DJ-1 is involved in epigenetic control of sphingosine-1-phosphate receptor expression in vascular neointima formation.

DJ-1 and sphingosine-1-phosphate (S1P) receptors (S1PRs) are implicated in the control of physiology and pathophysiology of cardiovascular systems such as blood pressure, atherosclerosis, and restenosis. Here, we investigated whether DJ-1 with antioxidant function participates in the regulation of S1PR1 and S1PR2 expression in vascular smooth muscle cells (VSMCs) and whether this response is related to vascular neointima formation. In vitro studies used cellular migration assay, western blot, reverse transcriptase and real-time PCR analysis, and immunocytochemistry. In vivo studies were performed using the carotid artery ligation model together with immunohistochemistry in DJ-1 knockout (DJKO) and corresponding wild-type (DJWT) mice. S1P stimulated migration of VSMCs from DJKO and DJWT mice. VSMC migration was suppressed by S1PR1 inhibitor but was elevated by S1PR2 inhibitor. Compared with DJWT mice, S1PR1 expression was higher in VSMCs and neointimal plaque from DJKO mice, but S1PR2 expression was lower. Overexpression of DJ-1 in DJKO VSMCs reduced S1PR1 expression and elevated S1PR2 expression. Compared with DJWT mice, histone deacetylase-1 recruitment and histone H3 acetylation at the S1PR1 promoter region were lower and higher, respectively, but this pattern was reversed at the S1PR2 promoter region in DJKO VSMCs. S1PR expressions and epigenetic changes at S1PR promoter regions in DJWT VSMCs treated with H2O2 showed similar patterns to those in DJKO VSMCs. Our findings suggest that DJ-1 may be involved in the regulation of S1PR1 and S1PR2 expression via H2O2-mediated histone modification in VSMCs. Consequently, this modification may affect S1P-induced VSMC migration and be related to vascular neointima formation.

Angiotensin II facilitates neointimal formation by increasing vascular smooth muscle cell migration: Involvement of APE/Ref-1-mediated overexpression of sphingosine-1-phosphate receptor 1.

Angiotensin II (Ang II) is implicated in the development of cardiovascular disorders including hypertension and atherosclerosis. However, the role of Ang II in the interaction between apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) and sphingosine-1-phosphate (S1P) signals in relation to vascular disorders remains to be clarified. This study aimed to determine whether APE/Ref-1 plays a role in epigenetic regulation of the S1P receptor (S1PR) in response to Ang II in vascular smooth muscle cell (VSMC) migration and vascular neointima formation. Ang II augmented the expression of S1PR1 in aortic smooth muscle cells of Sprague Dawley rats (RASMCs), which was attenuated by Ang II receptor (AT) 1 inhibitors, antioxidants, and APE/Ref-1 knockdown with small interference RNA. Ang II stimulation produced H2O2, and exogenous H2O2 elevated S1PR1 expression in RASMCs. Moreover, Ang II caused translocation of cytoplasmic APE/Ref-1 into the nucleus in RASMCs. H3 histone acetylation and APE/Ref-1 binding at the S1PR1 promoter were increased in RASMCs treated with Ang II. In addition, Ang II induced migration in RASMCs, which was suppressed by AT1 and S1PR1 inhibitors. The expression of S1PR1, and colocalization of APE/Ref-1 and acetylated histone H3 in vascular neointima, were greater in Ang II-infused rats compared with a control group. These findings demonstrate that Ang II stimulates the epigenetic regulation of S1PR1 expression via H2O2-mediated APE/Ref-1 translocation, which may consequently be involved in Ang II-induced VSMC migration and vascular neointima formation. Therefore, APE/Ref-1-mediated overexpression of S1PR1 may be implicated in the vascular dysfunction evoked by Ang II.

Lapabot: a compact telesurgical robot system for minimally invasive surgery: part II. Telesurgery evaluation.

As described in Part I, the Lapabot was developed considering telesurgery from the initial design stage. The robot configuration is based on the master-slave structure in which the operator can be separated spatially from the patient. The distributed control architecture communicating through high-speed network enables remote control of surgical robot manipulators. In this work, we added network communication modules using user datagram protocol/internet protocol for implementation of the telesurgical system. For a stable network environment, a dedicated research network was adopted. To characterize the network environment, a data packet sender and a repeater whose packet length and packet structure are similar to those of the real data packet were developed. The developed system was evaluated through in-vitro and in-vivo experiments. With the developed system, we have successfully performed remote control of the Lapabot. The roundtrip time delay for the control signal ranged from 1.4 to 4.1 ms. The total time delay for the operator including image signal acquisition and transmission delays was under 333 ms. It did not impede surgical procedures. Initial evaluation results demonstrate the feasibility of the developed telesurgical system.

Vertical Bone Augmentation Using Three-dimensionally Printed Cap in the Rat Calvarial Partial Defect.

BACKGROUND/AIM: Lost alveolar bone is commonly restored by distraction osteogenesis or bone blocks for substantial vertical bone augmentation (VBA), that is applied in conjunction with a barrier system. This study was performed to determine whether volume control of a three-dimensional (3D) printed nylon cap in the rat calvarial partial thickness bone defect would induce qualitative and quantitative differences in vertical bone regeneration. MATERIALS AND METHODS: A rat calvarial partial thickness bone defect was prepared and the 3D cap covered the defect to induce VBA, while the control group was left without cap placement. After six weeks the animals were sacrificed, and the calvaria were prepared for micro-CT (µCT) and histology. RESULTS: Quantitative µCT results showed that our cap system has significant osteoconductive properties, and the histology slide revealed new bone filled inside the cap. CONCLUSION: The results clearly showed that this system was successful for VBA in a research animal model.

Nootropic nanocomplex with enhanced blood-brain barrier permeability for treatment of traumatic brain injury-associated neurodegeneration.

Traumatic brain injury (TBI) is an intracranial injury which can induce immediate neuroinflammation and long-term neurological deficits. Methylene blue (MB) as a nootropic has a great potential to treat neurodegeneration after TBI because of its anti-inflmmatory and neuroprotective functions. However, its limited accumulation to the brain across the blood-brain barrier (BBB) remains a major hurdle to be overcome. In this paper, we present a polymer surfactant-encapsulated nanocomplex of MB as a delivery system with high BBB permeability for efficacious treatment of TBI-induced neurodegeneration. MB was formulated via electrostatically/hydrophobically directed assembly with fatty acid and Pluronic surfactant (F-127 or F-68) to construct nanocomplexes of two different colloidal sizes (<10 nm and ~108 nm in hydrodynamic diameter for NanoMB-127 and NanoMB-68, respectively). Compared to uncomplexed free MB, formulation into the ultrasmall nanocomplex (NanoMB-127) significantly enhanced the uptake of MB by blood-brain vascular endothelial bEnd3 cells in vitro, and indeed improved its BBB penetration upon systemic administration to normal mice in vivo. However, large-size NanoMB-68 showed negligible BBB crossing despite the efficient bEnd3 cell internalization in vitro, probably due to the unfavorable pharmacokinetic profile associated with its large particle size. By virtue of the efficient BBB penetration and cellular uptake, ultrasmall NanoMB-127 was shown to distinctively reduce the expression level of an inflammatory cytokine with no notable toxicity in vitro and also considerably prevent the neurodegeneration after TBI in mice at much lower doses than free MB. Overall, the Pluronic-supported nanocomplexation method allows efficient brain delivery of MB, offering a novel way of enhancing the efficacy of neurotherapeutics to treat brain diseases.

Recording nerve signals in canine sciatic nerves with a flexible penetrating microelectrode array.

OBJECTIVE: Previously, we presented the fabrication and characterization of a flexible penetrating microelectrode array (FPMA) as a neural interface device. In the present study, we aim to prove the feasibility of the developed FPMA as a chronic intrafascicular recording tool for peripheral applications. APPROACH: For recording from the peripheral nerves of medium-sized animals, the FPMA was integrated with an interconnection cable and other parts that were designed to fit canine sciatic nerves. The uniformity of tip exposure and in vitro electrochemical properties of the electrodes were characterized. The capability of the device to acquire in vivo electrophysiological signals was evaluated by implanting the FPMA assembly in canine sciatic nerves acutely as well as chronically for 4 weeks. We also examined the histology of implanted tissues to evaluate the damage caused by the device. MAIN RESULTS: Throughout recording sessions, we observed successful multi-channel recordings (up to 73% of viable electrode channels) of evoked afferent and spontaneous nerve unit spikes with high signal quality (SNR > 4.9). Also, minor influences of the device implantation on the morphology of nerve tissues were found. SIGNIFICANCE: The presented results demonstrate the viability of the developed FPMA device in the peripheral nerves of medium-sized animals, thereby bringing us a step closer to human applications. Furthermore, the obtained data provide a driving force toward a further study for device improvements to be used as a bidirectional neural interface in humans.

Development of an intrafascicular neural interface for peripheral nerve implantation.

This paper describes an intrafascicular neural interface for peripheral nerve implantation. The flexible penetrating microelectrode array with varying lengths (vl-FPMA), interconnection cable, wireless recording and stimulator modules were designed and fabricated to detect neural signals from the peripheral nerves or to stimulate them. The vl-FPMA consisted of silicon needles and polydimethylsiloxane (PDMS) platform supporting the needles. The length of electrode needles varied from 600 to 1000 µm. The interconnection cable was fabricated as parylene-metal-parylene sandwiched structure. The wireless recording/stimulation modules were also developed and connected with the electrodes. The integrated system was implanted in the sciatic nerve of beagles and the recording capability of the integrated system was demonstrated successfully.

Laboratory Animal Laws, Regulations, Guidelines and Standards in China Mainland, Japan, and Korea.

China, Japan, and Korea have spent decades developing and amending laws, regulations, and guidelines to address the humane care and use of laboratory animals. This process began in 1983 in China, 1973 in Japan, and 1991 in Korea and has continued to the present. The governmental oversight of research varies between these countries, ranging from regulations by multiple levels of government in China to self-regulation under multiple government guidelines in Japan. Common to all is incorporation of the internationally recognized principles of the 3Rs: replacement, reduction and refinement. This paper reviews how the framework of laws, regulations, and guidelines evolved in each of these countries, their current status, and the expectation that they will continue to evolve.

Effect of ischemic preconditioning based on different epicardial branching patterns of the left coronary artery in the rabbit heart.

The aim of this study was to characterize the effect of increasing numbers of preconditioning cycles on the different branching patterns (bifurcation/trifurcation) of the coronary artery in the rabbit heart. Fifty-six NZW rabbits were assigned to a bifurcation group and subjected to 0, 1, 3, 5, or 7 (B0, B1, B3, B5, or B7 subgroup, respectively) cycles of preconditioning (PC) (5 min of regional ischemia plus 10 min of reperfusion/cycle) and this was followed by 45 min of sustained ischemia and 72 h of reperfusion; 16 rabbits were assigned to a trifurcation group and subjected to 0, 1, 3, or 5 (T0, T1, T3, or T5 subgroup, respectively) cycles of PC. The ratio of necrotic zone (NZ) to ischemic zone (IZ) was calculated. The bifurcation group showed higher mortality (28.6%) than the trifurcation group (0%). The volume of the ischemic zone (expressed as a percentage of volume of the left ventricle) and the volume of the necrotic zone were larger in the bifurcation group than in the trifurcation group. The ratio of the necrotic zone to the ischemic zone was significantly lower in the B5 and B7 subgroups than in the B0, B1, and B3 subgroups. In the trifurcation group, the ratio of the necrotic zone to the ischemic zone showed a diminishing tendency in the subgroups as the PC cycle number increased, but without statistical significance. Thus, in the trifurcation pattern of the rabbit coronary artery there could be little effect on preconditioning, but in the bifurcation pattern the number of preconditioning is recommended to be 5 to 7 cycles. In this regard, different branching patterns of the epicardial branching of the rabbit coronary artery should be considered when interpreting experimental results on ischemic preconditioning.

Tracheal gas isufflation-aided mechanical ventilation during carbon dioxide-induced pneumoperitoneum in rabbits.

Despite numerous benefits of laparoscopic procedures, the serious hypercapnia and respiratory acidosis in hypercapnic patients with decreased pulmonary compliance during carbon dioxide-induced pneumoperitoneum (CDP) may be developed. Tracheal gas insufflation (TGI) has been shown to be a useful adjunct to controlled mechanical hypoventilation. This study was undertaken to identify whether TGI superimposed on controlled mechanical ventilation (CMV) improve ventilatory efficiency during CDP in rabbits. Sixteen paralyzed and anesthetized rabbits were used. The animals were assigned to two groups-CMV group: CMV alone; TGI group: CMV superimposed by TGI with flow rate of 2L/min. The animals were insufflated to intra-abdominal pressure of 8 mmHg with CO2 gas. Then, tidal volume (V(T)) was changed to maintain the set peak inspiratory pressure (PIP) value, while other ventilatory settings were kept constant. The set PIP value corresponding to 30, 60, and 90 min after the start of peritoneal insufflation of CO2 were 15, 22, and 25 cm H2O, respectively. During CDP with TGI, PaCO2 decreased significantly (p<0.01) from CMV without TGI of 82.1 +/- 14.1 to 47.5 +/- 5.5, 58.1 +/- 9.9 to 40.0 +/- 4.6, 47.1 +/- 9.4 to 32.7 +/- 5.1 mmHg at PIP of 15, 22, and 25 cm H2O, respectively. The inspired V(T) decreased significantly (p<0.05) from CMV without TGI of 18.4 +/- 3.9 to 12.8 +/- 2.8 ml at PIP of 15 cm H2O. TGI superimposed on CMV is more effective than CMV alone in enhancing ventilatory efficiency during CDP in rabbits.

A case of patent ductus arteriosus in a holstein calf.

This report describes a case of patent ductus arteriosus (PDA) which was found in a calf during an experiment on an implantable ventricular assist device. This is the first case report of PDA confirmed in the calf in Korea.

Increased HoxB4 Inhibits Apoptotic Cell Death in Pro-B Cells.

HoxB4, a homeodomain-containing transcription factor, is involved in the expansion of hematopoietic stem cells and progenitor cells in vivo and in vitro, and plays a key role in regulating the balance between hematopoietic stem cell renewal and cell differentiation. However, the biological activity of HoxB4 in other cells has not been reported. In this study, we investigated the effect of overexpressed HoxB4 on cell survival under various conditions that induce death, using the Ba/F3 cell line. Analysis of phenotypical characteristics showed that HoxB4 overexpression in Ba/F3 cells reduced cell size, death, and proliferation rate. Moreover, the progression from early to late apoptotic stages was inhibited in Ba/F3 cells subjected to HoxB4 overexpression under removal of interleukin-3-mediated signal, leading to the induction of cell cycle arrest at the G2/M phase and attenuated cell death by Fas protein stimulation in vitro. Furthermore, apoptotic cell death induced by doxorubicin-treated G2/M phase cell-cycle arrest also decreased with HoxB4 overexpression in Ba/F3 cells. From these data, we suggest that HoxB4 may play an important role in the regulation of pro-B cell survival under various apoptotic death environments.

Effects of various anesthetic protocols on 18F-flurodeoxyglucose uptake into the brains and hearts of normal miniature pigs (Sus scrofa domestica).

This study used positron emission tomography-computed tomography (PET-CT) to evaluate the effects of 4 anesthetic protocols on 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) accumulation in the brains and hearts of miniature pigs (Sus scrofa domestica). The 18F-FDG standard uptake value was quantified by dividing the brain into 6 regions: cerebellum, brainstem, and frontal, parietal, temporal, and occipital lobes. Five (2 female and 3 male) clinically normal miniature pigs were premedicated with medetomidine (200 µg/kg IM) after which the following 4 anesthetic protocols were administered by using a crossover design: 1) propofol (4 mg/kg IV)-isoflurane inhalation; 2) propofol (4 mg/kg IV); 3) ketamine (5 mg/kg IV); 4) tiletamine-zolazepam (4.4 mg/kg IM). Compared with levels after other protocols, brain accumulation of 18F-FDG increased during propofol anesthesia but decreased with tiletamine-zolazepam. Relative to that due to other protocols, heart accumulation of 18F-FDG increased with propofol-isoflurane anesthesia but decreased with tiletamine-zolazepam. Comparing glucose accumulation in the brain and heart of miniature pigs by using PET-CT, we found that glucose accumulation varied according to the anesthetic protocol and between the 2 organs. These results can be used to evaluate how different anesthetic agents affect glucose metabolism in brain and heart of miniature pigs. Furthermore, these data should be considered when selecting an anesthetic agent for miniature pigs that will undergo PET-CT imaging with 18F-FDG.

In vitro evaluation of hydraulic characteristics of prototype implantable intrathecal infusion pump.

The use of the intrathecal infusion pump for therapeutic treatment and pain management is increasing. For example, one such application is the pain treatment of cancer patients suffering from severe chronic pain, where all other treatment methods have failed. This method is gaining popularity because of its high cure effect with low dosage. In this study, we developed a prototype implantable intrathecal infusion pump and evaluated its mechanical and hydraulic characteristics in vitro to determine how its performance varied under different environmental conditions. The data are reported as means (standard deviations). In the experiments, the prototype pump could control the micro-scale infusion amount, and its performance was affected by ambient temperature and pressure conditions. In a temperature change test, at a constant pressure of 1.0 atm, the minimal amounts of a bolus were 4.44 (1.07), 5.06 (1.17), and 5.54 (0.90) uL for the temperature of 27.5, 36.5, and 42°C, respectively. In a pressure change test, at a constant temperature of 36.5°C, the minimal amounts of a bolus were 5.06 (1.17), 5.94 (0.67), and 6.13 (0.39) uL for pressures of 1.0, 0.9 and 0.8 atm, respectively. These experimental results demonstrate the possibility of using the prototype pump as an implantable microvolumetric infusion device. However, this prototype pump will have to undergo further design enhancement before being clinically feasible for such an application.

Apical rotation assessed by speckle-tracking echocardiography as an index of global left ventricular contractility.

BACKGROUND: Left ventricular (LV) apical rotation and twist can be estimated noninvasively by speckle-tracking echocardiography (STE). In this study, we tested whether apical rotation is an accurate index of LV contractility. METHODS AND RESULTS: We measured LV basal and apical rotation by STE in 11 open-chest anesthetized mongrel dogs under 8 different inotropic stages before and after ligation of either left anterior descending (n=6) or circumflex coronary artery (n=5). We measured LV pressure simultaneously with a high-fidelity pressure catheter and calculated LV ejection fraction (EF) with the biplane Simpson method and 2D echocardiography. Maximal positive dP/dt (dP/dt(max)) was used as the gold standard measurement of LV contractility. We compared LV twist and apical rotation and EF against dP/dt(max) by linear mixed model. LV apical rotation and twist showed dose-dependent increases and decreases after dobutamine and esmolol infusion, respectively. However, basal rotation did not change significantly during different inotropic conditions. There was a stronger association between dP/dt(max) and LV twist (R(2)=0.747, P<0.001) and apical rotation (R(2)=0.726, P<0.001) than between dP/dt(max) and EF (R(2)=0.408, P<0.001), and this trend was more apparent with coronary ligation irrespective of the ligation site. There was also a high association between dP/dt(max) and apical rotation alone, both with (R(2)=0.805, P<0.001) and without (R(2)=0.748, P<0.001) coronary ligation. Apical rotation alone showed comparable accuracy to LV twist. Apical rotational velocity also showed a high association with dP/dt(max) (R(2)=0.669, P<0.001) and LV twist (R(2)=0.892, P<0.001). CONCLUSIONS: Apical rotation assessed by STE is an effective noninvasive index of global LV contractility and is more closely related to dP/dt(max) than LV EF.

Evaluation of a novel doppler index of combined systolic and diastolic myocardial performance in Newfoundland dogs with familial prevalence of dilated cardiomyopathy.

A Doppler index of myocardial performance (IMP) has been recently proposed in human cardiology, which is calculated from the isovolumic contraction time (ICT), isovolumic relaxation time (IRT), and the ejection time (ET) using the following formula: (ICT+IRT)/ET. In this study, IMP was measured and evaluated in Newfoundland dogs categorized in four groups: Normal dogs (n = 31), dilated cardiomyopathy (DCM) (n = 34), depressed fractional shortening (dFS) (n = 27), and left ventricular enlargement (LVE) (n = 7). IMP was found to be independent of age, sex, body surface area, and the R-R interval in the Normal group. There were significant differences in IMP between the DCM group and the Normal and dFS groups (P < 0.05) and between Newfoundlands with overt vs. occult DCM. IMP is a Doppler index which appears to correlate with severity of disease and may be of use in the early diagnosis of affected dogs during screening for the presence of DCM.

Chronic heart failure model in rabbits based on the concept of the bifurcation/trifurcation coronary artery branching pattern.

The aim of this study was to develop a reliable chronic heart failure model by coronary artery ligation in the rabbit on the basis of the new concept of the bifurcation/trifurcation classification system of the epicardial branching pattern of the left coronary artery (LCA). New Zealand White rabbits (n = 37) were divided into 3 experimental groups: a posterolateral division of the bifurcation pattern of the LCA was ligated at the 75% level from the apex along the course of the division (B75 group, n = 15); a lateral division of the trifurcation pattern at the 75% level (T75 group, n = 11); and a posterolateral division of the bifurcation pattern at the 50% level (B50 group, n = 11). The infarct size and the lung and liver water content were determined at 4 weeks following ligation. The Q or QS wave on electrocardiogram (ECG) and the left ventricular (LV) dimensions (LVIDs and LVIDd), fractional shortening (FS), and mitral E-point to septal separation on ultrasonography were assessed at 10 min and at preligation and at 1, 2, and 4 weeks following ligation. The B75 group showed higher mortality (46.7%) than the T75 and B50 groups. The mean infarct size in the B75 group was 22.55 +/- 5.34% which was significantly larger than in the B50 (13.84 +/- 5.46%) and T75 (12.90 +/- 2.67%) groups (p < 0.001). All 3 groups had significantly greater Q or QS wave amplitudes on ECG at 1, 2, and 4 weeks than at 10 min after ligation. At 1 and 2 weeks after ligation, LVIDd, LVIDs, and FS showed significant dfferences in the B75 group as compared with the other groups. The level of ligation of the LCA for the development of a reliable chronic heart failure model in the rabbit is recommended to be 50% from the apex along the course of the posterolateral division in the bifurcation pattern and 75% from the apex along the course of the lateral division in the trifurcation pattern.