Long-term treatment with the streptococcal exotoxin streptolysin O inhibits vascular smooth muscle contraction by inducing iNOS expression in endothelial cells.

Streptolysin O (SLO), a bacterial toxin produced by common hemolytic streptococci, including Streptococcus pyogenes and resident microbiota, may be associated with inflammation in the cardiovascular system. We previously reported that short-term treatment with SLO at relatively high concentrations (10-1000 ng/mL) diminished acetylcholine-induced, endothelial-dependent relaxation in a concentration-dependent manner. However, the vascular function effects of long-term exposure to SLO at lower concentrations are poorly understood. In this study, treatment of rat aorta with endothelium with SLO (0.1-10 ng/mL) for 72 h inhibited contractions in response to norepinephrine and phenylephrine in a concentration-dependent manner, and this effect was abolished by endothelium denudation. We also observed decreased endothelium-dependent relaxation in aorta treated with a lower concentration of SLO (10 ng/mL) for 72 h. Long-term treatment with SLO (10 ng/mL) increased the expression of iNOS in aorta with endothelium but not aorta without endothelium, and the SLO-induced decrease in contraction was restored by treatment with NOS inhibitors. Pharmacologic and gene-mutant analyses further indicated that SLO-induced vascular dysfunction and iNOS upregulation are mediated through the TLR4/NOX2/ROS/p38 MAPK pathways. In vivo SLO treatment (46.8 pg/kg/min) for 7 days also diminished vascular contraction and relaxation activity in aorta with endothelium. We concluded that long-term treatment with SLO inhibits vascular contractile responses, primarily due to increased iNOS expression in the endothelium through TLR4-mediated pathways. Our present results, together with those of our previous study, suggest that endothelial cells play a key role in the pathophysiologic changes in cardiovascular function associated with long-term exposure to SLO. Significance Statement In the present study, we showed that long-term exposure to streptococcal exotoxin SLO inhibits agonist-induced contraction in rat aorta with endothelium, driven primarily by elevated iNOS production via NOX2-mediated ROS production through TLR4 activation on endothelial cells. In vivo treatment with SLO for 7 days also diminished vascular contraction and relaxation, providing evidence of possible pathophysiologic roles of SLO in endothelium-dependent vascular homeostasis.

Hypertension is linked to enhanced lymphatic contractile response via RGS16/RhoA/ROCK pathway.

Lymph capillary network can be expected to alter blood pressure via regulating interstitial electrolyte and volume balance. However, the pathophysiology of lymphatic vessel in hypertension is poorly understood. In this study, we examined lymph vessel function focusing on contractile response in hypertensive rats. It was found that thoracic ducts isolated from adult (10-14 wk old) spontaneously hypertensive rats (SHRs) exhibited increased agonist-mediated contraction compared with age-matched Wistar-Kyoto (WKY) rats, whereas lymphatic contractions in younger (4 wk old) SHRs, exhibiting normal blood pressure, were no different compared with age-matched control rats. Tight regulation of blood pressure with antihypertensive drugs (hydrochlorothiazide/hydralazine) did not prevent the augmented lymphatic contraction in adult SHRs; however, treatment of SHRs with angiotensin II (ANG II) type 1 receptor blocker (losartan) for 6 wk abolished the augmentation of lymphatic contractions. In addition, ANG II infusion in Wistar rat caused augmented lymphatic contractile responses in the thoracic duct. The augmented contractions in adult SHRs were diminished by a ROCK inhibitor (Y-27632). Consistently, the thoracic ducts in SHRs showed significantly higher phosphorylation of myosin phosphatase targeting protein-1 than WKY rats. Furthermore, gene expression profiling of adult SHR lymphatics showed marked loss of regulator of G-protein signaling 16 (RGS16) mRNA, which was confirmed by the real-time PCR. Treatment with the RGS inhibitor CCG-63808 enhanced contractions in thoracic ducts from Wistar rats, which were abolished by the ROCK inhibitor. It is concluded that lymphatic contractile function was enhanced in hypertensive model rats, which could be mediated by dysregulation of the ROCK pathway possibly through RGS16.NEW & NOTEWORTHY Lymph capillary controls interstitial electrolyte and volume balance, which may blunt increased blood pressure. However, the function of lymphatic vessel in hypertension is poorly understood. Our study showed that the lymphatic smooth muscle contractility is hyperreactive in two different hypertensive models. The lymphatic dysfunction could be mediated by dysregulation of ROCK pathway possibly through RGS16. The present finding supports a new concept showing the functional relationship between lymphatic contractile activity and hypertension.

Streptococcal Exotoxin Streptolysin O Causes Vascular Endothelial Dysfunction Through PKCß Activation.

Streptolysin O (SLO) is produced by common hemolytic streptococci that cause a wide range of diseases from pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. Although the importance of SLO in invasive hemolytic streptococcus infection has been well demonstrated, the role of circulating SLO in noninvasive infection remains unclear. The aim of this study was to characterize the pharmacological effect of SLO on vascular functions, focusing on cellular signaling pathways. In control Wistar rats, SLO treatment (1-1000 ng/ml) impaired acetylcholine-induced endothelial-dependent relaxation in the aorta and second-order mesenteric artery in a dose-dependent manner without any effects on sodium nitroprusside-induced endothelium-independent relaxation or agonist-induced contractions. SLO also increased phosphorylation of the endothelial NO synthase (eNOS) inhibitory site at Thr495 in the aorta. Pharmacological analysis indicated that either endothelial dysfunction or eNOS phosphorylation was mediated by protein kinase Cß (PKCß), but not by the p38 mitogen-activated protein kinase pathway. Consistent with this, SLO increased phosphorylation levels of protein kinase C substrates in the aorta. In vivo study of control Wistar rats indicated that intravenous administration of SLO did not change basal blood pressure but significantly counteracted the acetylcholine-induced decrease in blood pressure. Interestingly, plasma anti-SLO IgG levels were significantly higher in 10- to 15-week-old spontaneously hypertensive rats compared with age-matched control rats (P < 0.05). These findings demonstrated that SLO causes vascular endothelial dysfunction, which is mediated by PKCß-induced phosphorylation of the eNOS inhibitory site. SIGNIFICANCE STATEMENT: This study showed for the first time that in vitro exposure of vascular tissues to SLO impairs endothelial function, an effect that is mediated by protein kinase C ß-induced phosphorylation of the endothelial NO synthase inhibitory site. Intravenous administration of SLO in control and hypertensive rats blunted the acetylcholine-induced decrease in blood pressure, providing evidence for a possible role of SLO in dysregulation of blood pressure.

Treatment with Ligilactobacillus murinus lowers blood pressure and intestinal permeability in spontaneously hypertensive rats.

One feature of hypertension is a microbial imbalance with increased intestinal permeability. In this study, we examined whether an alteration in the microbiota affects blood pressure and intestinal permeability in spontaneously hypertensive rats (SHRs). We performed a 16S metagenome analysis of feces from 10- to 15-week-old SHRs using a synthetic long-read sequencing approach, and found a candidate for the microbiome treatment, Ligilactobacillus murinus (L. murinus), that was robustly decreased. Oral administration of L. murinus to SHRs for 2 weeks significantly inhibited blood pressure elevation and improved endothelium-dependent vasodilation but did not attenuate enhanced vascular contraction in SHR mesenteric arteries. The proximal colon of SHRs exhibited increased intestinal permeability with decreased levels of the tight junction protein claudin 4, morphological changes such as decreased intestinal crypts and elevated TNF-α levels, which was reversed by treatment with L. murinus. Consistent with these intestinal phenotypes, plasma lipopolysaccharides levels were elevated in SHR but decreased following L. murinus administration. We concluded that oral administration of L. murinus to SHRs exerts protective effects on intestinal permeability via restoration of claudin 4 expression and reversal of morphologic disorder, which may improve low-grade endotoxemia and thus reduce development of hypertension via recovery of endothelial vasodilating functions.

[Gut microflora and metabolic syndrome: new insight into the pathogenesis of hypertension].

Emerging evidences indicate that a microbial imbalance (dysbiosis) is linked to several diseases including metabolic cardiovascular diseases. A fecal microbiota transplantation from hypertensive human donor to germ-free mice caused blood pressure elevation. In addition, there is a report demonstrating that angiotensin II-induced hypertension and vascular dysfunction were attenuated in germ-free mice, suggesting that gut microbiome may mediate development of hypertension. Although detailed mechanism by which the dysbiosis induces an increased blood pressure remains unknown, changes in microbiome may modify host immune systems and induce inflammatory dysfunction in cardiovascular system, resulting in dysregulation of blood pressure. Some cohort studies demonstrated an association between a higher abundance of Streptococcaceae spp. and blood pressure. One recent report demonstrated that an increasing number of gram-positive Streptococcus was found in the feces of adult spontaneously hypertensive rats with an increased intestinal permeability. We hypothesized that increased bacterial toxin levels derived from gut Streptococcus may be a factor inducing blood pressure dysregulation. In this review, we discuss the possible role of microbiome in cardiovascular disease, especially hypertension.

Increased Blood Pressure Causes Lymphatic Endothelial Dysfunction via Oxidative Stress in Spontaneously Hypertensive Rats.

The lymphatic system is involved in the pathogenesis of edema, inflammation, and cancer metastasis. Because lymph vessels control fluid electrolytes and volume balance, changes in lymphatic activity can be expected to alter systemic blood pressure. This study examined possible changes in lymphatic contractile properties in spontaneously hypertensive rats (SHR). Thoracic ducts isolated from 10- to 12-week-old SHR exhibited either decreased acetylcholine-induced endothelium-dependent relaxation or sodium nitroprusside-induced endothelium-independent relaxation compared with age-matched Wister-Kyoto rats. The impairment in acetylcholine responsiveness was more pronounced than sodium nitroprusside responsiveness. N-Nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor blunted acetylcholine-induced relaxation in Wister-Kyoto rats, indicating an involvement of endothelial nitric oxide production. Endothelial dysfunction in lymph vessels of SHR was attenuated by tempol (a superoxide dismutase mimetic), apocynin, or VAS-2870 (NADPH oxidase inhibitors). Consistent with these observations, nitrotyrosine levels were significantly elevated in SHR, indicative of increased oxidative stress. In addition, protein expression of NADPH oxidase 2 and phosphorylation of p47phox (Ser345) were significantly increased in SHR. Further, SB203580 (a p38 MAPK inhibitor) restored the acetylcholine-induced relaxation in SHR. It is notable that 4-week-old SHR, which exhibited normal blood pressure, did not show any decreased activity of acetylcholine- or sodium nitroprusside-induced relaxation. Additionally, antihypertensive treatment of 4-week-old SHR with hydrochlorothiazide and reserpine or hydrochlorothiazide and hydralazine for 6 weeks completely restored lymphatic endothelial dysfunction. We conclude that contractile activity of lymphatic vessels is functionally impaired with the development of increasing blood pressure, which is mediated through increased oxidative stress via the p38 MAPK/NADPH oxidase 2 pathway.

Salt causes aging-associated hypertension via vascular Wnt5a under Klotho deficiency.

Aging is associated with a high prevalence of hypertension due to elevated susceptibility of BP to dietary salt, but its mechanism is unknown. Serum levels of Klotho, an anti-aging factor, decline with age. We found that high salt (HS) increased BP in aged mice and young heterozygous Klotho-knockout mice and was associated with increased vascular expression of Wnt5a and p-MYPT1, which indicate RhoA activity. Not only the Wnt inhibitor LGK974 and the Wnt5a antagonist Box5 but Klotho supplementation inhibits HS-induced BP elevation, similarly to the Rho kinase inhibitor fasudil, associated with reduced p-MYPT1 expression in both groups of mice. In cultured vascular smooth muscle cells, Wnt5a and angiotensin II (Ang II) increased p-MYPT1 expression but knockdown of Wnt5a with siRNA abolished Ang II-induced upregulation of p-MYPT1, indicating that Wnt5a is indispensable for Ang II-induced Rho/ROCK activation. Notably, Klotho inhibited Wnt5a- and Ang II-induced upregulation of p-MYPT1. Consistently, Klotho supplementation ameliorated HS-induced augmentation of reduced renal blood flow (RBF) response to intra-arterial infusion of Ang II and the thromboxane A2 analog U46619, which activated RhoA in both groups of mice and were associated with the inhibition of BP elevation, suggesting that abnormal response of RBF to Ang II contributes to HS-induced BP elevation. Thus, Klotho deficiency underlies aging-associated salt-sensitive hypertension through vascular non-canonical Wnt5a/RhoA activation.

High Salt Diet Impacts the Risk of Sarcopenia Associated with Reduction of Skeletal Muscle Performance in the Japanese Population.

The World Health Organization has recommended 5 g/day as dietary reference intakes for salt. In Japan, the averages for men and women were 11.0 g/day and 9.3 g/day, respectively. Recently, it was reported that amounts of sodium accumulation in skeletal muscles of older people were significantly higher than those in younger people. The purpose of this study was to investigate whether the risk of sarcopenia with decreased muscle mass and strength was related to the amount of salt intake. In addition, we investigated its involvement with renalase. Four groups based on age and salt intake ("younger low-salt," "younger high-salt," "older low-salt," and "older high-salt") were compared. Stratifying by age category, body fat percentage significantly increased in high-salt groups in both younger and older people. Handgrip strength/body weight and chair rise tests of the older high-salt group showed significant reduction compared to the older low-salt group. However, there was no significant difference in renalase concentrations in plasma. The results suggest that high-salt intake may lead to fat accumulation and muscle weakness associated with sarcopenia. Therefore, efforts to reduce salt intake may prevent sarcopenia.

Critical roles of VEGF-C-VEGF receptor 3 in reconnection of the collecting lymph vessels in mice.

Molecular mechanisms of reconnection of collecting lymph vessels were analyzed by using murine popliteal prenodal lymph vessels. At 1 and 2 weeks after being divided by cutting the lymph vessel, lymphatic reconnection was frequently observed accompanied by mesh-like lymphatic channels. Electron microscopic study also showed a monolayer of endothelial cells in the newly developed lymph vessels. Smooth muscle markers were immunofluorescently demonstrated in the wall of the new vessels. At 1 week after the procedure of cutting, augmented expressions of VEGF receptors 1, 2 and 3 were found immunohistochemically at the site of the reconnected lymph vessels. The expression of mRNA for VEGF receptor 3 was enhanced at 5 days and 1 week in small pieces of the tissues containing the reconnected lymph vessels, compared with that in the corresponding tissues obtained with sham operated ones. The administration of VEGF-C at the cutting site of the collecting lymph vessel significantly increased the rate of the reconnected lymph vessels, whereas additional treatment with Flt4/Fc chimera protein significantly reduced the rate of the reconnected ones. These results suggest that activation of VEGF-C-VEGF receptor 3 has critical roles in reconnection of the collecting lymph vessels in adult mice.

Head-down tilt posture elicits transient lymphocyte mobilization from the iliac, but not mesenteric, lymph nodes of rats.

The effects of short-term simulated microgravity on the lymph dynamics of rat lymph nodes were investigated using a combination of Bollman's cage and head-down tilt (HDT). Efferent lymphatics of the iliac and mesenteric lymph nodes were cannulated for the collection of lymph. There was no significant difference in lymph flow rate from the iliac lymph nodes between non-HDT (control) and HDT rats. Lymph flow rate from the mesenteric lymph nodes in HDT rats was slightly higher than that obtained with the control. The cell count obtained from the iliac lymph nodes in HDT rats was significantly larger than those of the controls, while no significant difference in the number of cells from the mesenteric lymph nodes was observed between the control and HDT groups. The cells from the iliac lymph nodes in the control and HDT rats were mostly lymphocytes. The distribution of subsets of lymphocytes (CD3+, CD4+, CD8a+, and CD45R+) from the iliac lymph nodes in HDT rats was not significantly different from the subsets of lymphocytes in the control. Immunization did not affect the distribution of lymphocyte subsets from the iliac lymph nodes in the control and HDT groups. There was no significant difference in the concentrations of lymph albumin in iliac afferent or efferent lymphatics between the control and HDT groups. These findings suggest that HDT posture in Bollman's cage induces transient output of lymphocytes from the iliac lymph nodes of rats in vivo without changing the flow rate, lymphocyte subsets, or concentration of albumin.

Stromal interaction molecule 1 modulates blood pressure via NO production in vascular endothelial cells.

In vascular endothelial cells, store-operated calcium entry (SOCE) activates endothelial NO synthase (eNOS) and regulates nitric oxide (NO) production as well as flow-dependent mechanical stimuli. Stromal interaction molecule 1, or STIM1, was recently identified to be essential for SOCE, acting as a calcium sensor for intracellular calcium stores. However, how STIM1 affects endothelial function and blood pressure (BP) remains unclear. We generated STIM1 fl/fl mice and vascular endothelial cell-specific STIM1 knockout mice using the Cre-loxP system, and conducted experiments using these mice to clarify the physiological role of STIM1 in vascular endothelial function and BP as follows: (1) SOCE was analyzed in isolated aortic endothelial cells by calcium add-back with fluorescent Ca2+ indicators. Phosphorylation of eNOS and NO production were evaluated by immunoblotting and the NO indicator, respectively. (2) Tension of aortic rings was measured in 10-week-old mice in response to acetylcholine. (3) BP was measured in 10-week-old mice by the telemetry system. The results were: (1) SOCE, eNOS activation, and NO production were suppressed by ~50-60% in endothelial cells from STIM1 knockout. (2) Endothelium-dependent vasodilation was decreased in aortic rings from STIM1 knockout mice, whereas endothelium-independent relaxation was not altered. (3) STIM1 knockout mice exhibited significant BP elevation, especially in nighttime. (124.3 ± 2.5/99.2 ± 3.9 vs. 114.1 ± 3.2/83.6 ± 1.7 (nighttime, mmHg), 109.7 ± 1.7/83.0 ± 3.0 vs. 104.8 ± 3.3/73.7 ± 1.6 (daytime, mmHg), knockout vs. control, respectively). In conclusion, STIM1 in vascular endothelial cell modulates vascular function through NO production and has a major role in regulating BP, especially in the active time.

Immunohistochemical demonstration of proliferating lymphatic vessels in colorectal carcinoma and its clinicopathological significance.

Lymphatic metastasis to the regional lymph nodes through the lymphatic vessels is an important indicator of poor prognosis in many types of malignant tumors. Recently, much attention has been paid to lymphangiogenesis for its possible role on tumor progression in various carcinomas. However, morphological evidence that lymphatic vessels actively proliferate in colorectal carcinoma has not been reported. Here, we first devised a triple immunostaining method to detect proliferating lymphatic vessels utilizing antibody to Ki-67 antigen as a marker of cell proliferation, antibody to cytokeratin as an epithelial cell marker, and antibody to podoplanin as a lymphatic vessel-specific marker. Ki-67/podoplanin-immunoreactivity enabled us to identify proliferating lymphatic vessels, while cytokeratin immunoreactivity allowed us to distinguish proliferating lymphatic vessels from Ki-67/cytokeratin-positive carcinoma cells in lymphatic lumens. Analyzing 64 colorectal carcinoma patients' samples using this technique, we showed that both lymphatic vessel density and proliferating activity of lymphatic vessels were significantly increased in colorectal carcinoma tissues compared with their normal counterparts. We then examined the correlation between the degree of lymphangiogenesis and patients' prognosis or clinicopathological variables, but no statistically significant differences were obtained in these analyses. Thus, these results combined together indicate that extensive lymphangiogenesis occurs in colorectal carcinoma, but that the degree of lymphangiogenesis alone is not an independent prognostic factor for this disease.

Characterization and microarray analysis of genes in human lymphatic endothelial cells from patients with breast cancer.

We successfully isolated human lymphatic endothelial cells from afferent lymph vessels (HALEC) of sentinel lymph nodes in patients with breast cancer by using trypsin digestion. The cells were cultured in EGM-2 medium with 10% FBS under the condition of 5% O2, 5% CO2, and 90% N2 at 37 degrees C. The cultured cells exhibited a monolayer with cobblestone appearance and a marked phagocytosis of Dil-Ac-LDL. Immunohistochemical lymphatic vessel markers were also found, such as podoplanin, LYVE-1, VEGF receptor 3, and Prox-1. Quantitative RT-PCR analysis also showed that podoplanin, VEGF R3, and Prox-1 mRNA were expressed more selectively in the cultured cells. The cells had marked immunoreactivity to antisera of ecNOS, iNOS, COX1, and weak reactivity of COX2. Constitutively expressed cell-type specific genes of the cultured cells were also analyzed by oligonucleotide microarray methods. Compared with human umbilical vein endothelial cells (HUVEC), the cells selectively expressed 88 known genes such as angiopoietin-like 4, oxygen radicals-related enzymes, and adhesion molecules and the related proteoglycans. The findings suggest that the cultured cells seem to be human lymphatic endothelial cells. In conclusion, the isolated, cannulated and enzymatic digested method we adopted for culture of human lymphatic endothelial cells may be easy and useful for investigating cellular, molecular biological, and genomic properties of the cells.

Examiner's finger-mounted near-infrared spectroscopy is feasible to analyze cerebral and skeletal muscle oxygenation in conscious Chihuahuas.

To measure regional saturation of oxygen ( rSO 2 ) of hemoglobin and total hemoglobin index (HbI) in the brain (through the molera of the head) and skeletal muscle (musculus gracilis) of conscious Chihuahua dogs using an examiner's finger-mounted near-infrared spectroscopy (NIRS) device, Toccare, we investigated brain and skeletal muscle NIRS in 48 Chihuahuas without severe disease. To measure rSO 2 and total HbI, a Toccare probe was placed on the molera of the head and musculus gracilis of each dog for real-time recording. Stable NIRS values were obtained within 10 s. We also examined the effect of anesthesia on rSO 2 and total HbI of a Chihuahua. Cerebral rSO 2 values ( 59 % ± 7 % ) were significantly lower than those obtained at femoral regions ( 67 % ± 6 % ), whereas total HbI values in the brain ( 0.38 ± 0.09 ) were significantly higher than those of the musculus gracilis ( 0.20 ± 0.05 ). Sedation with a combination of medetomidine and ketamine decreased cerebral rSO 2 along with a corresponding reduction in heart rate. Sevoflurane anesthesia with 100% O 2 maintained rSO 2 in the brain with a

Current topics of physiology and pharmacology in the lymphatic system.

We have reviewed physiological significance of rhythmical spontaneous contractions of collecting lymph vessels, which play a pivotal role in lymph transport and seem to control lymph formation through changing the pacemaker sites of the rhythmic contractions and contractile patterns of the lymphangions. A characteristic feature that the rhythmic pump activity works in vivo physiologically under the specific environment of lower oxygen tension in lymph (25-40 mm Hg) has been evaluated. With the characteristic feature, generation of endogenous nitric oxide (NO) from lymphatic endothelial cells and/or activation of ATP-sensitive potassium channels (K(ATP)) are reviewed to play crucial roles in the regulation of lymph transport at physiological or pathophysiological conditions. Chemical substances released from malignant tumor cells and tumor-derived parathyroid hormone-related peptide (PTHr-P) are also shown to cause a significant reduction of lymphatic pump activity through generation of endogenous NO and activation of K(ATP) channels. Finally, we have discussed physiological significance and roles of the lower oxygen tension in lymph, generation of endogenous NO, and activation of K(ATP) in lymph formation, lymph transport, and the functions of lymph nodes.

Effects of exercise intensity, posture, pressure on the back and ambient temperature on palmar sweating responses due to handgrip exercises in humans.

We have, by using newly developed ratemeters, attempted to examine the effects of exercise intensity, posture, pressure on the skin of the back, and ambient hyperthermic conditions (approximately 30 degrees C) on the 5-s handgrip exercise-mediated responses of active palmar sweating in humans. Thirty-five right-handed male (n=5) and female (n=30) volunteer students (20.2+/-1.3 years old) participated in the present study. Oral explanation of only the isometric handgrip exercise (IHG) caused a rapid and oscillatory response (pre-operational) of active palmar sweating in almost all subjects (10 of 14 subjects). Performing the IHG for 5-s caused a significant increase in active sweating rate (operation-mediated response) in both ipsi- and contra-lateral palmar surfaces of the thumbs of all subjects. The operation-mediated responses of active palmar sweating to the IHG were reproducible, resulting in no habituation. The increase of operation-mediated responses to the IHG was dependent upon exercise intensity (100-25% maximal voluntary contractions). The IHG-mediated ipsi- and contra-lateral responses of active palmar sweating were significantly decreased by changing the body posture from a seated to a supine position or by pressing the skin of the back. Ambient hyperthermic conditions (approximately 30 degrees C) for 60 min also resulted in a significant decrease in the back-pressure-dependent reduction of the operation-mediated responses of active palmar sweating to the IHG. In conclusion, in order to optimize the precision and reproducibility of clinical tests involving palmar sweating responses, it is important that subjects maintain a constant handgrip force and posture and that ambient temperature be kept under normothermic conditions.

A new preparation for visualizing lymphatic flow pathway in isolated rat lymph nodes.

BACKGROUND: There is no reported study in which the lymphatic flow pathway in lymph nodes has been visualized and investigated in in vitro studies. The purpose of the present study is first to develop an isolated rat lymph node preparation circulated through the afferent and efferent lymph vessels in order to visualize lymphatic flow pathway within the node by using a video microscope system, and then to evaluate lymphatic flow dynamics by using fluorescence-labeled substances. METHODS AND RESULTS: In the present study, rat iliac lymph node was isolated. The afferent and efferent lymph vessels of the lymph node were cannulated with glass micropipettes in an organ chamber, while the small-sized arteries and veins connected to the lymph node were secured with sutures. Krebs-bicarbonate solution with or without fluorescent probes [FITCdextran, mol. weight; 77,000 and/or fluoresbrite carboxylate-microspheres (FC-microspheres) mean diameter of 1 microm] was circulated through the lymph vessels of the node. The time-dependent lymphatic pathway of fluorescent probes was investigated with a video microscope system. FTIC-dextran first spread through the cortex and subsequently reached the medulla of the lymph node. A follicle-like structure became evident in the cortex. FITC-dextran appeared in the efferent lymph vessel at 109 +/- 21 sec after its circulation began, while FC-microspheres distributed in the lymph node did not emerge from the node within 20 min after their introduction. CONCLUSIONS: In vitro preparations constructed in the present study will enable us to visualize the lymphatic flow pathway of fluorescent substances within the isolated iliac lymph nodes of rats in the absence of blood circulation.

Arachidonic Acid-Induced COX-1 and COX-2-Mediated Vasodilation in Rat Gingival Arterioles In Vivo.

The roles of cyclooxygenase (COX) and prostaglandins (PGs) in the regulation of vasoreactivity of rat gingival arterioles in vivo were evaluated by sing an intravital microscope. The superfusion of indomethacin (a nonselective COX inhibitor) or SC-560 (a selective COX-1 inhibitor) onto the gingiva significantly constricted the arterioles, though NS-398 (a selective COX-2 inhibitor) did not affect the diameter of the arterioles. The SC-560-mediated constriction of the arterioles was completely reversed by an additional treatment with arachidonic acid (AA). The superfusion of AA, beraprost-Na (an analogue of PGI2) or PGE2 onto the gingival significantly dilated the arterioles dose-dependently. The AA-induced dilation of the arterioles was significantly reduced by the treatment with SC-560 or NS-398. The expression of COX-1 and COX-2 were positive in the endothelium, but not the smooth muscles, of the arterioles. The expression of PGE synthase (PGES) was found only in the smooth muscles, but not the endothelium, of the arterioles. Neither the endothelium nor the smooth muscles of the arterioles expressed PGI synthase (PGIS). These findings suggest that the COX-2-mediated PG cascade may collaborate with the COX-1 pathway in the regulation of arteriolar myogenic activity in rat gingiva in the case of the supply of a large amount of AA.