Rat Cremaster Muscle Research Articles

Advanced glycated end-products inhibit dilation through constitutive endothelial RAGE and Nox1/4 in rat isolated skeletal muscle arteries.

This study investigated the actions of advanced glycated end-products (AGE), their receptors (RAGE), and NAD(P)H oxidase (Nox) subtypes 1, 2, and 4 on mechanisms of endothelium-dependent dilation of the rat cremaster muscle artery (CMA). Immunofluorescence studies were used to examine expression of RAGE in rat arteries. ROS accumulation was measured using luminescence and fluorescence assays. Functional studies were performed using pressure myography. High levels of RAGE expression were shown in the endothelial cells of the CMA, compared with low endothelial expression in middle cerebral and mesenteric arteries and the aorta. Exogenous AGE (in vitro glycated bovine serum albumin) stimulated H2O2 accumulation in CMA, which was prevented by the RAGE antagonist FPS-ZM1, the NAD(P)H oxidase (Nox) inhibitor apocynin and inhibited by the Nox1/4 inhibitor setanaxib, but not the Nox2 inhibitor GSK2795039. In functional studies, AGE inhibited vasodilation of CMA stimulated by acetylcholine, sodium nitroprusside, and the BKCa activator NS1619, but not adenosine-induced dilation. FPS-ZM1, apocynin, and setanaxib prevented the inhibitory effects of AGE on responses to acetylcholine and NS-1619. These observations suggest RAGE are constitutively expressed in the endothelium of the rat CMA and may be activated by AGE to stimulate Nox1/4 and ROS formation with resulting inhibition of NO and BKCa-mediated endothelium-dependent dilation.

5-HT7 receptors mediate dilation of rat cremaster muscle arterioles in vivo.

Serotonin (5-HT) infusion in vivo causes hypotension and a fall in total peripheral resistance. However, the vascular segment and the receptors that mediate this response remain in question. We hypothesized that 5-HT7 receptors mediate arteriolar dilation to 5-HT in skeletal muscle microcirculation. Cremaster muscles of isoflurane-anesthetized male Sprague-Dawley rats were prepared for in vivo microscopy of third- and fourth-order arterioles and superfused with physiological salt solution at 34°C. Quantitative real-time PCR (RT-PCR) was applied to pooled samples of first- to third-order cremaster arterioles (2-4 rats/sample) to evaluate 5-HT7 receptor expression. Topical 5-HT (1-10 nmols) or the 5-HT1/7 receptor agonist, 5-carboxamidotryptamine (10-30 nM), dilated third- and fourth-order arterioles, responses that were abolished by 1 μM SB269970, a selective 5-HT7 receptor antagonist. In contrast, dilation induced by the muscarinic agonist, methacholine (100 nmols) was not inhibited by SB269970. Serotonin (10 nmols) failed to dilate cremaster arterioles in 5-HT7 receptor knockout rats whereas arterioles in wild-type litter mates dilated to 1 nmol 5-HT, a response blocked by 1 μM SB269970. Quantitative RT-PCR revealed that cremaster arterioles expressed mRNA for 5-HT7 receptors. 5-HT7 receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to 5-HT-induced hypotension, in vivo.

Impact of cyanidin 3-O-glucoside on rat micro-and systemic circulation, possibly thorough angiogenesis.

Cyanidin 3-O-glucoside (C3G), an antioxidant, is one of the most abundant anthocyanin in plant foods. Intervention trials and subsequent meta-analyses have suggested that anthocyanins could reduce the risks of cardiovascular diseases. This study investigated hemodynamic alterations following a single intragastric dose of C3G by measuring blood flow in rat cremaster muscle arteriole for 60 min. Next, in excised aortas, we performed western blotting to measure the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). A single oral dose of C3G significantly increased blood flow soon after ingestion, and it was maintained throughout the experimental period. In addition, aortic Akt phosphorylation increased. Then, we examined the impact of repeated oral administrations of C3G for 14 days. The mean blood pressure was significantly reduced at 7 and 14 days after treatment, with a slight increase in aortic eNOS expression. Immunohistochemical analyses of the soleus showed that the level of CD31, an angiogenesis-marker protein, was significantly increased with C3G. These results suggested that an oral dose of C3G increased blood flow, which promoted angiogenesis within skeletal muscle, and consequently, blood pressure was reduced.

5‐HT7 Receptors Mediate Dilation of Rat Cremaster Muscle Arterioles in vivo

Serotonin (5‐HT) infusion in vivo causes hypotension and a fall in total peripheral resistance (TPR) that is mediated by 5‐HT7 receptors. 5‐HT dilates small arterioles in skeletal muscle, a microcirculation that importantly contributes to TPR. However, the receptors that mediate this dilation have not been established. Therefore, we tested the hypothesis that 5‐HT7 receptors mediate the arteriolar dilation in the skeletal muscle microcirculation. Cremaster muscles of isoflurane‐anesthetized male Sprague‐Dawley rats were prepared for digital in vivo microscopy and superfused with physiological salt solution (PSS) at 34 °C. Bolus (1‐10 nmols) application of 5‐HT into the superfusate caused substantial dilation of 3rd to 5th order arterioles from 10 ± 1 to 27 ± 1 μm (n = 10 arterioles from 4 rats, p <0.0001) that recovered within 2‐3 min. Dilation to bolus application of 5‐HT was inhibited by superfusion with 1 μM SB269970, a selective 5‐HT7 receptor antagonist (diameter in SB = 9 ± 1 μm; diameter in SB + 5‐HT = 10 ± 1 μm; n = 10 arterioles from 4 rats; p = 0.8763). Similarly, steady‐state superfusion with 10‐30 nM of the 5‐HT1/7 receptor agonist 5‐carboxamidotryptamine (5‐CT) dilated 3rd‐5th‐order arterioles from 16 ± 3 to 37 ± 5 μm (n = 7 arterioles from 3 rats, p = 0.0004). This dilation was reversed (diameter in 1 μM SB269970 = 13 ± 2 μm, n = 7, p = 0.1479 vs. baseline) or blocked (diameter in 1 μM SB269970 + 10‐30 nM 5‐CT = 10 ± 2 μm, n = 7, p = 0.0005 vs. 5‐CT alone) by 1 μM SB269970. Dilation of arterioles to the muscarinic agonist, methacholine (MCH) was not inhibited by SB269970 verifying the specificity of the antagonist; arterioles dilated from 13 ± 2 μm to 25 ± 2 μm to 100 nmols MCH and dilated from 9 ± 2 μm to 32 ± 2 μm in response to MCH in the presence of 1 μM SB269970; n = 10 arterioles from 4 rats; p = 0.7232 vs. dilation in the absence of SB269970. Consistent with these data, pooled samples (4‐5 rats) of 1st and 2nd – order cremaster arterioles expressed message for 5‐HT7 receptors by quantitative real‐time PCR (CT = 36.4 ± 0.74, n = 3 pooled samples, p = 0.039 vs CT = 40). These data support our hypothesis that 5‐HT7 receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to the observed hypotension when these receptors are engaged in vivo.

Effect of β1 /β2 -adrenoceptor blockade on β3 -adrenoceptor activity in the rat cremaster muscle artery.

The physiological role of vascular β3 -adrenoceptors is not fully understood. Recent evidence suggests cardiac β3 -adrenoceptors are functionally effective after down-regulation of β1 /β2 -adrenoceptors. The functional interaction between the β3 -adrenoceptor and other β-adrenoceptor subtypes in rat striated muscle arteries was investigated. Studies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. β-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of β3 -adrenoceptor agonists and antagonists and other β-adrenoceptor ligands were measured using pressure myography. All three β-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The β3 -adrenoceptor agonists mirabegron and CL 316,243 had no effect on the diameter of pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the β3 -adrenoceptor agonist SR 58611A and the nonselective β-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of β1/2 -adrenoceptor antagonists nadolol (10 μM), atenolol (1 μM) and ICI 118,551 (0.1 μM) both mirabegron and CL 316,243 were effective in causing vasodilation and the potency of SR 58611A was enhanced, while responses to isoprenaline were inhibited. The β3 -adrenoceptor antagonist L 748,337 (1 μM) inhibited vasodilation caused by β3 -adrenoceptor agonists (in the presence of β1/2 -adrenoceptor blockade), but L 748,337 had no effect on isoprenaline-induced vasodilation. All three β-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but β3 -adrenoceptor mediated vasodilation was only evident after blockade of β1/2 -adrenoceptors. This suggests constitutive β1/2 -adrenoceptor activity inhibits β3 -adrenoceptor function in the endothelium of skeletal muscle resistance arteries.

Redox Regulation of Microvascular Permeability: IL-1β Potentiation of Bradykinin-Induced Permeability Is Prevented by Simvastatin.

Antioxidant effects of statins have been implicated in the reduction in microvascular permeability and edema formation in experimental and clinical studies. Bradykinin (Bk)-induced increases in microvascular permeability are potentiated by IL-1β; however, no studies have examined the protection afforded by statins against microvascular hyperpermeability. We investigated the effects of simvastatin pretreatment on albumin–fluorescein isothiocyanate conjugate (FITC-albumin) permeability in post-capillary venules in rat cremaster muscle. Inhibition of nitric oxide synthase with L-NAME (10µM) increased basal permeability to FITC-albumin, which was abrogated by superoxide dismutase and catalase. Histamine-induced (1 µM) permeability was blocked by L-NAME but unaffected by scavenging reactive oxygen species with superoxide dismutase (SOD) and catalase. In contrast, bradykinin-induced (1–100 nM) permeability increases were unaffected by L-NAME but abrogated by SOD and catalase. Acute superfusion of the cremaster muscle with IL-1β (30 pM, 10 min) resulted in a leftward shift of the bradykinin concentration–response curve. Potentiation by IL-1β of bradykinin-induced microvascular permeability was prevented by the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) inhibitor apocynin (1 µM). Pretreatment of rats with simvastatin (5 mg·kg−1, i.p.) 24 h before permeability measurements prevented the potentiation of bradykinin permeability responses by IL-1β, which was not reversed by inhibition of heme oxygenase-1 with tin protoporphyrin IX (SnPP). This study highlights a novel mechanism by which simvastatin prevents the potentiation of bradykinin-induced permeability by IL-1β, possibly by targeting the assembly of NADPH oxidase subunits. Our findings highlight the therapeutic potential of statins in the prevention and treatment of patients predisposed to inflammatory diseases.

Implications of Systemic Hematocrit on the Radial Distribution of Red Cells in Arterioles

It is well‐established that the tube hemacocrit (HctT) in blood vessels and glass tubes with an internal diameter less than 200‐microns is significantly lower than feed or discharge Hct (the Fahraeus Effect). Microscopic imaging of the flow stream reveals a decrease in Hct in the vicinity of the wall (the plasma layer, PL) but the mechanism of the effect is unclear. To address this question, we recorded high speed videos (3000 fps) of blood flow in 35‐micron diameter arterioles of the rat cremaster muscle at systemic Hct (HctS) levels of 20% to 72%. We found that the PL is an annulus bordering the wall that occupies 12% to 50% of the flow stream cross section, and that it is inversely related to HctS. In addition, results indicate that the Hct in the core region of the flow stream is not significantly different from HctS. The position of the PL coincides with the region of maximal shear stress in the flow stream, which may strip away the red cell monolayer adjacent to the wall, creating the PL while the monolayer moves toward the faster‐flowing center of the flow stream. In conclusion, while volumetric flow is related to the cross section of the flow stream, the PL is related to the circumference, thus reducing HctT as radius decreases.Support or Funding InformationThis work was supported by the NIH Heart Lung and Blood Institute under Grants R01HL126945 and R01HL138116.

Numerical Model for the Determination of Erythrocyte Mechanical Properties and Wall Shear Stress in vivo From Intravital Microscopy.

The mechanical properties and deformability of Red Blood Cells (RBCs) are important determinants of blood rheology and microvascular hemodynamics. The objective of this study is to quantify the mechanical properties and wall shear stress experienced by the RBC membrane during capillary plug flow in vivo utilizing high speed video recording from intravital microscopy, biomechanical modeling, and computational methods. Capillaries were imaged in the rat cremaster muscle pre- and post-RBC transfusion of stored RBCs for 2-weeks. RBC membrane contours were extracted utilizing image processing and parametrized. RBC parameterizations were used to determine updated deformation gradient and Lagrangian Green strain tensors for each point along the parametrization and for each frame during plug flow. The updated Lagrangian Green strain and Displacement Gradient tensors were numerically fit to the Navier-Lame equations along the parameterized boundary to determined Lame's constants. Mechanical properties and wall shear stress were determined before and transfusion, were grouped in three populations of erythrocytes: native cells (NC) or circulating cells before transfusion, and two distinct population of cells after transfusion with stored cells (SC1 and SC2). The distinction, between the heterogeneous populations of cells present after the transfusion, SC1 and SC2, was obtained through principle component analysis (PCA) of the mechanical properties along the membrane. Cells with the first two principle components within 3 standard deviations of the mean, were labeled as SC1, and those with the first two principle components greater than 3 standard deviations from the mean were labeled as SC2. The calculated shear modulus average was 1.1±0.2, 0.90±0.15, and 12 ± 8 MPa for NC, SC1, and SC2, respectively. The calculated young's modulus average was 3.3±0.6, 2.6±0.4, and 32±20 MPa for NC, SC1, and SC2, respectively. o our knowledge, the methods presented here are the first estimation of the erythrocyte mechanical properties and shear stress in vivo during capillary plug flow. In summary, the methods introduced in this study may provide a new avenue of investigation of erythrocyte mechanics in the context of hematologic conditions that adversely affect erythrocyte mechanical properties.

Minimally invasive gas embolization using acoustic droplet vaporization in a rodent model of hepatocellular carcinoma

Hepatocellular carcinoma is the third leading cause of cancer-related deaths worldwide. Many patients are not eligible for curative therapies, such as surgical resection of the tumor or a liver transplant. Transarterial embolization is one therapy clinically used in these cases; however, this requires a long procedure and careful placement of an intraarterial catheter. Gas embolization has been proposed as a fast, easily administered, more spatially selective, and less invasive alternative. Here, we demonstrate the feasibility and efficacy of using acoustic droplet vaporization to noninvasively generate gas emboli within vasculature. Intravital microscopy experiments were performed using the rat cremaster muscle to visually observe the formation of occlusions. Large gas emboli were produced within the vasculature in the rat cremaster, effectively occluding blood flow. Following these experiments, the therapeutic efficacy of gas embolization was investigated in an ectopic xenograft model of hepatocellular carcinoma in mice. The treatment group exhibited a significantly lower final tumor volume (ANOVA, p = 0.008) and growth rate than control groups – tumor growth was completely halted. Additionally, treated tumors exhibited significant necrosis as determined by histological analysis. To our knowledge, this study is the first to demonstrate the therapeutic efficacy of gas embolotherapy in a tumor model.

Visualization of the intrarenal distribution of capillary blood flow.

This study describes a modified technique to fill the renal vasculature with a silicon rubber (Microfil) compound and obtain morphologic information about the intrarenal distribution of capillary blood flow under a variety of conditions. Kidneys and cremaster muscles of rats were perfused in vivo with Microfil using a perfusion pressure equal to the animal's mean arterial pressure at body temperature. Microfil did not alter arteriolar diameter or the pattern of flow in the microcirculation of the cremaster muscle. The modified protocol reproducibly filled the renal vasculature, including; glomerular, peritubular, and vasa recta capillaries. We compared the filling of the renal circulation in control rats with that seen in animals subjected to maneuvers reported to alter the intrarenal distribution of blood flow. Infusion of angiotensin II, hypotension, volume expansion, and mannitol‐ or furosemide‐induced diuresis redistributed flow between renal cortical and medullary capillaries. The advantage of the current technique is that it provides anatomical information regarding the number, diameter, and branching patterns of capillaries in the postglomerular circulation critical in determining the intrarenal distribution of cortical and medullary blood flow.

Alpha1 -adrenergic stimulation selectively enhances endothelium-mediated vasodilation in rat cremaster arteries.

We have systematically investigated how vascular smooth muscle α 1‐adrenoceptor activation impacts endothelium‐mediated vasodilation in isolated, myogenically active, rat cremaster muscle 1A arteries. Cannulated cremaster arteries were pressurized intraluminally to 70 mmHg to induce myogenic tone, and exposed to vasoactive agents via bath superfusion at 34°C. Smooth muscle membrane potential was measured via sharp microelectrode recordings in pressurized, myogenic arteries. The α 1‐adrenergic agonist phenylephrine (25–100 nmol/L) produced further constriction of myogenic arteries, but did not alter the vasorelaxant responses to acetylcholine (0.3 μmol/L), SKA‐31 (an activator of endothelial Ca2+‐dependent K+ channels) (3 μmol/L) or sodium nitroprusside (10 μmol/L). Exposure to 0.25–1 μmol/L phenylephrine or 1 μmol/L norepinephrine generated more robust constrictions, and also enhanced the vasodilations evoked by acetylcholine and SKA‐31, but not by sodium nitroprusside. In contrast, the thromboxane receptor agonist U46619 (250 nmol/L) dampened responses to all three vasodilators. Phenylephrine exposure depolarized myogenic arteries, and mimicking this effect with 4‐aminopyridine (1 mmol/L) was sufficient to augment the SKA‐31‐evoked vasodilation. Inhibition of L‐type Ca2+ channels by 1 μmol/L nifedipine decreased myogenic tone, phenylephrine‐induced constriction and prevented α 1‐adrenergic enhancement of endothelium‐evoked vasodilation; these latter deficits were overcome by exposure to 3 and 10 μmol/L phenylephrine. Mechanistically, augmentation of ACh‐evoked dilation by phenylephrine was dampened by eNOS inhibition and abolished by blockade of endothelial KCa channels. Collectively, these data suggest that increasing α 1‐adrenoceptor activation beyond a threshold level augments endothelium‐evoked vasodilation, likely by triggering transcellular signaling between smooth muscle and the endothelium. Physiologically, this negative feedback process may serve as a “brake” to limit the extent of vasoconstriction in the skeletal microcirculation evoked by the elevated sympathetic tone.

Numerical Determination of Red Blood Cell Membrane Deformability from Intravital Microscopy

IntroductionRed cells in microvessels deform extensively in order to flow, thus resulting in high observed apparent viscosities and high stress concentrations localized to the red cell membrane. Flow through microvessels is consequently aided by the high compliance in the red cell membrane. However, changes in the mechanical properties of Red Blood Cells (RBC) as a result of storage lesions, can increase stress concentrations in the membrane, impairing oxygen transport and resulting in increased vesiculation and likely cell lysis. The objective of this study is to determine the mechanical properties and thus wall shear stress experienced by a RBC membrane during capillary flow by applying numerical methods and image analysis to videos obtained using cremaster muscle preparation.MethodsThe RBC membrane is modeled as a two dimensional Hookean Isotropic Solid. Videos of capillary flow of both fresh RBCs and RBCs stored for 2 weeks were taken in the rat cremaster muscle. The mechanical properties, including the elastic modulus, shear stress, and shear strain, of both conditions were compared for both groups. In order to determine strain, each RBC membrane will be parameterized by N (X,Y) points determined by the Moore Neighbor Tracing algorithm of videos of capillary flow obtained by intravital microscopy of the rat cremaster muscle, from which N deformation gradient tensors, [F], can be obtained numerically for each frame of the video. From these deformation gradient tensors, the updated Cauchy Strain Tensor will be determined as a function of position on the Cartesian plane relative to the centroid of the RBC. The strains determined from the updated Langrangian Green Strain are numerically fit to the Navier‐Lame Equations along the boundary of the RBC in order to experimentally fit the two Lame's Constants during capillary flow. These constants will then be used to determine the Cauchy Stress Tensor, using Hooke's Law, along the boundary of the RBC, allowing for the calculation of the Wall Shear Stress.Results and DiscussionThe average Elastic Modulus of fresh cells was found to be 5.4 ×106 ± 3.6 × 106 Pa, while of that of RBCs stored for two weeks was found to be statistically larger and was calculated to be 6.0 ×106 ± 9.7 × 107 Pa. Additionally, stored RBCs experienced higher shear strains and wall shear strains over larger areas of the membrane as compared with stored cells. Additionally, stress concentrators in the membrane were observed at the points of contact with the capillary wall. However, for stored cells, the high stress acted on a larger segment of the membrane. This algorithm for determining wall shear stress experienced by RBC membranes in vivo during capillary flow was thus able to detect statistically significant differences in the both the mechanical properties and stress concentrations between fresh cells and stored cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelium Mediated Dilation Does Not Blunt α1‐adrenergic Vasoconstriction in First Order Arterioles

In contracting skeletal muscle, several vasoactive compounds have been proposed to override sympathetic vasoconstriction, a phenomenon termed functional sympatholysis. Adenosine triphosphate (ATP) has emerged as a candidate molecule capable of mediating these effects, however, the precise source and mechanisms have not been fully elucidated. We tested the hypothesis that endothelium mediated mechanisms are responsible for attenuating α‐adrenergic vasoconstriction. Experiments were conducted on 1A arterioles surgically dissected from the rat cremaster muscle. Sequential sections of arteriole were cannulated on glass micropipettes, pressurized to 70 mmHg, and warmed to 34°C in physiological saline solution. We measured changes in isolated vessel diameter in response to logarithmic concentrations of the α1‐adrenergic receptor specific agonist phenylephrine following dilatory responses to intraluminal flow (flow mediated dilation, n=12), intraluminal adenosine (n=10), or intraluminal ATP (n=11). All vessels tested spontaneously developed myogenic tone (>40% from initial diameter) and displayed functioning endothelium (>40% dilation to acetylcholine). Vessels dilated equally 177±19 μm, 183±10 μm, and 181±22 μm during flow mediated dilation, adenosine, and ATP, respectively. There were no significant differences between treatments following concentration‐response curves generated by phenylephrine administration and the EC50 was not different −5.90±0.02, −6.12±0.18, and −6.00±0.06 for flow mediated dilation, adenosine, or ATP (p=NS). Thus, there was no attenuation of α1‐adrenergic vasoconstriction in response to intraluminal flow mediated dilation, adenosine, or ATP in 1A arterioles. These results suggest that endothelium mediated dilation does not blunt α1‐adrenergic vasoconstriction in first order skeletal muscle arterioles.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Implications‐Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle

BackgroundThe endothelial glycocalyx is a complex network of glycoproteins, proteoglycans, and glycosaminoglycans; it lines the vascular endothelial cells facing the lumen of blood vessels forming the endothelial glycocalyx layer (EGL).ObjectiveThis study aims to investigate the microvascular hemodynamics implication of the EGL by quantifying changes in blood flow hydrodynamics post enzymatic degradation of the glycocalyx layer.MethodsHigh‐speed intravital microscopy videos of small arteries (around 35 μm) of the rat cremaster muscle were recorded at various time points after enzymatic degradation of the EGL. The thickness of the cell free layer (CFL), blood flow velocity profiles, and volumetric flow rates were quantified. Hydrodynamic effects of the presence of the EGL were observed in the differences between the thickness of CFL in microvessels with an intact EGL and glass tubes of similar diameters.ResultsMaximal changes in the thickness of CFL were observed 40 min post enzymatic degradation of the EGL. Analysis of the frequency distribution of the thickness of CFL allows for estimation of the thickness of the endothelial surface layer (ESL), the plasma layer, and the glycocalyx. Peak flow, maximum velocity, and mean velocity were found to statistically increase by 24%, 27%, and 25%, respectively, after enzymatic degradation of the glycocalyx. The change in peak‐to‐peak maximum velocity and mean velocity were found to statistically increase by 39% and 32%, respectively, after 40 min post enzymatic degradation of the EGL. The bluntness of blood flow velocity profiles was found to be reduced post degradation of the EGL, as the exclusion volume occupied by the EGL increased the effective volume impermeable to RBCs in microvessels.ConclusionThis study presents the effects of the EGL on microvascular hemodynamics. Enzymatic degradation of the EGL resulted in a decrease in the thickness of CFL, an increase in blood velocity, blood flow, and decrease of the bluntness of the blood flow velocity profile in small arterioles. In summary, the EGL functions as a molecular sieve to solute transport and as a lubrication layer to protect the endothelium from red blood cell (RBC) motion near the vessel wall, determining wall shear stress.Support or Funding InformationThis work was supported by NIH grants from the Heart Lung and Blood Institute, P01‐HL11090, R01HL126945, and R01‐ HL138116.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Implications Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle.

The endothelial glycocalyx is a complex network of glycoproteins, proteoglycans, and glycosaminoglycans; it lines the vascular endothelial cells facing the lumen of blood vessels forming the endothelial glycocalyx layer (EGL). This study aims to investigate the microvascular hemodynamics implications of the EGL by quantifying changes in blood flow hydrodynamics post-enzymatic degradation of the glycocalyx layer. High-speed intravital microscopy videos of small arteries (around 35 μm) of the rat cremaster muscle were recorded at various time points after enzymatic degradation of the EGL. The thickness of the cell free layer (CFL), blood flow velocity profiles, and volumetric flow rates were quantified. Hydrodynamic effects of the presence of the EGL were observed in the differences between the thickness of CFL in microvessels with an intact EGL and glass tubes of similar diameters. Maximal changes in the thickness of CFL were observed 40 min post-enzymatic degradation of the EGL. Analysis of the frequency distribution of the thickness of CFL allows for estimation of the thickness of the endothelial surface layer (ESL), the plasma layer, and the glycocalyx. Peak flow, maximum velocity, and mean velocity were found to statistically increase by 24, 27, and 25%, respectively, after enzymatic degradation of the glycocalyx. The change in peak-to-peak maximum velocity and mean velocity were found to statistically increase by 39 and 32%, respectively, after 40 min post-enzymatic degradation of the EGL. The bluntness of blood flow velocity profiles was found to be reduced post-degradation of the EGL, as the exclusion volume occupied by the EGL increased the effective volume impermeable to RBCs in microvessels. This study presents the effects of the EGL on microvascular hemodynamics. Enzymatic degradation of the EGL resulted in a decrease in the thickness of CFL, an increase in blood velocity, blood flow, and decrease of the bluntness of the blood flow velocity profile in small arterioles. In summary, the EGL functions as a molecular sieve to solute transport and as a lubrication layer to protect the endothelium from red blood cell (RBC) motion near the vessel wall, determining wall shear stress.

Does Vascular Endothelial Cell or Smooth Muscle Affect the Decrease in Oxygen Consumption of Arteriolar Wall During Vasodilation?

Our previous studies demonstrated that vascular wall oxygen consumption rate (QO2) of arteriole under in vivo functional condition was dramatically greater than that reported by isolated vascular segment. Furthermore, such high QO2 of vascular wall decreases during vasodilation. However, it is unknown whether such a reduction in QO2 during vasodilation would be caused by a decrease in consumption of vascular endothelial cell (EC) or smooth muscle. In this study, we aimed to explain whether vascular EC or smooth muscle affects the decrease in vascular wall QO2 during vasodilation. QO2 during EC-dependent and EC-independent vasodilation in rat cremaster muscle was determined using the intra- and peri-vascular PO2 measured by phosphorescence quenching microscopy. EC-dependent vasodilation was induced by increased NO production due to increased blood flow, while EC-independent vasodilation was induced by topical administration of papaverine. EC-dependent and EC-independent vasodilation increased arteriolar diameters by 13% and 17%, respectively, relative to the values under a normal condition. Vascular wall QO2 decreased significantly during both EC-dependent and EC-independent vasodilation as compared with that under a normal condition. Vascular wall QO2 during EC-independent vasodilation was lower than that during EC-dependent vasodilation. However, there was no significant difference between the energy efficiency, which is defined as the variable ratio of circumferential wall tension (amount of mechanical work) to vascular wall QO2 (energy cost) between normal and vasodilated conditions. These results suggest that the decrease in vascular wall QO2 during vasodilation is related to reduced mechanical work of vascular smooth muscle.

Passivating Injured Endothelium with Kinexins in Thrombolytic Therapy.

Without conjunctive administration of an anticoagulant, endothelial injury-induced thrombosis is resistant to thrombolysis and prone to re-thrombosis. We hypothesized that co-delivery of recombinant tissue plasminogen activator (rtPA) with annexin V-containing anticoagulants that specifically target the injured endothelium may passivate the thrombogenic elements of the vascular injury site and enhance rtPA-induced thrombolysis. In this study, the effects of conjunctive administration of Kinexins (Kunitz inhibitor-annexin V fusion proteins) with rtPA on thrombolysis were determined in vitro and in vivo. Thromboelastometry showed that both TAP-A (tick anticoagulant peptide-annexin V fusion protein; an inhibitor of factor Xa [FXa] and prothrombinase) and A-6L15 (annexin V-6L15 fusion protein; an inhibitor of tissue factor/FVIIa) exerted concentration-dependent (10-100 nM) effects on clot formation, with TAP-A being several folds more potent than A-6L15 in whole blood. Combination of TAP-A or A-6L15 with rtPA (1 μg/mL) led to decrease in lysis index, suggesting conjunctive enhancement of thrombolysis by combined use of rtPA with TAP-A or A-6L15. In a rat cremaster muscle preparation subjected to photochemical injury, conjunctive administration of rtPA and TAP-A significantly restored tissue perfusion to 56%, which is approximately two fold of that by rtPA or TAP-A alone. Near-infrared fluorescence images demonstrated local retention of a fluorescent A-6L15-S288 at the injury site, suggesting a targeting effect of the fusion protein. Pharmacokinetic analysis showed that 123I-labelled TAP-A and A-6L15 had initial distribution half-lives (T1/2α) of approximately 6 minutes and elimination half-lives (T1/2β) of approximately 2.3 hours. In conclusion, Kinexins were potentially useful adjunctive agents with rtPA thrombolytic therapy especially for thrombosis induced by endothelial injury.

Ringer's lactate solution enhances the inflammatory response during fluid resuscitation of experimentally induced haemorrhagic shock in rats.

IntroductionHemorrhagic shock leads to systemic oxygen deficit (hypoxaemia) that results in systemic inflammatory response syndrome (SIRS), a recognised cause of late mortality in this case. The aim of this study was to analyse the impact of fluid resuscitation, using two Ringer solutions, on the microcirculation changes that take place during experimentally induced haemorrhagic shock.Material and methodsA model of the rat cremaster muscle was used to assess microcirculation in vivo. The experimental groups (n = 10 each) included: control (CTRL); shock (HSG); Ringer’s acetate (RAG); and Ringer’s lactate (RLG). Microhaemodynamic parameters were measured during the experiment.ResultsA statistically significantly higher level of leukocytes, both those attached to the endothelium and those located in the extravascular space (p < 0.05), was reported in the lactate Ringer (LR) group compared with the AR group. There were significant differences in the activity of A3 arterioles compared with A1 and A2 arterioles. Ringer’s lactate solution seemed to the inflammation response during fluid resuscitation from haemorrhagic shock. A3 arterioles are likely to play a role as a pre-capillary sphincter in the skeletal muscle.ConclusionsThe present study revealed that fluid resuscitation with Ringer’s lactate solution exacerbates inflammation in the skeletal muscle. It is worth noting that Ringer’s acetate solution reduces local inflammation and could therefore be recommended as the “first line” crystalloid of the fluid resuscitation during haemorrhagic shock.

Altered post-capillary and collecting venular reactivity in skeletal muscle with metabolic syndrome.

With the development of the metabolic syndrome, both post-capillary and collecting venular dilator reactivity within the skeletal muscle of obese Zucker rats (OZR) is impaired. The impaired dilator reactivity in OZR reflects a loss in venular nitric oxide and PGI2 bioavailability, associated with the chronic elevation in oxidant stress. Additionally, with the impaired dilator responses, a modest increase in adrenergic constriction combined with an elevated thromboxane A2 production may contribute to impaired functional dilator and hyperaemic responses at the venular level. For the shift in skeletal muscle venular function with development of the metabolic syndrome, issues such as aggregate microvascular perfusion resistance, mass transport and exchange within with capillary networks, and fluid handling across the microcirculation are compelling avenues for future investigation. While research into vascular outcomes of the metabolic syndrome has focused on arterial/arteriolar and capillary levels, investigation into venular function and how this impacts responses has received little attention. Using the in situ cremaster muscle of obese Zucker rats (OZR; with lean Zucker rats (LZR) as controls), we determined indices of venular function. At ∼17weeks of age, skeletal muscle post-capillary venular density was reduced by ∼20% in LZR vs. OZR, although there was no evidence of remodelling of the venular wall. Venular tone at ∼25μm (post-capillary) and ∼75μm (collecting) diameter was elevated in OZR vs. LZR. Venular dilatation to acetylcholine was blunted in OZR vs. LZR due to increased oxidant stress-based loss of nitric oxide bioavailability (post-capillary) and increased α1 - (and α2 -) mediated constrictor tone (collecting). Venular constrictor responses in OZR were comparable to LZR for most stimuli, although constriction to α1 -adrenoreceptor stimulation was elevated. In response to field stimulation of the cremaster muscle (0.5, 1, 3Hz), venular dilator and hyperaemic responses to lower frequencies were blunted in OZR, but responses at 3Hz were similar between strains. Venous production of TxA2 was higher in OZR than LZR and significantly higher than PGI2 production in either following arachidonic acid challenge. These results suggest that multi-faceted alterations to skeletal muscle venular function in OZR may contribute to alterations in upstream capillary pressure profiles and the transcapillary exchange of solutes and water under conditions of metabolic syndrome.

Numerical Determination of Wall Shear Stress in Red Blood Cell Membranes

Red cells in microvessels deform extensively in order to flow, thus resulting in high observed apparent viscosities and high stress concentrations localized to the red cell membrane. Flow through microvessels is consequently aided by the high compliance in the red cell membrane. However, changes in the mechanical properties of Red Blood Cells (RBC) as a result of storage lesions, can increase stress concentrations in the membrane, impairing oxygen transport and resulting in increased vesiculation and likely cell lysis. The objective of this study is to determine the mechanical properties and thus wall shear stress experienced by a RBC membrane during capillary flow. The RBC membrane is modeled as a two dimensional Hookean Isotropic Solid. Videos of capillary flow of both fresh RBCs and RBCs stored for 2 weeks were taken in the rat cremaster muscle. The mechanical properties, including the elastic modulus, shear stress, and shear strain, of both conditions were compared for both groups. In order to determine strain, each RBC membrane will be parameterized by N (X,Y) points determined by the Moore Neighbor Tracing algorithm of videos of capillary flow obtained by intravital microscopy of the rat cremaster muscle, from which N deformation gradient tensors, [F], can be obtained numerically for each frame of the video. From these deformation gradient tensors, the updated Cauchy Strain Tensor will be determined as a function of position on the Cartesian plane relative to the centroid of the RBC. The strains determined from the updated Langrangian Green Strain are numerically fit to the Navier‐Lame Equations along the boundary of the RBC to experimentally fit the two Lame's Constants during capillary flow. These constants will then be used to determine the Cauchy Stress Tensor, using Hooke's Law, along the boundary of the RBC, allowing for the calculation of the Wall Shear Stress. The average Elastic Modulus of fresh cells was found to be 5.4 ± 0.4 × 106 N/m2, while of that of RBCs stored for two weeks was found to be statistically larger and was calculated to be 6.0 ± 1.0 × 106 N/m2. Additionally, stored RBCs experienced higher shear strains and wall shear strains over larger areas of the membrane as compared with stored cells. Additionally, stress concentrators in the membrane were observed at the points of contact with the capillary wall. However, for stored cells, the high stress acted on a larger segment of the membrane. This algorithm for determining wall shear stress experienced by RBC membranes in vivo during capillary flow was thus able to detect statistically significant differences in the both the mechanical properties and stress concentrations between fresh cells and stored cells.Support or Funding InformationThis work was supported by NIH grants from the Heart Lung and Blood Institute, P01‐HL110900, R56‐HL123015, and R01‐HL126945.