indispensable for cardiac surgery but leads to systemic inflammatory responses and leukocyte activation, possibly due to blood contact with the surface of the CPB unit, surgical, ischemic reperfusion injury, etc. Systemic inflammatory responses during CPB result in increased morbidity and mortality. Activation of leukocytes is an important part of this process and directly contributes to coagulopathy and hemorrhage. This inflammatory response may contribute to the development of postoperative complications, including myocardial dysfunction, respiratory failure, renal and neurologic dysfunction, altered liver function and ultimately, multiple organ failure. Various pharmacologic and mechanical strategies have been developed to minimize the systemic inflammatory response during CPB. For example, leukocyte removal filters were developed in the 1990s for incorporation into the CPB circuit. However, studies of this approach have yielded conflicting findings. The purpose of this was to review the studies of a novel leukocyte removal filter in patients undergoing CPB.

Chronic Rho-kinase inhibition improves left ventricular contractile dysfunction in early type-1 diabetes by increasing myosin cross-bridge extension.

BACKGROUND: Impaired actin-myosin cross-bridge (CB) dynamics correlate with impaired left ventricular (LV) function in early diabetic cardiomyopathy (DCM). Elevated expression and activity of Rho kinase (ROCK) contributes to the development of DCM. ROCK targets several sarcomeric proteins including myosin light chain 2, myosin binding protein-C (MyBP-C), troponin I (TnI) and troponin T that all have important roles in regulating CB dynamics and contractility of the myocardium. Our aim was to examine if chronic ROCK inhibition prevents impaired CB dynamics and LV dysfunction in a rat model of early diabetes, and whether these changes are associated with changes in myofilament phosphorylation state. METHODS: Seven days post-diabetes induction (65 mg/kg ip, streptozotocin), diabetic rats received the ROCK inhibitor, fasudil (10 mg/kg/day ip) or vehicle for 14 days. Rats underwent cardiac catheterization to assess LV function simultaneous with X-ray diffraction using synchrotron radiation to assess in situ CB dynamics. RESULTS: Compared to controls, diabetic rats developed mild systolic and diastolic dysfunction, which was attenuated by fasudil. End-diastolic and systolic myosin proximity to actin filaments were significantly reduced in diabetic rats (P < 0.05). In all rats there was an inverse correlation between ROCK1 expression and the extension of myosin CB in diastole, with the lowest ROCK expression in control and fasudil-treated diabetic rats. In diabetic and fasudil-treated diabetic rats changes in relative phosphorylation of TnI and MyBP-C were not significant from controls. CONCLUSIONS: Our results demonstrate a clear role for ROCK in the development of LV dysfunction and impaired CB dynamics in early DCM.

Pulmonary Macrophages Attenuate Hypoxic Pulmonary Vasoconstriction via ß3AR/iNOS Pathway in Rats Exposed to Chronic Intermittent Hypoxia.

Chronic intermittent hypoxia (IH) induces activation of the sympathoadrenal system, which plays a pivotal role in attenuating hypoxic pulmonary vasoconstriction (HPV) via central ß1-adrenergic receptors (AR) (brain) and peripheral ß2AR (pulmonary arteries). Prolonged hypercatecholemia has been shown to upregulate ß3AR. However, the relationship between IH and ß3AR in the modification of HPV is unknown. It has been observed that chronic stimulation of ß3AR upregulates inducible nitric oxide synthase (iNOS) in cardiomyocytes and that IH exposure causes expression of iNOS in RAW264.7 macrophages. iNOS has been shown to have the ability to dilate pulmonary vessels. Hence, we hypothesized that chronic IH activates ß3AR/iNOS signaling in pulmonary macrophages, leading to the promotion of NO secretion and attenuated HPV. Sprague-Dawley rats were exposed to IH (3-min periods of 4-21% O2) for 8 h/d for 6 weeks. The urinary catecholamine concentrations of IH rats were high compared with those of controls, indicating activation of the sympathoadrenal system following chronic IH. Interestingly, chronic IH induced the migration of circulating monocytes into the lungs and the predominant increase in the number of pro-inflammatory pulmonary macrophages. In these macrophages, both ß3AR and iNOS were upregulated and stimulation of the ß3AR/iNOS pathway in vitro caused them to promote NO secretion. Furthermore, in vivo synchrotron radiation microangiography showed that HPV was significantly attenuated in IH rats and the attenuated HPV was fully restored by blockade of ß3AR/iNOS pathway or depletion of pulmonary macrophages. These results suggest that circulating monocyte-derived pulmonary macrophages attenuate HPV via activation of ß3AR/iNOS signaling in chronic IH.

CCN1 (Cyr61) Is Overexpressed in Human Osteoarthritic Cartilage and Inhibits ADAMTS-4 (Aggrecanase 1) Activity.

OBJECTIVE: ADAMTS-4, also called aggrecanase 1, is considered to play a key role in aggrecan degradation in human osteoarthritic (OA) cartilage, but information about regulators of ADAMTS-4 aggrecanase activity remains limited. We undertook this study to search for molecules that modulate ADAMTS-4 activity. METHODS: Molecules copurified with ADAMTS-4 from ADAMTS-4-transfected chondrocytic cells were sequenced by nanoscale liquid chromatography tandem mass spectrometry. Binding activity was determined by immunoprecipitation and solid-phase binding assay. Effects on ADAMTS-4 activity were examined by aggrecan digestion assay. Expression of the binding molecule in OA cartilage and chondrocytes was examined by immunohistochemistry and reverse transcription-polymerase chain reaction. RESULTS: We identified CCN1 (Cyr61) as an ADAMTS-4-binding protein and showed specific binding to the ADAMTS-4 cysteine-rich domain. Aggrecanase activity of ADAMTS-4 was inhibited by interaction with CCN1. Expression of messenger RNA for CCN1 was significantly higher in human OA cartilage than in normal cartilage. CCN1 was immunolocalized to chondrocytes in OA cartilage, showing direct correlations of immunoreactivity with the Mankin score of cartilage lesions and chondrocyte cloning. CCN1 and ADAMTS-4 were commonly coexpressed in clustered chondrocytes. CCN1 expression in OA chondrocytes was down-regulated by interleukin-1α (IL-1α) and up-regulated by transforming growth factor ß (TGFß). ADAMTS-4 expression was induced by treatment with IL-1α or TGFß, but aggrecanase activity was detected only under stimulation with IL-1α. TGFß-treated chondrocytes exhibited aggrecanase activity when CCN1 expression was knocked down. CONCLUSION: Our findings provide the first evidence that CCN1 suppresses ADAMTS-4 activity and that CCN1 overexpression is directly correlated with chondrocyte cloning in OA cartilage. Our results suggest that the TGFß/CCN1 axis plays a role in chondrocyte cluster formation through inhibition of ADAMTS-4.

Functional and Electrical Integration of Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Myocardial Infarction Rat Heart.

In vitro expanded beating cardiac myocytes derived from induced pluripotent stem cells (iPSC-CMs) are a promising source of therapy for cardiac regeneration. Meanwhile, the cell sheet method has been shown to potentially maximize survival, functionality, and integration of the transplanted cells into the heart. It is thus hypothesized that transplanted iPSC-CMs in a cell sheet manner may contribute to functional recovery via direct mechanical effects on the myocardial infarction (MI) heart. F344/NJcl-rnu/rnu rats were left coronary artery ligated (n = 30), followed by transplantation of Dsred-labeled iPSC-CM cell sheets of murine origin over the infarct heart surface. Effects of the treatment were assessed, including in vivo molecular/cellular evaluations using a synchrotron radiation scattering technique. Ejection fraction and activation recovery interval were significantly greater from day 3 onward after iPSC-CM transplantation compared to those after sham operation. A number of transplanted iPSC-CMs were present on the heart surface expressing cardiac myosin or connexin 43 over 2 weeks, assessed by immunoconfocal microscopy, while mitochondria in the transplanted iPSC-CMs gradually showed mature structure as assessed by electron microscopy. Of note, X-ray diffraction identified 1,0 and 1,1 equatorial reflections attributable to myosin and actin-myosin lattice planes typical of organized cardiac muscle fibers within the transplanted cell sheets at 4 weeks, suggesting cyclic systolic myosin mass transfer to actin filaments in the transplanted iPSC-CMs. Transplantation of iPSC-CM cell sheets into the heart yielded functional and electrical recovery with cyclic contraction of transplanted cells in the rat MI heart, indicating that this strategy may be a promising cardiac muscle replacement therapy.

Changes in inflammatory response during and after cardiopulmonary bypass using a rat extracorporeal circulation model.

Cardiopulmonary bypass (CPB) is indispensable for cardiac surgery. Since the ethical and technical difficulties associated with clinical research limit the acquisition of new knowledge, it is desirable to have a miniature extracorporeal circulation (ECC) system for small animals. We aimed to establish a miniature ECC system and apply the system to the rat for investigating systemic inflammatory response changes during and after CPB. The ECC system consisted of a membranous oxygenator (polypropylene, 0.03 m(2)), tubing line (polyvinyl chloride) and a roller pump. Priming volume of this system is only 7 ml. Rats were divided into the SHAM (received surgical preparation only without CPB) group and the CPB group. ECC pump flow was initiated and maintained at 70 ml/kg/min. We measured the serum cytokine levels of tumor necrosis factor-α, interleukin (IL)-6, and IL-10, and biochemical markers (lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase) before, 30, and 60 min after the initiation of CPB, in addition, 30, 60, and 120 min after the CPB weaning. During CPB, blood pressure and hemoglobin were maintained around 80 mmHg and 10g/dl, the serum cytokine levels and biochemical markers were significantly elevated in the CPB group compared with the SHAM group. These data suggest that CPB promotes organ damage and a systemic inflammatory response. This rat ECC model is considered to be equivalent to the already established human CPB and useful for studying the mechanism of pathophysiological changes during and after CPB.

A novel small animal extracorporeal circulation model for studying pathophysiology of cardiopulmonary bypass.

Extracorporeal circulation (ECC) is indispensable for cardiac surgery. Despite the fact that ECCcauses damage to blood components and is non-physiologic, its pathophysiology has not been fully elucidated. This is because difficulty in clinical research and animal experiments keeps the knowledge insufficient. Therefore, it is desirable to have a miniature ECC model for small animals, which enables repetitive experiments, to study the mechanism of pathophysiological changes during ECC. We developed a miniature ECC system and applied it to the rat. We measured changes in hemodynamics, blood gases and hemoglobin (Hb) concentration, serum cytokines (TNF-α, IL-6, IL-10), biochemical markers (LDH, AST, ALT), and the wet-to-dry weight (W/D) ratio of the lung for assessing whether the rat ECC model is comparable to the human ECC. The ECC system consisted of a membranous oxygenator (polypropylene, 0.03 m(2)), tubing line (polyvinyl chloride), and roller pump. Priming volume of this system is only 8 ml. Rats (400-450 g) were divided into the SHAM group (n = 7) and the ECC group (n = 7). Blood samples were collected before, 60 and 120 min after initiation of ECC. During ECC, blood pressure and Hb were maintained around 80 mmHg and 10 g/dL, respectively. The levels of the inflammatory and biochemical markers and the W/D ratio were significantly elevated in the ECC group, indicating some organ damages and systemic inflammatory responses during ECC. We successfully established the ECC for the rat. This miniature ECC model could be a useful approach for studying the mechanism of pathophysiology during ECC and basic assessment of the ECC devices.

Impact of bypass flow rate and catheter position in veno-venous extracorporeal membrane oxygenation on gas exchange in vivo.

The clinical use of veno-venous extracorporeal membrane oxygenation (VVECMO) in adult patients with respiratory failure is rapidly increasing. However, recirculation of blood oxygenated by ECMO back into the circuit may occur in VVECMO, resulting in insufficient oxygenation. The cannula position and bypass flow rate are two major factors influencing recirculation, but the relationship and ideal configuration of these factors are not fully understood. In the present study, we attempted to clarify these parameters for effective gas exchange. VVECMO was performed in eight adult goats under general anesthesia. The position of the drainage cannula was fixed in the inferior vena cava (IVC), but the return cannula position was varied between the IVC, right atrium (RA), and superior vena cava (SVC). At each position, the recirculation rates calculated, and the adequacy of oxygen delivery by ECMO in supplying systemic oxygen demand was assessed by measuring the arterial oxygen saturation (SaO2) and pressure (PaO2). Although the recirculation rates increased as the bypass flow rates increased, SaO2 and PaO2 also increased in any position of return cannula. The recirculation rates and PaO2 were 27 ± 2% and 162 ± 16 mmHg, 36 ± 6% and 139 ± 11 mmHg, and 63 ± 6% and 77 ± 9 mmHg in the SVC, RA and IVC position at 4 L/min respectively. In conclusion, the best return cannula position was the SVC, and a high bypass flow rate was advantageous for effective oxygenation. Both the bypass flow rates and cannula position must be considered to achieve effective oxygenation.

ß2-Adrenergic receptor-dependent attenuation of hypoxic pulmonary vasoconstriction prevents progression of pulmonary arterial hypertension in intermittent hypoxic rats.

In sleep apnea syndrome (SAS), intermittent hypoxia (IH) induces repeated episodes of hypoxic pulmonary vasoconstriction (HPV) during sleep, which presumably contribute to pulmonary arterial hypertension (PAH). However, the prevalence of PAH was low and severity is mostly mild in SAS patients, and mild or no right ventricular hypertrophy (RVH) was reported in IH-exposed animals. The question then arises as to why PAH is not a universal finding in SAS if repeated hypoxia of sufficient duration causes cycling HPV. In the present study, rats underwent IH at a rate of 3 min cycles of 4-21% O2 for 8 h/d for 6 w. Assessment of diameter changes in small pulmonary arteries in response to acute hypoxia and drugs were performed using synchrotron radiation microangiography on anesthetized rats. In IH-rats, neither PAH nor RVH was observed and HPV was strongly reversed. Nadolol (a hydrophilic ß(1, 2)-blocker) augmented the attenuated HPV to almost the same level as that in N-rats, but atenolol (a hydrophilic ß1-blocker) had no effect on the HPV in IH. These ß-blockers had almost no effect on the HPV in N-rats. Chronic administration of nadolol during 6 weeks of IH exposure induced PAH and RVH in IH-rats, but did not in N-rats. Meanwhile, atenolol had no effect on morphometric and hemodynamic changes in N and IH-rats. Protein expression of the ß1-adrenergic receptor (AR) was down-regulated while that of ß2AR was preserved in pulmonary arteries of IH-rats. Phosphorylation of p85 (chief component of phosphoinositide 3-kinase (PI3K)), protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS) were abrogated by chronic administration of nadolol in the lung tissue of IH-rats. We conclude that IH-derived activation of ß2AR in the pulmonary arteries attenuates the HPV, thereby preventing progression of IH-induced PAH. This protective effect may depend on the ß2AR-Gi mediated PI3K/Akt/eNOS signaling pathway.