Visualising peripheral arterioles and venules through high-resolution and large-area photoacoustic imaging.

Photoacoustic (PA) imaging (PAI) has been shown to be a promising tool for non-invasive blood vessel imaging. A PAI system comprising a hemispherical detector array (HDA) has been reported previously as a method providing high morphological reproducibility. However, further improvements in diagnostic capability will require improving the image quality of PAI and fusing functional and morphological imaging. Our newly developed PAI system prototype not only enhances the PA image resolution but also acquires ultrasonic (US) B-mode images at continuous positions in the same coordinate axes. In addition, the pulse-to-pulse alternating laser irradiation shortens the measurement time difference between two wavelengths. We scanned extremities and breasts in an imaging region 140 mm in diameter and obtained 3D-PA images of fine blood vessels, including arterioles and venules. We could estimate whether a vessel was an artery or a vein by using the S-factor obtained from the PA images at two wavelengths, which corresponds approximately to the haemoglobin oxygen saturation. Furthermore, we observed tumour-related blood vessels around breast tumours with unprecedented resolution. In the future, clinical studies with our new PAI system will help to elucidate various mechanisms of vascular-associated diseases and events.

Photoacoustic mammography capable of simultaneously acquiring photoacoustic and ultrasound images.

We have constructed a prototype photoacoustic mammography system (PAM-02) capable of simultaneously acquiring photoacoustic (PA) and ultrasound (US) images. Each PA, US, and fused PA/US image can be acquired over a wide area of the breast using the scanning module of a US transducer, a PA detector, and optical prisms. The resolution of the PA images exhibits improvement from 2 to 1 mm compared to images acquired using our previous prototype. The maximum scan area of PAM-02 is 90 mm along the horizontal axis and 150 mm along the vertical axis. In a phantom experiment, the available depth was at least 45 mm. A representative example of the application of the PAM-02 prototype in clinical research at Kyoto University is presented and shows S-factor images, which are considered an approximation parameter related to hemoglobin saturation of tumor-related blood vessels. We confirmed the applicability of the system for anatomical and biological research.

Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells.

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.

Effects of purple sweet potato anthocyanins on development and intracellular redox status of bovine preimplantation embryos exposed to heat shock.

The development of cleavage stage preimplantation embryos is disrupted by exposure to heat shock, such as high temperatures in the summer season. In this study, we investigated whether addition of anthocyanins, which are strong scavengers of reactive oxygen species (ROS), improves development and intracellular redox status of heat-exposed bovine preimplantation embryos by reduction of heat shock-derived oxidative stress. After in vitro fertilization (IVF), embryos were cultured at 38.5 C through Day 8 (Day 0=day of IVF) with 0, 0.1, 1 and 10 microg/ml anthocyanins (non-heat-shocked group). On Day 2, embryos were cultured at 41.5 C for 6 h with 0, 0.1, 1 and 10 microg/ml anthocyanins followed by culture at 38.5 C until Day 8 (HS group). After exposure to heat shock, the intracellular ROS and glutathione (GSH) contents of individual embryos were measured in the non-heat-shocked and HS groups using fluorescent probes. On Day 8, the blastocysts formation rates of the embryos and total cell numbers of blastocysts were evaluated. Embryos exposed to heat shock without anthocyanins showed a significant decrease in blastocyst formation rate and GSH content (P<0.05) and an increase in intracellular ROS (P<0.05) compared with non-heat-shocked embryos. In contrast, addition of 0.1 microg/ml anthocyanins significantly (P<0.05) improved the blastocyst formation rate of the heat-shocked embryos. Addition of any dose of anthocyanins produced a significant decrease in the ROS levels (P<0.05) and tended to increase the GSH levels under heat-shock conditions. However, addition of higher concentrations (1 and 10 microg/ml) of anthocyanins to the culture media under heat shock did not improve the development of embryos. These results indicate that anthocyanins maintain the intracellular redox balance of heat-shocked bovine embryos by reducing intracellular oxidative stress and increasing the GSH levels. Thus, alterations of the redox state using natural antioxidative polyphenols is a useful approach for reducing heat shock-derived oxidative stress.

Left ventricular mechanoenergetics after hyperpolarized cardioplegic arrest by nicorandil and after depolarized cardioplegic arrest by KCl.

We hypothesized that there are no differences in left ventricular (LV) mechanoenergetics between after hyperpolarized cardioplegic arrest by nicorandil (nicorandil arrest) and after depolarized one by high potassium chloride (KCl arrest). The aim of the present study was to test this hypothesis using LV curved end-systolic pressure-volume relation (ESPVR) and linear pressure-volume area (PVA)-myocardial oxygen consumption per beat (VO2) relation. All hearts underwent 30 min global ischemia (30 degrees C) after infusion of 5 ml of cardioplegia. Cardioplegia consisted of either 30 mmol/l KCl (7 hearts) or nicorandil (100 micromol/l) in Tyrode solution (6 hearts). After a 30-min blood reperfusion, ESPVR and VO2-PVA relation were assessed again. Mean end-systolic pressure (ESP(mLVV)) and mean PVA at midrange LV volume (PVA(mLVV)) significantly (P < 0.05) decreased to 79.1 +/- 13.4% and 85.4 +/- 17.1% of control after KCl arrest and to 85.3 +/- 14.8% and 86.4 +/- 16.9% of control after nicorandil arrest. There were no significant differences in both decreases of mean ESP(mLVV) and PVA(mLVV) between each arrest. The slopes of VO2-PVA relations were also unchanged after each arrest. There was a significant (P < 0.005) difference in the decreases of mean VO2 intercepts of VO2-PVA relations between post-KCl arrest (73.9 +/- 8.2% of control) and post-nicorandil arrest (99.2 +/- 10.1% of control), however. Proteolysis of alpha-fodrin due to Ca2+ overload was significantly marked after KCl arrest. The present results indicate that the total calcium handling in excitation-contraction coupling is transiently impaired after KCl arrest, whereas it is unchanged after nicorandil arrest. This suggests the possibility that nicorandil is a better cardioplegia than KCl.

Effects of heat shock on in vitro development and intracellular oxidative state of bovine preimplantation embryos.

We investigated the effects of heat shock on developmental competence of bovine embryos and intracellular oxidative state. After in vitro fertilization, embryos were exposed to heat shock at 41 degrees C for 6 hr on days 0, 2, 4, and 6, respectively. On day 2, cleavage rate was not significantly different in all groups. However, the percentage of embryos developing to blastocyst stage after exposure to heat shock on day 0 (18.8 +/- 4.3%) and day 2 (23.6 +/- 3.7%) were significantly decreased compared with control (37.5 +/- 4.0%), day 4 (40.0 +/- 7.4%), and day 6 (38.1 +/- 2.0%). In addition, the total cell number of blastocysts was significantly decreased by heat shock on day 0 (107.5 +/- 6.6) and day 2 (112.8 +/- 5.7) compared with the control (143.2 +/- 9.4). To evaluate intracellular oxidative state by heat shock, embryos exposed to heat shock on days 0, 2, 4, and 6 were incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCHFDA) and fluorescence of oxidized DCHFDA by reactive oxygen species (ROS) was detected under fluorescent microscope. The intensity of fluorescence was significantly increased when embryos were exposed to heat shock on days 0 and 2. However, heat shock on day 4 and day 6 did not increase the fluorescence intensity. These results indicate that (1) heat shock to earlier stage embryos causes a decrease in development to blastocysts and cell proliferation and (2) the decrease in development by heat shock could be involved in an increase of intracellular oxidative stress. Mol. Reprod. Dev. 67: 77-82, 2004.

Relative changes in mRNA expression of angiopoietins and receptors tie in bovine corpus luteum during estrous cycle and prostaglandin F2alpha-induced luteolysis: a possible mechanism for the initiation of luteal regression.

Local angiogenesis and angiolysis in the corpus luteum (CL) relate to the luteal function. Recent studies indicate that angiopoietins (ANPT) and their receptors Tie regulate remodeling of microvasculature. We therefore examined 1) the relative changes in the expression of mRNA for ANPT-1, ANPT-2, Tie1 and Tie2 in bovine CL by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) during the estrous cycle and prostaglandin F2alpha (PGF2alpha)-induced luteolysis, and 2) the effect of ANPT-2 on progesterone (P4) release from CL at the late stage of the estrous cycle by an in vitro microdialysis system (MDS). The CLs were classified into 4 stages (early: Day 2-5, n=7, mid: Day 8-12, n=15, late: Day 15-17, n=9, regressing: Day >18, n=19). The levels of ANPT-1 mRNA in early and regressing CL were lower than those in mid and late CL, whereas ANPT-2 mRNA expression did not change during the estrous cycle. The Tie2 mRNA expression decreased as the CL aged. During PGF2alpha-induced luteolysis, ANPT-2 mRNA expression was acutely and temporally increased at 2 h after PGF2alpha injection. The expression of ANPT-1 mRNA was decreased from 4 h after PGF2alpha injection and kept low levels. In the experiment with the in vitro MDS, an infusion of ANPT-2 (100 ng/ml) acutely inhibited P4 release from late CL. Overall, results suggest that decrease of ANPT-1 mRNA is a basic mechanism of vascular remodeling in CL. In addition, ANPT-2 might play a role in regulation of P4 secretion in CL during luteolysis.

Changes in prostaglandin secretion by the regressing bovine corpus luteum.

Secretion of prostaglandins (PGs) by the regressing corpus luteum (CL) was investigated in the cow. Six cows were implanted with microcapillary dialysis membranes of a microdialysis system (MDS) into the CL during Days 8-9 (Day 0 = estrus), and a prostaglandin (PG) F2alpha analogue (Estrumate) was injected intramuscularly (i.m.) to induce luteolysis. Acute increases in intraluteal release of PGF2alpha and PGE2 were observed during the first 4 h, followed by decreases over the next 8 h. Intraluteal release of both PGs gradually increased again during the period 48-72 h. Concentrations of PGF2alpha in ovarian venous plasma (OVP) were 4-13 times higher than those of jugular venous plasma (JVP) (P < 0.001) during the period of the experiment, and increased from 24 h after treatment with Estrumate (P < 0.05). Cyclooxygenase (COX)-2 mRNA expression increased (P < 0.05) at 2 and 24 h after treatment with Estrumate. The results indicated that local release of PGF2alpha and PGE2, and COX-2 mRNA expression were increased by Estrumate in the regressing CL at the later stages of luteolysis. Thus, luteal secretion of PGs may be involved in the local mechanism for structural rather than functional luteolysis.

Hericium erinaceum (yamabushitake) extract-induced acute respiratory distress syndrome monitored by serum surfactant proteins.

This is the first report suggesting a causal relationship between acute respiratory distress syndrome and Hericium erinaceum extract, which is commercialized as a diet food. A 63-year-old man was admitted to our hospital for intensive care of severe acute respiratory failure with diffuse infiltration in both lungs. Bronchoalveolar lavage fluid revealed a high percentage of lymphocytes. Lymphocyte stimulation test showed a strong reactivity against extract formulation of Hericium erinaceum (Yamabushitake), which he had taken four months before onset. He recovered with successful steroid pulse therapy under mechanical ventilation. Concentrations of surfactant protein (SP)-A and SP-D in sera reflected the clinical features.

Oxygen costs of left ventricular contractility for dobutamine and Ca(2+) in normal rat hearts and the cost for dobutamine in Ca(2+) overload-induced failing hearts.

The aim of the present study was to compare the oxygen (O(2)) cost of left ventricular (LV) contractility (equivalent maximal elastance; eEmax, an index for contractility) for dobutamine (a beta-receptor stimulant) with that for calcium (Ca(2+)) in normal rat hearts and to assess the O(2) cost of LV eEmax for dobutamine in Ca(2+) overload-induced failing rat hearts. The mean O(2) cost of LV eEmax (x10(-4) microl O(2) x beat(-1) x mmHg(-1) x ml x g(-2)) for Ca(2+) was 1.30+/-0.37 in 12 normal hearts, and for dobutamine it was 1.26+/-0.30 in eight different normal hearts. In the same five normal hearts, the mean O(2) cost of LV eEmax for dobutamine was 1.15+/-0.27, and for Ca(2+) it was 0.81+/-0.36. These mean values showed no significant differences between Ca(2+) and dobutamine. In five Ca(2+) overload-induced failing hearts, the mean O(2) cost of LV eEmax for Ca(2+) could not be assessed, but the mean O(2) cost of LV eEmax for dobutamine was 1.04+/-0.40. This mean value for dobutamine did not differ significantly from those (see above 1.26+/-0.30 or 1.15+/-0.27) in the normal hearts. The present results indicate, in terms of the coupling of mechanical work and energetics of the heart, that the total Ca(2+) handling VO(2) in excitation-contraction coupling against unit LV contractility change for dobutamine in the contractile failing hearts does not differ from that in the normal hearts. This suggests that in the Ca(2+) overload-induced contractile failing hearts, there were no changes in the sensitivity of the contractile machinery for Ca(2+), in the Ca(2+)/ATP in the total Ca(2+) handling, and in the ATP/VO(2) in the mitochondria.

Effects of LH and PGF2alpha in equine dominant follicles observed by MDS.

Microdialysis System (MDS) is a novel technique used for investigation of molecule secretion between different cell populations. Local hormonal secretion at follicular wall has been still unclear. This MDS study was used to determine progesterone (P4), androstenedione (A4), estradiol-17beta (E2) and Prostaglandin F2alpha (PGF2alpha) release in mare pre-ovulatory follicles. Follicles larger than 30 mm were isolated from the ovary and follicular fluid aspirated for hormone assay. Follicular fluid collected from small, middle and large follicles were analyzed by EIA. The concentrations of P4 and PGF2alpha were similar among the different sizes of follicles. The release of A4 was observed in middle and large follicles. E2 concentration was observed in middle follicles and was higher in large follicles compared with middle follicles. Follicular wall was cut and incubated for MDS and when LH was infused, there was an increase in P4 and A4 release. PGF2alpha release was considerably high after LH infusion compared to the control group. Infusion of PGF2alpha increased P4 and A4 release but there was no change in E2 release. This results suggest that in pre-ovulatory follicles, LH stimulates theca interna cells and also PGF2alpha seemed to have a mediator role to induce steroid hormone production and luteinization of follicular cells. The nature of the mechanisms involved in selection of large follicles is still a perplexing research problem in reproduction.

Calcium-dependent activation of Pyk2 by hypoxia.

The Pyk2 tyrosine kinase can be activated by both calcium-dependent and calcium-independent mechanisms. Exposure to moderate hypoxia (5% O(2)) induced a rapid and persistent tyrosine phosphorylation of Pyk2 in pheochromocytoma (PC12) cells. Hypoxia and KCl-depolarization increased the phosphotyrosine content of Pyk2 by twofold and fourfold, respectively. Both of these effects were abolished in the absence of extracellular calcium. There was a modest activation of MAPK in parallel with the onset of Pyk2 phosphorylation. However, there was no detectable activation of either JNK or c-src, two other known downstream targets of Pyk2. Thus, exposure to hypoxia may selectively target specific subsets of Pyk2 signalling pathways.

Left ventricular diastolic dysfunction in type 2 diabetes mellitus model rats.

To gain insight into the pathogenesis of diabetic cardiomyopathy, we investigated cardiac function in terms of the coupling of left ventricular mechanical work and the energetics in Otsuka Long-Evans Tokushima Fatty rats, which are well known as a model of type 2 diabetes mellitus (DM). Neither left ventricular systolic function and mean coronary flow nor coronary flow reserve differed even in late DM rats. The amount of oxygen required for mechanical work and contraction was unaltered, although myosin isozyme was finally transformed from V(1) to V(3). The maximum pacing rate was decreased from 300 to 240 beats/min, and the left ventricular relaxation rate was significantly (P < 0.05) slower only in late DM rats, resulting in decreased oxygen consumption per minute for total Ca(2+) handling in excitation-contraction coupling mainly consumed by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) without significant changes in basal metabolism or in mitochondrial oxidative phosphorylation. The protein level of SERCA2 in membranes was significantly (P < 0.001) lower in severe DM rats. We conclude that the only lusitropic dysfunction due to the depressed expression of SERCA2 is related to generating diabetic cardiomyopathy even in the present type 2 diabetic rats.

Intraluteal release of angiotensin II and progesterone in vivo during corpora lutea development in the cow: effect of vasoactive peptides.

The newly formed corpus luteum (CL) develops rapidly and has the features of active vascularization and mitosis of steroidogenic cells. Such local mechanisms must be strictly regulated by the complex relationship between angiogenic growth factors and vasoactive peptides such as angiotensin (Ang) II, atrial natriuretic peptide (ANP), and endothelin (ET)-1. Thus, the objective of the present study was to determine 1) the changes in vasoactive peptides and progesterone (P) concentrations within the developing CL, along with the changes in concentration in ovarian venous plasma (OVP) and jugular venous plasma (JVP) in the cow, 2) the effects of CL exposure to vasoactive peptides on Ang II and P secretion, and 3) the expression of mRNA for ANP type C receptor in the bovine CL and endothelial cells (ETC) from bovine developing CL. A microdialysis system (MDS) was surgically implanted into multiple CL of six cows on Day 3 after a GnRH injection that induced superovulation, and a catheter was simultaneously inserted into the ovarian vein. The Ang II concentration in OVP was higher than that in JVP throughout the experiment, while the intraluteal release of Ang II was stable. During the experimental period, the concentrations of other vasoactive peptides (ANP and ET-1) showed no clear changes in plasma and were below detectable levels in the MDS perfusate. Exposure of CL to Ang II using the MDS stimulated P release, while exposure to ANP enhanced Ang II release within the developing CL. However, ET-1 had no effect on either P or Ang II release. The expression of mRNA for ANP type C receptor was mainly observed in early CL and ETC. The results suggest that the ET-Ang-ANP system in the preovulatory follicle switches to an Ang-ANP system to enhance both the angiogenesis and steroidogenesis that are actively occurring in developing CL.