Hamster Cheek Pouch Microcirculation Research Articles

Protective effects of Ruscus extract in combination with ascorbic acid and hesperidine methylchalcone on increased leukocyte-endothelial interaction and macromolecular permeability induced by ischemia reperfusion injury.

Despite the well-recognized effectiveness of Ruscus aculetus extract combined or not with ascorbic acid (AA) and hesperidine methyl chalcone (HMC) on ischemia reperfusion (I/R) injury protection, little is known about the contribution of each constituent for this effect. To investigate the effects of AA and HMC combined or not with Ruscus extract on increased macromolecular permeability and leukocyte-endothelium interaction induced by I/R injury. Hamsters were treated daily during two weeks with filtered water (placebo), AA (33, 100 and 300 mg/kg/day) and HMC (50, 150 and 450 mg/kg/day) combined or not with Ruscus extract (50, 150 and 450 mg/kg/day). On the day of experiment, the cheek pouch microcirculation underwent 30 min of ischemia, and the number of rolling and adherent leukocytes and leaky sites were evaluated before ischemia and during 45 min of reperfusion. Ruscus extract combined with AA and HMC (Ruscus extract mixture) significantly prevented post-ischemic increase in leukocyte rolling and adhesion and macromolecular permeability compared to placebo and these effects were more prominent than AA and HMC alone on leukocyte adhesion and macromolecular leakage. Ruscus extract mixture were more effective than its isolated constituents in protect the hamster cheek pouch microcirculation against I/R injury.

Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract

The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract.Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2+ liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking.Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50–100μg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2+ liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction.Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors.

Microvascular responses to aldosterone in hamster cheek pouch microcirculation

The aim of the present study was to assess the in vivo effects of aldosterone topically applied on the hamster cheek pouch microcirculation under baseline conditions or during ischemia-reperfusion. Male Syrian hamsters were anesthetized, tracheotomized and intubated. They were studied under baseline conditions or submitted to ischemia-reperfusion. Cheek pouch microvessels were visualized by fluorescence microscopy. Microvascular parameters were determined by computerized methods. Under baseline conditions, aldosterone (0.2, 0.5, 2.4 μM/L/2 min) induced dose-dependent constriction of all arterioles within 2.0 ± 0.5 min of administration. Diameter reduction was in the same range in smaller arterioles: A3 ones constricted by 24 ± 3% of baseline (at the highest dose). Aldosterone applied prior to ischemia and at reperfusion caused arteriolar constriction, marked microvascular permeability (0.66 ± 0.03 Normalized Grey Level), reduction in perfused capillary (-70 ± 4% of baseline) and leukocyte adhesion. All changes were statistically significant compared with ischemic animals. Potassium canrenoate (mineralcorticoid receptor inhibitor) prior to aldosterone did not abolish the aldosterone-induced effects, while valsartan (angiotensin II AT1 receptor inhibitor) prior to aldosterone ameliorated microvascular ischemia-reperfusion injury. In conclusion, aldosterone determined dose-dependent arteriolar constriction likely by angiotensin II type-1 receptor activation (non-genomic mechanism) worsening the effects of ischemia-reperfusion on capillary perfusion, while protecting from free radical formation.

Effects of babassu nut oil on ischemia/reperfusion-induced leukocyte adhesion and macromolecular leakage in the microcirculation: Observation in the hamster cheek pouch

BackgroundThe babassu palm tree is native to Brazil and is most densely distributed in the Cocais region of the state of Maranhão, in northeastern Brazil. In addition to the industrial use of refined babassu oil, the milk, the unrefined oil and the nuts in natura are used by families from several communities of African descendants as one of the principal sources of food energy. The objective of this study was to evaluate the effects of babassu oil on microvascular permeability and leukocyte-endothelial interactions induced by ischemia/reperfusion using the hamster cheek pouch microcirculation as experimental model.MethodsTwice a day for 14 days, male hamsters received unrefined babassu oil (0.02 ml/dose [BO-2 group], 0.06 ml/dose [BO-6 group], 0.18 ml/dose [BO-18 group]) or mineral oil (0.18 ml/dose [MO group]). Observations were made in the cheek pouch and macromolecular permeability increase induced by ischemia/reperfusion (I/R) or topical application of histamine, as well as leukocyte-endothelial interaction after I/R were evaluated.ResultsThe mean value of I/R-induced microvascular leakage, determined during reperfusion, was significantly lower in the BO-6 and BO-18 groups than in the MO one (P < 0.001). In addition, histamine-induced increase of microvascular permeability was significantly less pronounced in BO groups compared to MO one. No significant differences among groups in terms of leukocyte adhesion, concentrations of tumor necrosis factor alpha, interleukin 1, and interleukin 6 were found.ConclusionsOur findings suggest that unrefined babassu oil reduced microvascular leakage and protected against histamine-induced effects in postcapillary venules and highlights that these almost unexploited nut and its oil might be secure sources of food energy.

Preconditioning of the response to ischemia/reperfusion-induced plasma leakage in hamster cheek pouch microcirculation

OBJECTIVE:Ischemic preconditioning and some drugs can protect tissues from injury by preserving microcirculation. This study evaluated vascular permeability in a hamster cheek pouch preparation using either short ischemic periods or bradykinin as preconditioning stimuli followed by 30 min of ischemia/reperfusion.METHOD:Sixty-six male hamsters were divided into 11 groups: five combinations of different ischemic frequencies and durations (one, three or five shorts periods of ischemia, separated by one or five minutes) with 10 min intervals between the ischemic periods, followed by 30 min ischemia/reperfusion; three or five 1 min ischemic periods with 10 min intervals between them followed by the topical application of histamine (2 µM); bradykinin (400 nM) followed by 30 min of ischemia/reperfusion; and three control groups (30 min of ischemia/reperfusion or histamine or bradykinin by themselves). Macromolecular permeability was assessed by injection of fluorescein-labeled dextran (FITC-dextran, MW = 150 kDa; 250 mg/Kg body weight), and the number of leaks/cm2 was counted using an intravital microscope and fluorescent light in the cheek pouch.RESULTS:Plasma leakage (number of leaks/cm2) was significantly reduced by preconditioning with three and five 1 min ischemic periods, one and three 5 min ischemic periods and by bradykinin. Histamine-induced macromolecular permeability was also reduced after three periods of 5 min of ischemia.CONCLUSION:Short ischemic periods and bradykinin can function as preconditioning stimuli of the ischemia/reperfusion response in the hamster cheek pouch microcirculation. Short ischemic periods also reduced histamine-induced macromolecular permeability.

Maxadilan, the Lutzomyia longipalpis vasodilator, drives plasma leakage via PAC1–CXCR1/2-pathway

Experiments were designed to determine if the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage through activation of leukocytes and to what extent these effects could be due to PAC1 and CXCR1/2 receptor stimulation. Intravital microscopy of hamster cheek pouches utilizing FITC-dextran and rhodamine, respectively, as plasma and leukocyte markers was used to measure arteriolar diameter, plasma leakage and leukocyte accumulation in a selected area (5mm2) representative of the hamster cheek pouch microcirculation. Our studies showed that the sand fly vasodilator maxadilan and PACAP-38 induced arteriolar dilation, leukocyte accumulation and plasma leakage in postcapillary venules. The recombinant mutant of maxadilan M65 and an antagonist of CXCR1/2 receptors, reparixin, and an inhibitor of CD11b/CD18 up-regulation, ropivacaine, inhibited all these effects as induced by maxadilan. Dextran sulfate, a complement inhibitor with heparin-like anti-inflammatory effects, inhibited plasma leakage and leukocyte accumulation but not arteriolar dilation as induced by maxadilan and PACAP-38. In vitro studies with isolated human neutrophils showed that maxadilan is a potent stimulator of neutrophil migration comparable with fMLP and leukotriene B4 and that M65 and reparixin inhibited such migration. The data suggest that leukocyte accumulation and plasma leakage induced by maxadilan involves a mechanism related to PAC1- and CXCR1/2-receptors on leukocytes and endothelial cells.

Effects of propionyl-L-carnitine on ischemia–reperfusion injury in hamster cheek pouch microcirculation

Background and purpose Propionyl-l-carnitine (pLc) exerts protective effects in different experimental models of ischemia–reperfusion (I/R). The aim of the present study was to assess the effects of intravenously and topically applied pLc on microvascular permeability increase induced by I/R in the hamster cheek pouch preparation. Methods The hamster cheek pouch microcirculation was visualized by fluorescence microscopy. Microvascular permeability, leukocyte adhesion to venular walls, perfused capillary length, and capillary red blood cell velocity (VRBC) were evaluated by computer-assisted methods. E-selectin expression was assessed by in vitro analysis. Lipid peroxidation and reactive oxygen species (ROS) formation were determined by thiobarbituric acid-reactive substances (TBARS) and 2′-7′-dichlorofluorescein (DCF), respectively. Results In control animals, I/R caused a significant increase in permeability and in the leukocyte adhesion in venules. Capillary perfusion and VRBC decreased. TBARS levels and DCF fluorescence significantly increased compared with baseline. Intravenously infused pLc dose-dependently prevented leakage and leukocyte adhesion, preserved capillary perfusion, and induced vasodilation at the end of reperfusion, while ROS concentration decreased. Inhibition of nitric oxide synthase prior to pLc caused vasoconstriction and partially blunted the pLc-induced protective effects; inhibition of the endothelium-derived hyperpolarizing factor (EDHF) abolished pLc effects. Topical application of pLc on cheek pouch membrane produced the same effects as observed with intravenous administration. pLc decreased the E-selectin expression. Conclusions pLc prevents microvascular changes induced by I/R injury. The reduction of permeability increase could be mainly due to EDHF release induce vasodilatation together with NO. The reduction of E-selectin expression prevents leukocyte adhesion and permeability increase.

Platelet‐activating factor (PAF) and bradykinin (BK) induce venular hyperpermeability in part by decreasing cAMP levels and Epac activity

We have established earlier that PAF causes hyperpermeability via eNOS‐derived NO. We investigate here the alternative hypothesis that PAF and BK diminish cAMP levels and Epac activity and thus reduce the barrier properties of the endothelium. We studied the hamster cheek pouch microcirculation by intravital microscopy and measured FITC‐Dx clearance (Cl‐FITC‐Dx) as an index of macromolecular transport. We homogenized the pouches and determined cAMP content (ELISA kit) at defined intervals related to changes in Cl‐FITC‐Dx. We applied 8‐CPT‐2′‐O‐Me‐cAMP (8‐CPT) topically 2 min after PAF application to stimulate Epac activity. PAF (10−7M) and BK (10−6M) elevated Cl‐FITC‐Dx from 2.66 + 0.4 and 2.21 + 0.76 to 112.28 + 43.02 and 67.76 + 10.03 nl/min, respectively. Application of 8‐CPT reduced the PAF and BK‐induced elevation in Cl‐FITC‐Dx by 50%. cAMP levels in tissues collected at 5 min, 15 min and 30 min after PAF application were both reduced by 30% compared to baseline (Basal; 29.5 ± 4.5, PAF 30 min; 18.7 ± 1.3 pmol cAMP/g). We speculate that PAF and BK stimulate hyperpermeability via two complementary mechanisms: a) eNOS‐derived NO and b) reduction of cAMP levels and Epac activity. We demonstrate that the PAF‐ and BK‐induced hyperpermeability can be deactivated by increasing Epac activity. (Supported by Fondecyt 1040816, Proyecto puente VRAID y NIH 5RO1 HL070634‐06).

The involvement of CGRP, adrenomedullin, and sensory nerves in remote vasomotor responses within the hamster cheek pouch microcirculation

Previous work from our laboratory demonstrated a role for sensory nerves in remote dilations to microapplied methacholine by blocking the response with CGRP8-37 and concluded CGRP was the neurotransmitter. Recently, a more specific CGRP receptor antagonist, BIBN4096BS, was developed. The goals of the present study are to characterize the effects of BIBN4096BS on vasomotor responses in the hamster cheek pouch microcirculation, and to verify the role of CGRP in remote dilations to capsaicin and methacholine and to test adrenomedullin as an alternative neurotransmitter. BIBN4096BS pretreatment inhibits dilation to CGRP while having no significant effect on baseline diameter, it shifts the EC 50 to superfused CGRP from 1.5 ± 0.3 pM to 2.5 ± 0.6 nM and it shifts the apparent EC 50 to capsaicin from 31.5 nM to 171 nM. Local and remote dilations caused by the microapplication of methacholine are not inhibited by 300 nM BIBN4096BS (Local: 9.7 ± 1.2 versus 9.7 ± 1.5; 500:5.5 ± 0.4 versus 5.7 ± 0.5; 1000:4.4 ± 0.6 versus 4.8 ± 0.5). Remote dilations to methacholine were significantly inhibited however when adrenomedullin receptor antagonist adrenomedullin-(26–52) was microapplied to the remote site. Perivascular neurons containing adrenomedullin can be detected with immunohistochemistry. The results, combined with previous work, suggest that adrenomedullin, and not CGRP, is involved in remote dilations to methacholine.

Neutrophil‐induced increase in vascular permeability involves activation of the contact system

Adhesion of polymorphonuclear leukocytes (PMN) to the endothelial cell (EC) lining leads to increased vascular permeability. Plasma prekallikrein (PK), factor XII (FXII) and H‐kininogen are principal components of the contact system assembling on the EC surface. Activated PK cleaves H‐kininogen, liberating bradykinin that triggers EC contraction and paracellular gap formation. Here, we investigated the role of the contact system in PMN‐evoked plasma extravasation. Thioglycollate‐induced pleurisy in the mouse was used to study PMN‐dependent plasma leakage. FITC‐dextran served as plasma marker. The fluorescence intensity and PMN content in the exudate was markedly increased 4 h after thioglycollate injection, whereas in neutropenic animals, the plasma leakage was abolished. Treatment with antagonists to bradykinin B1 and B2 receptors, or an inhibitor to PK/FXII largely reduced fluid extravasation by 79% and 74% respectively. Intravital microscopy of the hamster cheek pouch microcirculation revealed that treatment with the B1/B2 antagonists reduced plasma leakage without reducing PMN adhesion. These results demonstrate a critical role of contact system activation in PMN‐evoked increase in vascular permeability. Our data also indicate that PMN‐dependent permeability changes and PMN extravasation may be regulated independent of each other.

Intermittent hypoxia modulates nitric oxide-dependent vasodilation and capillary perfusion during ischemia-reperfusion-induced damage

The microvascular function of nitric oxide (NO) during ischemia-reperfusion (I/R) in intermittent hypoxia (IH)-pretreated hamsters was analyzed using 20 mg/kg of the nonselective NO inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and 5 mg/kg of the preferential inducible NO inhibitor S-methylisothiourea sulphate (SMT) injected before I/R. Studies were made in the hamster cheek pouch microcirculation (intravital fluorescence microscopy). IH consisted of 6 min of 8% O(2) breathing followed by 6 min of 21% O(2) for every 8 h for 21 days. Normoxia controls (NCs) were exposed to room air for the same period. The effects were characterized in terms of systemic hemodynamics, diameter, flow, wall shear stress in arterioles, capillary perfusion, and the concentrations of thiobarbituric acid-reactive substances (TBARS) and plasma NO, assessed as nitrite/nitrate (NOx) levels. IH did not change arterial blood pressure and increased hematocrit and shear stress. IH increased NOx and TBARS levels and reduced arterial diameter, blood flow, and capillary perfusion versus the NC. Conversely, TBARS and NOx were lower during I/R in IH-pretreated hamsters, resulting in vasodilation and the increase of capillary perfusion and shear stress. After IH, capillary perfusion was reduced by 24% (2.3%) and enhanced by 115% (1.7%) after I/R (P < 0.05). Both modalities of NO blockade decreased NOx generation and increased TBARS versus IH. l-NAME and SMT induced a significant decrease in arteriolar diameter, blood flow, and capillary perfusion (P < 0.05). l-NAME enhanced TBARS more than SMT and aggravated I/R damage. In conclusion, we demonstrated that preconditioning with IH greatly reduces oxidative stress and stimulates NO-induced vasodilation during I/R injury, thus maintaining capillary perfusion.

ITF1697, a Stable Lys-Pro-Containing Peptide, Inhibits Weibel-Palade Body Exocytosis Induced by Ischemia/Reperfusion and Pressure Elevation

A number of Lys-Pro-containing short peptides have been described as possessing a variety of biological activities in vitro. Because of limited metabolic stability, however, their efficacy in vivo is uncertain. To exploit the pharmacological potential of Lys-Pro-containing short peptides, we synthesized a series of chemically modified forms of these peptides. One of them, ITF1697 (Gly-(Nalpha-Et)Lys-Pro-Arg) was stable in vivo and particularly efficacious in experimental models of disseminated endotoxemia and of cardiovascular disorders. Using intravital fluorescence microscopy, we studied the peptide cellular and molecular basis of protection in the Syrian hamster cheek pouch microcirculation subjected to ischemia/reperfusion (I/R) and in pressure elevation-induced proinflammatory responses in isolated Sprague-Dawley rat lungs. Continuous intravenous infusion of ITF1697 at 0.1 to 100 mug/kg/min nearly completely protected the cheek pouch microcirculation from I/R injury as measured by decreased vascular permeability and increased capillary perfusion. Adhesion of leukocytes and platelets to blood vessels was strongly inhibited by the peptide. ITF1697 exerted its activity at the early stages of endothelial activation and inhibited P-selectin and von Willebrand factor secretion. Further mechanistic studies in the rat lung preparation revealed that the peptide inhibited the intracellular Ca(2+)-dependent fusion of Weibel-Palade bodies with the plasma membrane. The ability of ITF1697 to inhibit the early functions of activated endothelial cells, such as the exocytosis of Weibel-Palade bodies, represents a novel and promising pharmacological tool in model of pathologies of a variety of microvascular disorders.

Bradykinin- and substance P-induced edema formation in the hamster cheek pouch is tyrosine kinase dependent

The purpose of this study was to determine whether protein tyrosine kinase, a ubiquitous family of intracellular signaling enzymes that regulates endothelial cell function, modulates bradykinin- and substance P-induced increase in macromolecular efflux from the intact hamster cheek pouch microcirculation. Using intravital microscopy, I found that suffusion of bradykinin or substance P (each, 0.5 and 1.0 microM) onto the cheek pouch elicited significant, concentration-dependent leaky site formation and increase in clearance of fluorescein isothiocyanate-dextran (FITC-dextran; molecular mass, 70 kDa; P < 0.05). These responses were significantly attenuated by suffusion of genistein (1.0 microM) or tyrphostin 25 (10 microM), two structurally unrelated, nonspecific protein tyrosine kinase inhibitors (P < 0.05). Conceivably, the kinase(s) involved in this process could be agonist specific because genistein was more effective than tyrphostin 25 in attenuating bradykinin-induced responses while the opposite was observed with substance P. Both inhibitors had no significant effects on adenosine (0.5 M)-induced responses (P > 0.5). Collectively, these data suggest that the protein tyrosine kinase metabolic pathway modulates, in part, the edemagenic effects of bradykinin and substance P in the intact hamster cheek pouch microcirculation in a specific fashion.

Pharmacological action of the CGRP antagonist, BIBN4096BS, on vasomotor responses in the hamster cheek pouch microcirculation

Previous work from our laboratory demonstrated a role for capsaicin-sensitive sensory nerves in remote dilations to methacholine. In this previous study, the release of CGRP from sensory nerves was implicated based on the ability of the antagonist, CGRP8-37, to inhibit remote dilations to methacholine. However, the concentrations of CGRP8-37 in these experiments (30–300 μM) may inhibit neurotransmitters other than CGRP. Recently, a more specific CGRP antagonist, BIBN4096BS, was developed for clinical use. The goals of the present study are to characterize the effects of BIBN4096BS in the hamster cheek pouch microcirculation, and to determine the role of CGRP in remote dilations to capsaicin and methacholine. Dilation to 10−9 M CGRP (16.8±0.7μm to 35±2.7μm) is reversed by 10−7 BIBN4096BS within 5 minutes (18.2±1.6μm). Similarly, 10−7 BIBN4096BS pretreatment attenuates dilation to 10−9 M CGRP (control: 16±1.5 to 30.3±2.6; BIBN4096BS:18.9±1 to 20.42±1.1) while having no significant effect on baseline diameter (19.0±1 to 18.9±1). BIBN4096BS shifts the apparent EC50 to capsaicin from 31.5nM to 171nM. Microinjected capsaicin results in consistent local dilations and variable remote dilations which are inhibited by BIBN4096BS, however local and remote responses to microinjected methacholine are not affected by BIBN4096BS. The results suggest that CGRP is not the neurotransmitter involved in remote dilations to methacholine.

Longchain n-3 polyunsaturated fatty acids and microvascular reactivity: Observation in the hamster cheek pouch

Previous experiments in our laboratory, using the hamster cheek pouch microcirculation, have shown that precapillary vessels exhibit spontaneous rhythmic luminal variations, termed vasomotion, a myogenic activity sustained by a balance between membrane currents among which polarizing K + currents play an important role. In these microvessels, endothelium-derived relaxing factors (EDRFs) seem to regulate arteriolar diameter [via nitric oxide (NO) and cyclic GMP] and vasomotion [probably via endothelium-derived hyperpolarizing factor (EDHF)]. Fish or fish oil diet can decrease the risk of cardiovascular diseases, probably by modifying the conductance of selective ion channels, such as K + and/or Ca ++, and/or increasing the production of vasodilators, such as NO. To investigate its effect on microvascular reactivity, using the same preparation and an intravital microscope coupled to a closed circuit TV system, male hamsters were treated for 14 days, twice a day, with 0.4 mL/100 g body weight with fish or olive oil. An attempt was also undertaken to record in arterioles, in vivo, the membrane potential of smooth muscle cells during their vasomotor activity combining conventional microelectrode and intravital microscopy techniques. The effects of topical application of two vasodilators, acetylcholine [endothelium-dependent one, NO release and membrane hyperpolarization via Ca ++-activated K + channels (K Ca)] and sodium nitroprusside (endothelium-independent, NO donor and no change on membrane potential) and two vasoconstrictors which elicited membrane depolarization via Ca ++ channels, phenylephrine (α 1-adrenergic receptor agonist) and serotonin (5-hydroxi-tryptamine) on mean internal diameter of arterioles and venules, arteriolar blood flows, spontaneous arteriolar vasomotion frequency and amplitude and functional capillary density (FCD, number of capillaries with flowing red blood cells per unit area of tissue) were determined. Anesthesia was induced by sodium pentobarbital (i.p.) and maintained with α-chloralose through the femoral vein. In the presence of vasomotion, the membrane potentials are slowly oscillating by about 20 mV around values of approximately − 50 mV in perfect synchrony with vasomotor movements and depolarizing phases coincide with vasoconstrictions while polarizing ones with vasodilatations. Comparing all parameters, in control conditions, only the spontaneous vasomotion frequency was significantly higher (2.37 times higher) on the group treated with fish oil and persisted as such throughout all experiments. With topical application of the drugs mentioned above, the group treated with fish oil showed, for each drug concentration, a balance towards vasodilatation with consequent increase on arteriolar blood flow and on FCD, compared with the olive oil treated one. No significant changes on mean arterial pressure, spontaneous arteriolar vasomotion amplitude or venular diameter could be detected in the two groups. Our results support the concept that, in the hamster cheek pouch microcirculation, fish oil supplementation activates K + channels which act as the EDHF and might also increase the production of vasodilators, probably NO.

Acupuncture Reduces Experimental Renovascular Hypertension Through Mechanisms Involving Nitric Oxide Synthases

To test the hypothesis that acupuncture on stomach 36 point (ST-36) reduces hypertension by activating nitric oxide synthase signaling mechanisms. The authors used the two-kidney, one-clip renal hypertension (2K1C) hamster model with electroacupuncture treatment. Thirty-minute daily electroacupuncture treatment for 5 days reduced mean arterial pressure from 160.0 +/- 7.6 to 128.0 +/- 4.3 mmHg (mean +/- SEM), compared to 115.0 +/- 7.2 mmHg in sham-operated hamsters. Electroacupuncture increased periarteriolar NO concentration from 309.0 +/- 21.7 nM to 417.9 +/- 20.9 nM in the 2K1C hamster cheek pouch microcirculation when measured with NO-sensitive microelectrodes. Hypertension reduced endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) proteins relative to the sham-operated control, as measured by Western blotting. Electroacupuncture prevented the reduction of eNOS and nNOS associated with hypertension and showed even higher eNOS and nNOS expressions than sham-operated control in stomach and cheek pouch tissues, which are on the stomach meridian. Analysis of liver tissue, a non-stomach-meridian organ, indicated that electroacupuncture did not have a significant benefit in terms of enhanced expressions of eNOS and nNOS in the treated 2K1C hypertensive group. Activation of eNOS and nNOS is one of the mechanisms through which ST-36 electroacupuncture reduces blood pressure; this reduction works through the stomach meridian.

TRPV1 in microvascular regulation

Few studies have described the in vivo role of the capsaicin receptor, TRPV1, in the context of microcirculatory control. Recent in vitro studies have suggested a role for TRPV1 in both the initiation of myogenic tone, and vasodilation; however in vitro experiments may not fully reflect responses seen in vivo. In this study we characterize the microcirculatory response to TRPV1 activation in vivo using the hamster cheek pouch microcirculation. Superfusion of capsaicin, a TRPV1 agonist, results in dose dependent dilation with an EC50 of 31 nM. Superfusion with the TRPV1 antagonist, capsazepine (3x10−6M) or acute desensitization of TRPV1 by repeated superfusate application of 10−6 M capsaicin results in the loss of capsaicin induced vasodilation, but has no influence on myogenic tone. Similarly, chemical dennervation of the cheek pouch (4mg/ml capsaicin injected into the cheek pouch 3 days before experiments) abolishes vasodilator response to capsaicin but has no effect on the development of myogenic tone. Bupivacaine (10−4 M) does not prevent capsaicin induced vasodilation, suggesting that vasodilation can occur in the absence of neuronal sodium channel activation and afferent nerve conduction. Brief, perivascular micro-application of capsaicin (10−5M) causes local dilation which decays 500–700 μm from the activation site. Repeated capsaicin micro-application (10−5 M) results in localized loss of vasodilation at the application site, but preserves capsaicin-induced vasodilation in upstream or downstream segments of the arteriole and its branches. These data support a role for TRPV1 in arteriolar vasodilation, and do not support a role for TRPV1 in the development of myogenic tone.

Electroacupuncture on stomach 36 point (ST‐36) reduces hypertension through endothelial nitric oxide synthase (eNOS) activity in the stomach meridian

Acupuncture has been used in Asia to treat hypertension for a long time. We hypothesized that acupuncture on ST-36 activates eNOS signaling mechanisms to reduce hypertension. We tested the hypothesis using the hamster two-kidney, one-clip renal hypertension (2K1C) model. 30 minute daily electroacupuncture (EA) treatment for 5 days reduced mean arterial pressure from 160.0±7.6 to 129.5±8.8 mmHg (mean±SEM), compared to 115.0±7.2 mmHg in sham-operated hamsters. We used NO-sensitive microelectrodes to measure periarteriolar NO concentration. EA increased periarteriolar NO concentration from 82.3±10.2 nM to 135.1±12.6 nM in the 2K1C hamster cheek pouch microcirculation. Hypertension reduced eNOS protein relative to the sham-operated control, as measured by Western blotting. EA prevented the reduction of eNOS associated with hypertension and showed even higher eNOS expression than the sham-operated control in stomach and hamster cheek pouch tissues, which are on the stomach meridian. Analysis of liver tissue indicated that EA on ST-36 caused only a slight increase in eNOS expression in hypertensive animals compared with the non-treatment 2K1C group. The liver eNOS levels in 2K1C-treated hamsters were less than in sham-operated hamsters. We conclude 1) stimulation of eNOS is a mechanism stimulated by ST-36 elecroacupuncture to reduce blood pressure; 2) ST-36 elecroacupuncture-induced reduction of blood pressure works through the stomach meridian (supported by NIH grant 5RO1 HL 70634).

No Pain, No Gain

Myocardial ischemia, the major cause of mortality and morbidity in the United States, accounts for nearly 20% of all deaths.1 The prognosis of patients with acute myocardial infarction is strongly related to the amount of tissue destroyed during ischemia/reperfusion; hence, limiting this injury is of paramount importance for reducing the morbidity and mortality associated with ischemic heart disease.2,3 Over the past 40 years, several hundred pharmacological or nonpharmacological therapies have been reported to alleviate ischemia/reperfusion injury in experimental animal models; unfortunately, few of these results have been reproducible, and, with the exception of early reperfusion, none have been translated into clinical therapies.4 These sobering facts provide a cogent rationale for a reassessment of the models and methodologies that have been used heretofore to test the efficacy of putative cardioprotective interventions. The obvious translational failure in this field calls for a new paradigm in which preclinical studies must be done as rigorously as their clinical counterparts, that is, using clinically relevant animal models, appropriate statistical methods, and a multicenter, randomized, blinded design using centralized core laboratories for data analysis.4 This paradigm would also improve our understanding of the mechanism of cardiomyocyte death during ischemia/reperfusion. Article p 3617 One facet of ischemic biology that has received relatively little attention is the role of cardiac sensory nerves. Myocardial ischemia/reperfusion is known to cause the release of bradykinin and protons, both of which activate the vanilloid receptor 1 (VR1, also known as transient receptor potential vanilloid type 1 [TRPV1]) on the capsaicin-sensitive cardiac sensory nerves (unmyelinated [type C] or thinly myelinated [A-δ] fibers).5 The VR1 is a nonselective cation channel that plays an important role in the polymodal detection of noxious stimuli such as low pH and heat.6 It can also be activated by chemicals such as capsaicin …

Protective Effects of Insulin during Ischemia-Reperfusion Injury in Hamster Cheek Pouch Microcirculation

Objective: The effects of insulin (0.18 nM–0.18 µM) on reduced capillary perfusion, microvascular permeability increase and leukocyte adhesion induced by ischemia-reperfusion injury were investigated in the hamster cheek pouch microcirculation. To gain insight into the insulin’s mechanism of action, the effects of its higher concentration (0.18 µM) were investigated after inhibition of tyrosine kinase (TK), nitric oxide synthase (NOS), protein kinase C (PKC), phosphatidylinositol 3-kinase and K⁺_(ATP) channels, alone or in combination. Two concentrations for each inhibitor were used. Methods: Microcirculation was visualized by fluorescence microscopy. Perfused capillary length, microvascular permeability, leukocyte adhesion to venular walls, vessel diameter and capillary red blood cell velocity were assessed by computer-assisted methods. Measurements were made at baseline (B), after 30 min of ischemia (I), and after 30 min of reperfusion (R). Results: In control animals, perfused capillary length decreased by 63 ± 5% of baseline at R. Microvascular permeability increased at I and R, while leukocyte adhesion was most pronounced in V1 postcapillary venules at R. Insulin dose-dependently preserved capillary perfusion at R (–28 ± 6 and –15 ± 6% of baseline), but was unable to prevent the increase in permeability at I (0.25 ± 0.05 and 0.29 ± 0.06 Normalized Grey Levels, NGL) and R (0.49 ± 0.10 and 0.53 ± 0.09 NGL), according to the concentrations. Adhesion of leukocytes was observed mostly in V3 venules at R (9 ± 2 and 10 ± 2/100 µm venular length, with the lower and higher concentration, respectively). Nitric oxide synthase inhibition by N^G-nitro-L-arginine-methyl ester prior to insulin did not affect capillary perfusion at R (–18 ± 3% of baseline with higher concentration), but prevented permeability increase (0.20 ± 0.04 NGL, according to higher concentration) and reduced leukocyte adhesion in V3 venules at R (1.5 ± 1.0/100 µm of venular length, with higher concentration). Blockade of K⁺_(ATP) channels by glibenclamide prior to insulin decreased perfused capillary length at R (–58 ± 6% of baseline with higher concentration), attenuated leakage at R (0.30 ± 0.04 NGL, according to higher concentration) and caused leukocyte adhesion mainly in V1 venules at R (9.0 ± 1.5/100 µm of venular length, with higher concentration). Inhibition of either TK, PKC or phosphatidylinositol 3-kinase did not affect microvascular responses to insulin. Simultaneous inhibition of TK and NOS did not increase protection. Conclusions: Insulin prevents ischemia-reperfusion injury by promoting capillary perfusion through an apparent activation of K⁺_(ATP) channels and increase in nitric oxide release.