Rat Cremaster Muscle Research Articles

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation. The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of calcium signaling in the arteriolar endothelium.

Role of actin cytoskeleton and microtubules in passive diameter and viscoelastic charectistics of rat cremaster arterioles

In resistance arteries the role of actin fibers and microtubules on their passive viscoelastic charectistics are not clearly understood. Arterioles were isolated from rat cremaster muscles, and pressurized to 60mmHg and incubated at 34.5 °C. The arterioles were observed under a microscope where vessel diameter was recorded. After vessels developed tone they were washed and incubated in solution containing EGTA but not Ca++ for 30 minutes. After obtaining maximum passive diameter arterioles were subjected to different pressures before and after a 30‐minute incubation with Cytochalasin D, Latrunculin, or Demecolcine alone or in combination.ResultsPressure diameter curves were obtained before and after actin cytoskeleton and microtubule disruption. There was no significant difference in the pressure diameter curve before and after administration of DMSO, Cytochalasin D, Latrunculin or Demecolcine alone or in combinations (p>0.05). We have previously shown that Demecholcine disrupts the microtubular network. Confocal images further indicate that treatment with Cytochalasin D and or Latrunculin successfully disrupts the actin cytoskeleton.DiscussionBased on the above results, it can be considered that the actin fibers and microtubules do not play a major role in the passive viscoelastic characteristics of cremaster arterioles.

Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles: Effects of erythrocyte aggregation

Recently, we have shown that temporal variations in the cell-free layer width can potentially enhance nitric oxide (NO) bioavailability in small arterioles. Since the layer width variations can be augmented by red blood cell aggregation, we tested the hypothesis that an increase in the layer width variations due to red blood cell aggregation could provide an underlying mechanism to improve NO bioavailability in the endothelium and promote vasodilatory effects. Utilizing cell-free layer width data acquired from arterioles of the rat cremaster muscle before and after dextran infusion in reduced flow conditions (wall shear stress = 0.13–0.24 Pa), our computational model predicted exponential enhancements of NO bioavailability in the endothelium and soluble guanylyl cyclase (sGC) activation in the smooth muscle layer with increasing temporal variability of the layer width. These effects were mediated primarily by the transient responses of wall shear stress and NO production rate to the layer width variations. The temporal variations in the layer width were significantly enhanced ( P < 0.05) by aggregation, leading to significant improvements ( P < 0.05) in NO bioavailability and sGC activation. As a result, the significant reduction ( P < 0.05) of sGC activation due to the increased width of the layer after aggregation induction was diminished by the opposing effect of the layer variations. These findings highlighted the possible enhancement of NO bioavailability and vascular tone in the arteriole by the augmented layer width variations due to the aggregation.

An automated method for cell-free layer width determination in small arterioles

Histogram-based thresholding techniques utilized for cell-free layer width measurement in arteriolar flow may produce an overestimation of the layer width since they do not consider faint shaded regions near the vessel wall as part of the erythrocyte column. To address this problem, we developed a new method for detecting the boundary of the erythrocyte column based on an edge detection algorithm. This automated method (grayscale method) provides local detections of the inner vessel wall as well as the boundary between the cell-free layer and the erythrocyte column without binarization of grayscale images. The cell-free layer width measurements using the grayscale method and existing techniques (minimum method and Otsu's method) were compared with those determined manually in arteriolar flows of the rat cremaster muscle. In the absence of the shaded regions, values obtained by the grayscale method and minimum method were statistically in good agreement with the manual method but not in the case of Otsu's method. When the faint shaded regions were present, the grayscale method appeared to produce more accurate results than the minimum method and Otsu's method.

Plasma expander viscosity effects on red cell-free layer thickness after moderate hemodilution

The objective of the study was to investigate the effects of plasma viscosity after hemodilution on the thickness of the erythrocyte cell free layer (CFL) and on the interface between the flowing column of erythrocytes and the vascular endothelium. The erythrocyte CFL thickness was measured in the rat cremaster muscle preparation. Plasma viscosity was modified in an isovolemic hemodilution, in which the systemic hematocrit (Hctsys) was lowered to 30%. The plasma expanders (PE) of similar nature and different viscosities were generated by glutaraldehyde polymerization of human serum albumin (HSA) at various molar ratios glutaraldehyde to HSA: (i) unpolymerized HSA; (ii) PolyHSA24:1, molar ratio = 24 and (iii) PolyHSA60:1, molar ratio = 60. The HSA viscosities determined at 200 s(-1) were 1.1, 4.2 and 6.0 dyn x cm(-2), respectively. CFL thickness, vessel diameter and blood flow velocity were measured, while volumetric flow, shear rate and stress were calculated. Hemodilution with PolyHSA60:1 increased plasma viscosity and the blood showed marked shear thinning behavior. CFL thickness decreased as plasma viscosity increased after hemodilution; thus the CFL thickness with HSA and PolyHSA24:1 increased compared to baseline. Conversely, the CFL thickness of PolyHSA60:1 was not different from baseline. Blood flow increased with both PolyHSA's compared to baseline. Wall shear rate and shear stress increased for PolyHSA60:1 compared to HSA and PolyHSA24:1, respectively. In conclusion, PE viscosity determined plasma viscosity after hemodilution and affected erythrocyte column hydrodynamics, changing the velocity profile, CFL thickness, and wall shear stress. This study relates the perfusion caused by PolyHSA60:1 to hemodynamic changes induced by the rheological properties of blood diluted with PolyHSA60:1.

Involvement of YC-1 in extracellular signal-regulated kinase action in rat cremasteric muscle

The nitric oxide (NO)-soluble guanylate cyclase (sGC) signalling pathway is attributed to the prevention of ischaemia-reperfusion (I/R)-induced leucocyte-endothelium adhesive interactions. YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole), a NO-independent sGC activator, has been shown to exert cardiovascular benefits, but its action on leucocyte-endothelium interactions remains unknown. In this study, the direct effect and the underlying mechanism of the anti-adhesive action of YC-1 have been examined in cremasteric microcirculation. Rat cremaster muscle was subjected to 4 h pudic-epigastric artery ischaemia followed by 2 h reperfusion and intravital microscopy was used to observe leucocyte-endothelium interaction and to quantify functional capillaries in rat cremaster muscle flaps. The values for leucocyte rolling, adhering and transmigrating were 5.5-, 6.9- and 8.8-fold greater, respectively, in I/R than in sham-control animals. YC-1 treatment rescued functional capillary density and reduced leucocyte rolling, adhering and transmigrating in I/R injured cremaster muscles to levels observed in sham-controls. Interestingly, these effects were completely blocked by the MEK (extracellular signal-regulated kinase (ERK) kinase) inhibitor (PD98059) but not by sGC or protein kinase C inhibitors. Cotreatment of PD98059 with YC-1 caused a 3.3-, 7.5- and 8.3-fold increase in the values for leucocyte rolling, adhering and transmigrating, respectively, in postcapillary venules of I/R-injured cremaster muscle. This study has indicated that the anti-adhesive and functional capillary density rescue properties of YC-1 were mediated predominantly by the activation of ERK but not sGC, although YC-1 was identified to be a sGC activator. A better understanding of the action of YC-1 on the microvasculature may help shed light on its therapeutic potential for cardiovascular disease.

Modulation of NO Bioavailability by Temporal Variation of the Cell-Free Layer Width in Small Arterioles

The cell-free layer exhibits dynamic characteristics in the time domain that may be capable of altering nitric oxide (NO) bioavailability in small arterioles. However, this effect has not been fully elucidated. This study utilized a computational model on NO transport to predict how temporal variations in the layer width could modulate NO bioavailability in the arterioles. Data on the layer width was acquired from high-speed video recordings in arterioles (ID=48.4±1.8μm) of the rat cremaster muscle. We found that when wall shear stress response was not considered, the layer variability could lead to a slight decrease (1.6-6.6%) in NO bioavailability that was independent of transient changes in NO scavenging rate. Conversely, the transient response in wall shear stress and NO production rate played a dominant role in reversing this decline such that a significant augmentation (5.3-21.0%) in NO bioavailability was found with increasing layer variability from 24.6 to 63.8%. This study highlighted the importance of the temporal changes in wall shear stress and NO production rate caused by the layer width variations in prediction of NO bioavailability in arterioles.

Regional heterogeneity of α-adrenoreceptor subtypes in arteriolar networks of mouse skeletal muscle

Activation of vascular adrenoreceptors (ARs) governs the magnitude and distribution of muscle blood flow in accord with the distribution of AR subtypes. Functional studies in the rat cremaster muscle indicate that α1ARs predominate in proximal arterioles (first-order, 1A) while α2ARs predominate in distal arterioles (third-order, 3A). However, little is known of AR subtype distribution in arteriolar networks of locomotor skeletal muscles, particularly in the mouse. We tested the hypotheses that functional AR subtypes exhibit heterogeneity among branches of arteriolar networks in a locomotor muscle and that the nature of this heterogeneity can vary between muscles having diverse functions. In anaesthetized male C57BL/6J mice (3 months old), concentration-response curves (10(-9) m to 10(-5) m, 0.5 log increments) were evaluated in the gluteus maximus muscle superfused with physiological saline solution (35°C, pH 7.4; n ≥ 5 per group). Noradrenaline (NA, non-selective αAR agonist) constricted 1A, 2A and 3A with similar potency and efficacy. Phenylephrine (PE; α1AR agonist) evoked greater (P < 0.05) constriction in 3A (inhibited by 10(-8) m prazosin; α1AR antagonist) while UK 14304 (UK; α2AR agonist) evoked greater (P < 0.05) constriction in 1A (inhibited by 10(-7) m rauwolscine; α2AR antagonist). Isoproterenol (isoprenaline; βAR agonist) dilated 1A, 2A and 3A near-maximally with similar potency and efficacy; these dilatations were inhibited by 10(-7) m propranolol (βAR antagonist) which otherwise had no effect on responses to NA, PE, or UK. Complementary experiments in the mouse cremaster muscle revealed a pattern of αAR subtype distribution that, while distinct from the gluteus maximus muscle, was consistent with that reported for the rat cremaster muscle. We conclude that functional αAR subtype distribution in arteriolar networks of skeletal muscle varies with muscle function as well as vessel branch order.

A comparative study of histogram-based thresholding methods for the determination of cell-free layer width in small blood vessels

We have recently proposed a computer-based method utilizing a thresholding algorithm (the Otsu method) to provide a convenient way of measuring the cell-free layer width in vivo and in vitro. However, this method does not seem to be a universal method that can be applied to all microvascular studies. Thus, we examined four different histogram-based thresholding algorithms (Otsu, intermode, minimum and second peak) to provide a technical suggestion on the selection of a suitable thresholding algorithm for the cell-free layer measurement. All the measurements were taken in microvascular flows in the rat cremaster muscle recorded with a high-speed camera. The width of the cell-free layer manually measured was compared with that determined by the automated method utilizing the four thresholding algorithms. With our experimental system, results showed that the cell-free layer width determined by the minimum algorithm was in best accordance with the manual measurement. We concluded that the accuracy of the automated methods for determination of the cell-free layer width would depend on the image quality, in particular on the contrast between the red blood cell core and background, which might differ due to the different microscopic setup. Therefore, one may need to examine several appropriate thresholding methods when selecting the best suitable algorithm for the experimental conditions.

Effect of Cell-Free Layer Variation on Arteriolar Wall Shear Stress

Relationship between a cell-free layer and wall shear stress (WSS) in small arterioles has been of interest in microcirculatory research. However, influence of temporal variation in the cell-free layer width on the WSS in vivo has not been fully elucidated. In this study, we tested the hypothesis that the layer variation would increase the WSS, and this effect would be enhanced by red blood cell aggregation. The cell-free layer width in arterioles (29.5–67.1 μm ID) in rat cremaster muscles were obtained with a high-speed video camera, and the layer width data were introduced into WSS estimation. Dextran 500 was administrated to elevate the aggregation level of red blood cells to those seen in normal human blood. The variation of the layer was quantified by the variability (coefficient of variation), and its effect on WSS was studied under normal and reduced flow conditions. We found that the dextran-induced red blood cell aggregation significantly elevated the variability (p < 0.01) at low pseudoshear rates of 9.2 ± 0.6 s−1. The WSS estimated without taking account of the variability showed underestimation of its value than that of with consideration of the variability under all flow conditions, and this effect became more pronounced with increasing the variability. The variation of the cell-free layer should, therefore, be considered in the determination of the WSS particularly in the presence of red blood cell aggregation under reduced flow condition.

Functional adrenoreceptor distribution in arteriolar networks of mouse gluteus maximus muscle

Adrenoreceptor (AR) activation governs the magnitude and distribution of exercise hyperemia in accord with heterogeneity of AR subtype distribution. Functional studies in rat cremaster muscle (CM) indicate α1 AR predominate in proximal (1A) arterioles and α2 AR in distal (3A) arterioles yet little is known of arteriolar AR subtype distribution in locomotor muscles, particularly for the mouse. We tested the hypothesis that functional AR subtype exhibits regional heterogeneity in arteriolar networks of gluteus maximus muscle (GM). In anesthetized male C57BL/6J mice (3 mo), concentration‐response curves (1 nM – 1 μM, 0.5 log increments) were performed in superfused GM (35 °C, pH 7.4; n 5/group). Norepinephrine (NE, nonselective α AR agonist) constricted 1A, 2A and 3A with similar potency and efficacy. In contrast to rat CM, phenylephrine (PE; α1 AR agonist) evoked greater (p&lt;0.05) constriction in 3A (inhibited by prazosin, 10 nM; α1 AR antagonist) while UK14304 (UK; α2 AR agonist) evoked greater (p&lt;0.05) constriction in 1A (inhibited by rauwolscine, 0.1 μM; α2 AR antagonist). Isoproterenol (β AR agonist) dilated 1A, 2A, and 3A with similar potency and efficacy; each was inhibited by propanolol (β AR antagonist, 0.1 μM) which otherwise had no effect on responses to NE, PE, or UK. We conclude that functional AR distribution in arteriolar networks of mouse GM is heterogeneous and differs from previous reports. (HL086483)

Effect of advanced glycation end products on dilation of rat skeletal muscle arterioles

Post translational protein modification, including the formation and accumulation of advanced glycation endproducts (AGE), may contribute to vascular dysfunction in diabetes. This study investigated the effect of AGE on dilation of arterioles possessing myogenic tone. AGE were manufactured by incubating 250 mM glucose‐6‐phosphate with 10 mg/ml BSA in PBS for 60 days. The effects of the modified protein were studied on isolated, pressurized (70 mmHg) first‐order arterioles from rat cremaster muscle. The AGE preparation inhibited dilation of the arterioles caused by both acetylcholine (ACh; endothelium‐dependent) and sodium nitroprusside (SNP), but had no effect on adenosine‐induced vasodilation. Iberiotoxin (0.1 μM), an antagonist of large‐conductance, Ca2+‐activated K+ channels (BKCa), inhibited ACh‐induced dilation. The combination of iberiotoxin and AGE did not cause a greater inhibition of ACh‐induced vasodilation than the application of iberiotoxin or AGE alone. The inhibitory effect of AGE on ACh‐ and SNP‐induced vasodilation was abolished by pre‐incubation of the arterioles individually with the NOS inhibitor L‐NAME (100 μM), the anti‐oxidant lipoic acid (2 μM) or the NADPH oxidase inhibitor apocynin (500 μM). Taken together, these observations suggest that AGE inhibit ACh‐ and SNP‐induced dilation of the arterioles by inhibiting BKCa, through generation of reactive oxygen species.

Modulation of NO bioavailability by temporal variability of cell‐free layer width in the arterioles

The formation of a cell (erythrocyte)‐free layer near the vessel wall during blood flow is capable of attenuating NO consumption by the red blood cells and thus improves the preservation of NO in the smooth muscle for eliciting vasodilatory responses. This study utilized a transient NO transport mathematical model to predict how temporal variability exhibited by the layer width and its relevant transient physiological responses could modulate NO preservation by this diffusion barrier at high pseudoshear rates (291.5 ± 13.5 s−1). Data on cell‐free layer width was experimentally acquired from arterioles (48.4 ± 2.3 μm) of the rat cremaster muscle. We found that a time‐varying diffusion barrier could lead to a slight decrease (2% – 6%) in NO bioavailability and this effect was independent of transient changes in NO scavenging rate in the blood lumen. Conversely, transient changes in wall shear stress and accompanying NO release rate played a dominant role in reversing this decline such that an augmentation in NO bioavailability of between 3% – 15% was found. However this increase was not sufficient to induce any significant change in vascular tone. This study highlighted the importance of accounting for transient wall shear stress effect on NO release rate as mediated by the layer width temporal variability when assessing the physiological implication of the layer on NO bioavailability in arterioles.

Effect of erythrocyte aggregation and flow rate on cell-free layer formation in arterioles

Formation of a cell-free layer is an important dynamic feature of microcirculatory blood flow, which can be influenced by rheological parameters, such as red blood cell aggregation and flow rate. In this study, we investigate the effect of these two rheological parameters on cell-free layer characteristics in the arterioles (20-60 mum inner diameter). For the first time, we provide here the detailed temporal information of the arteriolar cell-free layer in various rheological conditions to better describe the characteristics of the layer variation. The rat cremaster muscle was used to visualize arteriolar flows, and the extent of aggregation was raised by dextran 500 infusion to levels seen in normal human blood. Our results show that cell-free layer formation in the arterioles is enhanced by a combination of flow reduction and red blood cell aggregation. A positive relation (P < 0.005) was found between mean cell-free layer widths and their corresponding SDs for all conditions. An analysis of the frequency and magnitudes of cell-free layer variation from their mean value revealed that the layer deviated with significantly larger magnitudes into the red blood cell core after flow reduction and dextran infusion (P < 0.05). In accordance, the disparity of cell-free layer width distribution found in opposite radial directions from its mean became greater with aggregation in reduced flow conditions. This study shows that the cell-free layer width in arterioles is dependent on both flow rate and red blood cell aggregability, and that the temporal variations in width are asymmetric with a greater excursion into the red blood cell core than toward the vessel wall.

Activation of AMP-Activated Protein Kinase by 5-Aminoimidazole-4-Carboxamide-1-β- d -Ribofuranoside in the Muscle Microcirculation Increases Nitric Oxide Synthesis and Microvascular Perfusion

To investigate the effects of activation of the AMP-activated protein kinase (AMPK) on muscle perfusion and to elucidate the mechanisms involved. In a combined approach, we studied the vasoactive actions of AMPK activator by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) on rat cremaster muscle resistance arteries ( approximately 100 mum) ex vivo and on microvascular perfusion in the rat hindlimb in vivo. In isolated resistance arteries, AICAR increased Thr172 phosphorylation of AMPK in arteriolar endothelium, which was predominantly located in microvascular endothelium. AICAR induced vasodilation (19+/-4% at 2 mmol/L, P<0.01), which was abolished by endothelium removal, inhibition of NO synthase (with N-nitro-L-arginine), or AMPK (with compound C). Smooth muscle sensitivity to NO, determined by studying the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP), was not affected by AICAR except at the highest dose. AICAR increased endothelial nitric oxide synthase activity, as indicated by Ser1177 phosphorylation. In vivo, infusion of AICAR markedly increased muscle microvascular blood volume (approximately 60%, P<0.05), as was evidenced by contrast-enhanced ultrasound, without effects on blood pressure, femoral blood flow, or hind leg glucose uptake. Activation of AMPK by AICAR activates endothelial nitric oxide synthase in arteriolar endothelium by increasing its Ser1177 phosphorylation, which leads to vasodilation of resistance arteries and recruitment of microvascular perfusion in muscle.

Comparison of the effects of inhalation, epidural, spinal, and combined anesthesia techniques on rat cremaster muscle flap microcirculation

This experimental study was designed to investigate and compare the effects of different anesthesia techniques on rat cremaster muscle flap microcirculation. Fifty male Sprague-Dawley rats (130-150 g body weight) were divided into five experimental groups containing ten animals each. Group I, group II, and group III were designated as inhalation, epidural, and spinal anesthesia groups, respectively. Group IV was designated as a combination group for inhalation and epidural anesthesia. Group V was a combination group of inhalation and spinal anesthesia. Group III and group V showed significant increases in the number of rolling and sticking leucocytes and in RBC volume (peripheral stasis) when compared with group I. Blood flow and velocity significantly increased without peripheral stasis in groups II and IV when compared with group I. Although there was no statistically significant difference in the numbers of rolling, sticking, and transmigrating leucocytes or in functional capillary perfusion, group IV had better flow hemodynamics in the peripheral microcirculation when compared with group I. The inhalation and epidural anesthesia combination was determined to be the ideal anesthesia technique for improved peripheral microcirculation. Spinal anesthesia, either separately or in combination with inhalation anesthesia, has adverse effects on microcirculation.

Diannexin Treatment Decreases Ischemia-Reperfusion Injury at the Endothelial Cell Level of the Microvascular Bed in Muscle Flaps

Ischemia-reperfusion injury (IRI) is a common and serious complication of reperfusion following vascular occlusion. We present a novel interpretation of the pathogenesis of IRI. According to this hypothesis, anoxia resulting from ischemia allows translocation of phosphatidylserine to the surface of endothelial cells (ECs), providing an attachment site for leukocytes and platelets. This attachment impedes blood flow through the microvasculature. During IRI mediators of increased vascular permeability are produced, resulting in edema. We have developed a recombinant homodimer of human Annexin V, Diannexin, to attenuate IRI. Annexin V (36 kDa) rapidly passes from the circulation into the urine. In Diannexin 2 annexin V molecules are joined by a short peptide linker to produce a 73 kDa protein, which exceeds the renal filtration threshold. Diannexin has a half-life of about 2.5 hours in the human circulation. Diannexin also has a higher affinity for phosphatidylserine on cell surfaces than the monomer has. Such binding inhibits leukocyte attachment to ECs, and inflammatory mediator formation, during IRI. The aim of the study now reported was to ascertain the effects of Diannexin on IRI in the rat cremaster muscle flap, as revealed by intravital microscopy. During IRI there was increased attachment of leukocytes to ECs, reduced blood flow and augmented vascular permeability. Administration of Diannexin before or just after ischemia prevented these effects. Diannexin inhibited transmigration of leukocytes during IRI. Edema complicates peripheral vascular surgery, stroke, and other clinical conditions. Diannexin has proven to be safe when administered to patients after major surgical operations, and it may be useful to prevent IRI associated with peripheral vascular surgery.

An ex vivo Study of the Correlation Between Acoustic Emission and Microvascular Damage

The objective of this study was to conduct an ex vivo examination of correlation between acoustic emission and tissue damage. Intravital microscopy was employed in conjunction with ultrasound exposure in cremaster muscle of male Wistar rats. Definity microbubbles were administered intravenously through the tail vein (80 μL.kg −1.min −1infusion rate) with the aid of a syringe pump. For the pulse repetition frequency (PRF) study, exposures were performed at four locations of the cremaster at a PRF of 1000, 500, 100 and 10 Hz (one location per PRF per rat). The 100-pulse exposures were implemented at a peak rarefactional pressure (P r) of 2 MPa, frequency of 2.25 MHz with 46 cycle pulses. For the pressure amplitude threshold study, 100-pulse exposures (46 cycle pulses) were conducted at various peak rarefactional pressures from 0.5 MPa to 2 MPa at a frequency of 2.25 MHz and PRF of 100 Hz. Photomicrographs were captured before and 2-min postexposure. On a pulse-to-pulse basis, the 10 Hz acoustic emission was considerably higher and more sustained than those at other PRFs (1000, 500, and 100 Hz) ( p < 0.05). Damage, measured as area of extravasation of red blood cells (RBCs), was also significantly higher at 10 Hz PRF than at 1000, 500 and 100 Hz ( p < 0.01). The correlation of acoustic emission to tissue damage showed a trend of increasing damage with increasing cumulative function of the relative integrated power spectrum (CRIPS; R 2 = 0.75). No visible damage was present at P r ≤ 0.85 MPa. Damage, however, was observed at P r ≥ 1.0 MPa and it increased with increasing acoustic pressure. (E-mail: ssamuel@umich.edu)

Experimental gas embolism and tissue oxygenation using intravital microscopy

Little is known about microhemodynamic responses to arterial gas embolism (AGE) and associated local O2 tensions (PO2) have never been evaluated in vivo. Therefore, we assessed the adequacy of intravital microscopy to study the microcirculation during AGE, and investigated how bubble‐induced vaso‐occlusion and blood flow redistribution were associated with changes in arteriolar and tissue PO2. Blood pressure and heart rate were not significantly different before and after local AGE in the rat cremaster muscle. The first attempt of embolization did not result in arteriolar occlusion and multiple attempts were needed. Control diameter and blood flow averaged 53 ± 25 and 13 ± 10 nl/s, respectively. After AGE, these parameters changed to 53 ± 24 µm and 1 ± 3 nl/s. Arteriolar PO2 averaged 59 ± 19 mmHg during control and dropped to 34 ± 27 mmHg (upstream) and 24 ± 22 mmHg (downstream) after AGE. Interstitial PO2 changed from 44 ± 22 to 23 ± 20 mmHg and did not follow the drop recorded in the embolized arteriole when the PO2 in nearby flowing vessels remained high. We also measured in vivo bubble reabsorption (volume ≤ 20pL). Our findings suggest that blood redistribution in the muscle prevented severe hypoxia in some tissue areas after AGE and that arteriolar entrapment of air bubbles is associated with prior endothelial cell damage caused by the first bubble passage.Support: NIH, ONR.

Effect of erythrocyte aggregation and flow rate on temporal variation of cell‐free layer width in arterioles

The cell‐free layer (CFL) is a dynamic feature of microcirculatory blood flow and its variability is commonly described in terms of standard deviation (SD). However, CFL width in an arteriole follows a non‐Gaussian distribution imposed by asymmetrical nature of its physical environment, which implies that more detailed statistical analyses are required to better interpret CFL characteristics. In this study, rat cremaster muscle was used to visualize arteriolar flows and erythrocyte aggregability was raised by dextran infusion to levels seen in normal human blood. The effects of aggregation and flow reduction on CFL characteristics in arterioles (ID 20‐50 μm) were investigated. A positive relation was found between mean CFL widths and their corresponding SD and was predominantly enhanced by flow reduction. Both normalized mean and SD of CFL width increased significantly at low pseudoshear rates (17.1 ± 6.1 s−1), especially in the presence of aggregation. The variability of the CFL width was further analyzed in terms of its frequency and magnitude of deviations from its mean value, extending either towards the vessel wall or into the erythrocyte core. There was no significant effect of either flow condition or aggregation on the frequency of CFL deviations, whereas the magnitude of deviations significantly increased after flow reduction and dextran infusion (P &lt; 0.05), especially into the erythrocyte core.