Gap junction uncouplers attenuate arteriolar response to distal capillary stimuli.
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Adenosine-5'-(N-ethylcarboxamide)↗
Biomedical subjects
K Tyml
Publications and source records attributed to K Tyml.
Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle.
Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.
Acetylcholine↗
Differential microvascular response to disuse in rat hindlimb skeletal muscles.
The aim of the study was to address discrepant findings in the literature regarding coupling between decreased functional demand during disuse and reduced capillarity. We previously reported [K. Tyml, O. Mathieu-Costello, and E. Noble. Microvasc. Res. 49: 17-32, 1995] that severe disuse of rat extensor digitorum longus (EDL) muscle caused by a 2-wk application of tetrodotoxin (TTX) on the sciatic nerve is not accompanied by capillary loss. Using the same animal model, the present study examined whether this absence of coupling could be explained in terms of 1) too short a duration of disuse and 2) muscle-specific response to disuse. Fischer 344 rats were exposed to either no treatment (control) or to 2- or 8-wk TTX applications. Fiber size, capillary density per fiber cross-sectional area, and capillary-to-fiber (C/F) ratio were determined by morphometry in the EDL muscle (control, 2- and 8-wk groups) and in the superficial portion of medial gastrocnemius (Gas) muscle (control, 2 wk). In both muscles, microvascular blood flow was evaluated by intravital microscopy [red blood cell velocity in capillaries (V(RBC))] and by laser Doppler flowmetry (LDF). Regardless of duration of TTX application or muscle type, TTX-induced disuse resulted in a significant reduction of fiber area (44-71%). However, capillary density increased in EDL muscle (both at 2 and 8 wk) but not in Gas muscle. C/F ratio decreased in EDL muscle at 8 wk (18%) and in Gas muscle (39%). This indicates that the effect on capillarity depended on duration of disuse and on muscle type. V(RBC) and LDF signal were significantly larger in EDL than in Gas muscle. Analysis of change in capillarity vs. V(RBC) suggested that the outcome of disuse may be modulated by blood flow. We conclude that the duration of skeletal muscle disuse per se does not dictate capillary loss, and we hypothesize that discrepant findings of coupling between functional demand and capillarity could be due to the presence/absence of flow-related angiogenesis superimposed on the capillary removal process during disuse.
Animals↗
Local L-NAME decreases blood flow and increases leukocyte adhesion via CD18.
Local inhibition of nitric oxide (NO) synthesis with L-arginine analogs such as NG-nitro-L-arginine methyl ester (L-NAME) decreased red blood cell velocity (VRBC) in capillaries and increased leukocyte adhesion in postcapillary venules in rat skeletal muscle. The goal of the present study was to determine the mechanism of this response to L-NAME. Using intravital videomicroscopy, we examined blood flow in the surface microvasculature of rat extensor digitorum longus muscle. L-NAME (30 mM in the pipette) locally applied to capillaries (300 microns from feeding arteriole) reduced VRBC [control VRBC = 244 +/- 53 (SE) microns/s; delta VRBC = -52 +/- 8%] and increased leukocyte adhesion (from 0.2 +/- 0.01 to 1.3 +/- 0.3 cells/100 microns) in control animals. Systemic pretreatment with fucoidan (selectin binder), superoxide dismutase and catalase (extracellular antioxidants), dimethylthiourea (intracellular antioxidant), or ketotifen (mast cell stabilizer) did not alter this response. Pretreatment with CL26, an anti-CD18 antibody, abolished the L-NAME response. Our results suggest that L-NAME increased leukocyte-endothelial interactions via an effect on CD11/CD18 or its ligand, intercellular adhesion molecule.
Animals↗
Capillary and arteriolar responses to local vasodilators are impaired in a rat model of sepsis.
Although sepsis is known to affect vascular function, little is known about changes at the capillary level. We hypothesized that sepsis attenuates the "upstream" arteriolar response to vasoactive agents applied locally to capillaries. Sepsis in rats was induced by cecal ligation and perforation. After 24 h, extensor digitorum longus muscle was prepared for intravital microscopy. Phenylephrine (PE, 10 mM) and acetylcholine (ACh, 10 mM) were applied iontophoretically on terminal arterioles and on their downstream daughter capillaries (300 micron from arteriole). There was no significant difference between control and septic rats in baseline arteriolar diameters [8.0 +/- 0.6 vs. 9.8 +/- 0.8 (SE) micron- or baseline red blood cell velocity (VRBC) in perfused daughter capillaries (255 +/- 10 vs. 264 +/- 13 micron/s). Application of PE onto arterioles resulted in comparable constrictions (i.e., -22% diameter change) and VRBC reductions (-100%) in control and septic rats. In contrast, arteriolar diameter and VRBC increases after application of ACh were attenuated in sepsis (diameter: from 41 to 14%; VRBC: from 67 to 24%). Application of PE onto the capillary reduced VRBC to the same level (-100%) in both groups, whereas application of ACh increased VRBC less in septic than in control rats (20 vs. 73%). On the basis of arteriolar-capillary pair stimulations, sepsis affected VRBC responses to ACh more in the capillary than in the arteriole. When the adenosine analog 5'-N-ethylcarboxamidoadenosine (0.1 mM) was used instead of ACh, similar effects of sepsis were seen. To test for a possible involvement of inducible NO synthase (iNOS) in sepsis-induced attenuated ACh responses, arterioles and capillaries in septic animals were locally pretreated with the iNOS blocker aminoguanidine (10 mM). In both microvessels, aminoguanidine restored the ACh response to the control level. We conclude that impaired capillary VRBC and arteriolar diameter responses to vasodilators applied to capillaries in septic rat skeletal muscle were due to dysfunction at arteriolar and capillary levels. The study underscores the significant role iNOS/NO may play in sepsis-induced alteration of vascular reactivity in vivo.
Acetylcholine↗
Comparable effects of arteriolar and capillary stimuli on blood flow in rat skeletal muscle.
Although the capillary wall represents an active interface between blood and tissue, the potential role of the capillary in blood flow control has not been determined. The goals were (i) to establish the presence of the capillary sensing and communication phenomenon (Dietrich and Tyml, Microvasc. Res. 43, 87-99, 1992) in mammalian microvasculature and (ii) to determine the relative sensitivity of the capillary and the arteriole to locally applied vasoactive agents. Using intravital video microscopy, norepinephrine (NE; 10(-7)-3 x 10(-3) M), acetylcholine (ACh; 10(-4)-10(-2) M), or bradykinin (BK; 10(-9)-10(-3) M) was applied via micropipettes on capillaries (300 microm downstream from feeding arterioles) or on arterioles, at the surface of the extensor digitorum longus muscle of anesthetized rats. Red blood cell velocity (VRBC) in capillaries and arteriolar diameters was measured from video recordings. The overall control VRBC and control diameter were 190 microm/sec and 8.3 microm, respectively. NE applied on the capillary caused a dose-dependent reduction in VRBC (up to 100%, i.e., 0 microm/sec) via a constriction of the feeding arteriole. Both ACh and BK applied on the capillary caused a dose-dependent increase in VRBC (up to 115%) via arteriolar dilation. Based on two different approaches, these responses could not be explained in terms of diffusion of agents from capillary to the arteriole. When testing for the relative sensitivity of the arteriole and the capillary, application of NE and ACh on arterioles caused VRBC and diameter responses similar to those of capillary stimulations. When testing for the speed of response in these two microvessels, the time of noticeable VRBC change after NE (i.e., 10% from control) was also similar. We concluded that (i) the rat skeletal muscle capillary could respond to a variety of locally applied materials and (ii) the capillary could have as profound an effect on microvascular flow as the arteriole. Thus capillary could have the potential to participate in microvascular flow control.
Acetylcholine↗
Capillary adrenoceptors in rat skeletal muscle.
The purpose of this study was to examine whether functional alpha- and beta-adrenoceptors exist on capillaries of rat skeletal muscle, and further to determine which subtype of these receptors predominates on these capillaries. Using intravital video microscopy, we measured red blood cell velocity (VRBC) responses in capillaries of rat extensor digitorum longus muscle (EDL) following a local application of these agonists: norepinephrine (NE; alpha 1, alpha 2; 10(-7) to 3 x 10(-3) M), phenylephrine (PE; alpha 1; 3 x 10(-4) to 10(-2) M), clonidine (CLO; alpha 2; 3 x 10(-3) to 10(-2) M), UK14304 (alpha 2; 3 x 10(-4) to 10(-2) M), and isoproterenol (IPR; beta 1, beta 2; 10(-7) to 3 x 10(-3) M). Responses to NE (10(-5) M) were also measured after a local pretreatment with prazosin (alpha 1 antagonist; 10(-5) to 10(-3) M) and rauwolscine (alpha 2 antagonist; 3 x 10(-4) to 3 x 10(-2) M), while responses to IPR (10(-5) M) were measured after local atenolol (ATE; beta 1 antagonist; 10(-3) to 10(-2) M) and butoxamine (BUT; beta 2 antagonist; 10(-3) to 10(-2) M) pretreatment. The overall control VRBC was 226 microns/sec. NE, PE, CLO, and UK14304 resulted in concentration-dependent decreases of VRBC (from -12 to -89%) from the control level, while IPR caused concentration-dependent increases (17 to 174%). PE reduced VRBC to a larger degree than CLO and UK14304. NE-induced VRBC responses tended to be attenuated more by prazosin than by rauwolscine. Both ATE (10(-2) M) and BUT (10(-3) and 10(-2) M) alone decreased VRBC. However, only ATE significantly attenuated the IPR-induced VRBC responses. These results suggest that the capillary of rat EDL muscle has alpha- and beta-adrenoceptors. From the two alpha-adrenoceptor subtypes, the capillary may be predominated by the alpha 1-adrenoceptors.
Adrenergic alpha-Agonists↗
Evidence for K+ channels involvement in capillary sensing and for bidirectionality in capillary communication.
Although the capillary sensing and communication phenomenon has been characterized, its mechanism is not clear. It has been hypothesized that capillary sensing involves a membrane potential change in the capillary endothelium and/or pericyte and that communication represents an electrotonic spread of this change along the capillary. The goal of the present study was to address this hypothesis by examining the presence of K+ channels on the capillary and by determining bidirectionality of communication. Using intravital microscopy, we locally applied K+ (100 mM), acetylcholine (ACh; 3 mM), and norepinephrine (NE; 0.3 mM) on capillaries, 400-500 microns downstream from the arteriole, at the surface of the sartorius muscle in anesthetized frogs. Responses were measured in terms of red blood cell velocity (VRBC) changes in the stimulated capillary (control prestimulation VRBC ranged from 110 to 770 microns/sec). K+ and ACh caused significant 19 and 38% increases in VRBC, while NE caused a -46% decrease, respectively. The K+ response was blocked by local pretreatment with K+ channel blocker BaCl2 (1 microM) and by pretreatment with tetraethyl ammonium chloride (TEA; 5 mM). Responses to ACh and NE were attenuated by pretreatment with 1 microM BaCl2 (to 1%) and with 50 mM TEA (to -25%), respectively. In a separate experiment, NE (3 mM) application on the capillary 500 microns away from the draining venule (capillary occluded) caused a 19% venular constriction (i.e., similar to a reported 21% arteriolar constriction caused by the NE stimulus). We concluded that (i) K+ channels were present on the capillary and (ii) capillary communication was bidirectional. We interpreted these results to be consistent with the above hypothesis of membrane potential change and electrotonic spread.
Acetylcholine↗
Nitric oxide release in rat skeletal muscle capillary.
Nitric oxide (NO) has been shown to be a potent vasodilator released from endothelial cells (EC) in large blood vessels, but NO release has not been examined in the capillary bed. Because the capillary bed represents the largest source of EC, it may be the largest source of vascular NO. In the present study, we used intravital microscopy to examine the effect of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on the microvasculature of the rat extensor digitorum longus muscle. L-NAME (30 mM) applied locally to a capillary (300 micron(s) from the feeding arteriole) reduced red blood cell (RBC) velocity [VRBC; control VRBC = 238 +/- 58 (SE) micron/s; delta VRBC = -76 +/- 8%] and RBC flux (4.4 +/- 0.7 to 2.8 +/- 0.7 RBC/s) significantly in the capillary, but did not change feeding arteriole diameter (Dcon = 6.3 +/- 0.7 micron, delta D = 5 +/- 7%) or draining venule diameter (Dcon = 10.1 +/- 0.6 micron, delta D = 4 +/- 2%). Because of the VRBC change, the flux reduction was equivalent to an increased local hemoconcentration from 1.8 to 5 RBCs per 100 micron capillary length. L-NAME also caused an increase in the number of adhering leukocytes in the venule from 0.29 to 1.43 cells/100 micron. L-NAME (30 mM) applied either to arterioles or to venules did not change capillary VRBC. Bradykinin (BK) locally applied to the capillary caused significant increases in VRBC (delta VRBC = 111 +/- 23%) and in arteriolar diameter (delta D = 40 +/- 5%). This BK response was blocked by capillary pretreatment with 30 mM L-NAME (delta VRBC = -4 +/- 27%; delta D = 5 +/- 9% after BK). We concluded that NO may be released from capillary EC both basally and in response to the vasodilator BK. We hypothesize that 1) low basal levels of NO affect capillary blood flow by modulating local hemoconcentration and leukocyte adhesion, and 2) higher levels of NO (stimulated by BK) may cause a remote vasodilation to increase microvascular blood flow.
Animals↗
Ascorbate uptake by microvascular endothelial cells of rat skeletal muscle.
OBJECTIVE: In several systems, exogenous ascorbate (reduced vitamin C) has been shown to protect against microvascular injury induced by reactive oxygen species. Since skeletal muscle is relatively resistant to oxidative injury, it is possible that under physiological conditions endogenous ascorbate in the muscle microvasculature affords such protection. To examine the ability of microvascular endothelium to accumulate ascorbate, we aimed (1) to develop an in vitro model of microvascular endothelial cells derived from rat hindlimb skeletal muscles and (2) to investigate the uptake and steady-state concentration of ascorbate in these cells. METHODS: Microvascular cells were enzymatically dissociated, isolated on a density gradient, and grown in serum-supplemented medium. After passaging, they were tested for formation of tube-like structures, coagulation factor VIII antigen expression, Griffonia simplicifolia lectin I-isolectin B4 binding, and acetylated low-density lipoprotein (LDL) uptake. Concentrations of reduced ascorbate were measured by high-performance liquid chromatography (HPLC) with electrochemical detection. Transport activity was assessed on the basis of the initial rate [14C]ascorbate uptake. RESULTS: The cultured cells tested positively for factor VIII antigen expression, lectin binding, LDL uptake, and tube formation. Although these cells did not synthesize ascorbate de novo, they accumulated reduced vitamin C when it was added to the culture medium. The initial rate of [14C]ascorbate uptake was 0.9 mumol/g cell protein 10 min when cells were incubated with 10 microM of the radiolabeled vitamin. This uptake was Na+-dependent and was blocked by the organic anion transport inhibitor sulfinpyrazone, but was not acutely affected by glucose. Following incubation with a physiological concentration of vitamin C (100 microM L-ascorbate), cells accumulated a high concentration of ascorbate within 6 h (approximately 16 mM at steady-state). Steady-state cellular ascorbate concentration was also dependent on extracellular Na+ and sensitive to sulfinpyrazone. CONCLUSIONS: Microvascular cells derived from rat hindlimb muscles demonstrated endothelial characteristics. These cells accumulated reduced vitamin C by means of Na+-dependent ascorbate transporters, which are distinct from hexose carriers. The high endothelial ascorbate concentration at steady-state is consistent with the role of ascorbate as a major antioxidant in the skeletal muscle microvasculature.
Animals↗
Microvascular response to ischemia, and endothelial ultrastructure, in disused skeletal muscle.
It has previously been demonstrated that muscle atrophy associated with aging and disuse is accompanied by changes in microvascular function including absolute loss of capillaries, increased mean red blood cell velocity (VRBC), and absence of reactive hyperemia. The purpose of the present study was to determine whether disuse could account for these changes. The right extensor digitorum longus muscle in male Fisher 344 rats was subjected to 15 days of disuse through the neural application of tetrodotoxin (TTX). Microvascular function, as assessed using intravital microscopy, was compared for muscles from control (n = 8) and TTX-treated (n = 5) animals. The TTX-induced disuse was associated with a 40.5% decrease in muscle weight, a 51.6% decrease in fiber cross-sectional area, a 62% decrease in mitochondrial volume density, and increased capillary damage (TTX, 11% control, 1.1%). Although capillary density in the disused muscle increased (by 139%), when corrected for muscle atrophy, the absolute number of capillaries was maintained. With TTX disuse, VRBC heterogeneity was not different from that in the control rats while the mean velocity increased 3.18x. TTX disuse did not alter the pattern of reactive hyperemia following 30 min of complete ischemia. These results suggest that short-term TTX-induced atrophy affects both microvascular structure and resting state blood flow in rat skeletal muscle, but it does not affect the vascular responsiveness following a metabolic challenge.
Animals↗
Heterogeneity of red blood cell velocity in skeletal muscle decreases with increased flow.
OBJECTIVE: Effective material exchange between blood and tissue depends on the heterogeneity of microvascular flow. The objective was to address inconsistencies between intravital studies regarding this dependency. We tested the hypothesis that heterogeneity of red blood cell velocity (VRBC) in capillary beds varies with the strength of metabolic stimulus and with capillary bed geometry. METHODS: We used videomicroscopy to measure VRBC in a bed of 10-24 capillaries at the surface of extensor digitorum longus (EDL) muscle in anesthetized rats. The coefficient of variation (CV = standard deviation/mean; an index of spatial heterogeneity) was computed in the same bed before and after (i) 1, 2, 4, or 8 Hz supramaximal muscle contraction or (ii) adenosine superfusion (10(-7)-10(-3) M). Beds with or without arteriolar-venular capillary shunts were used. RESULTS: Although control VRBC differed between beds (shunt: 232 microns/s; no shunt: 130 microns/s), the percentage increases in postcontraction VRBC did not (range: 111-326%). In both beds, control CV varied greatly (overall range: 28-117%) and 2-8 Hz muscle contractions reduced CV significantly by 25%. Similar results were obtained for adenosine. In confirmatory experiments using the rat cremaster muscle, contractions (4 Hz) and adenosine (10(-4) M) also reduced CV. Based on all data, CV = 63-0.022 VRBC (r = 0.82, P < 0.001). CONCLUSIONS: The heterogeneity of VRBC decreased with metabolic stress, regardless of capillary bed geometry. We propose that both the large variability in control CV and the relatively shallow dependence of CV on velocity could be responsible for the present inconsistencies between intravital studies.
Analysis of Variance↗
Evaluation of pulsatile and nonpulsatile flow in capillaries of goat skeletal muscle using intravital microscopy.
It is commonly believed that pulsatile flow generated by the pumping action of the heart is dampened out by the time it reaches the microcirculation. In clinical practice, most of the cardiopulmonary bypass pumps and ventricular assist devices are nonpulsatile. To test the hypothesis that pulsatile flow generated by the heart does exist at the microvascular level, intravital microscopy of a large animal model (goat) was developed to visualize and to videorecord the surface microcirculation of the flexor carpi ulnaris muscle from the right forelimb. Density of perfused capillaries and red blood cell velocity in capillaries were measured in five goats during pulsatile perfusion provided by the heart and during a subsequent 3-hr period of nonpulsatile perfusion provided by a centrifugal ventricular assist device (Centrimed, Sarns 3M) that bypassed the heart. Throughout the experiment, the heart rate, innominate artery mean blood pressure, and flow remained unchanged. During the pulsatile regimen, velocities showed regular fluctuations that coincided with the period of the cardiac cycle (range of periods: 0.5-0.8 sec). The peak velocity amplitudes (range: 0.25-0.55 mm/sec) correlated directly with the amplitude of the pulse pressure. During the nonpulsatile regimen, no such correlations were seen. During pulsatile flow and during the 3-hr nonpulsatile period, capillary density remained stable at 24 capillaries/mm of test line but there were significant increases in red cell velocity, from 0.8 to 1.2 mm/sec (P < 0.05), and in coefficient of variation of velocity (used as an index of flow heterogeneity), from 19 to 34% (P < 0.05). We conclude that (1) pulsatility exists in the capillary bed and that it directly correlates with the pumping action of the heart and (2) nonpulsatile flow produced by the ventricular assist device does not cause an acute deterioration in microvascular perfusion. We interpret the increase in heterogeneity of flow as an early sign of microvascular dysfunction. Prolonged use of the nonpulsatile device may, therefore, lead to deterioration in perfusion that could compromize the function of the organ.
Animals↗
Microvascular perfusion is impaired in a rat model of normotensive sepsis.
We hypothesized that normotensive sepsis affects the ability of the microcirculation to appropriately regulate microregional red blood cell (RBC) flux. An extensor digitorum longus muscle preparation for intravital study was used to compare the distribution of RBC flux and the functional hyperemic response in SHAM rats and rats made septic by cecal ligation and perforation (CLP). Using intravital microscopy, we found that sepsis was associated with a 36% reduction in perfused capillary density (from 35.3 +/- 1.5 to 22.5 +/- 1.0 capillaries/mm of test line) and a 265% increase in stopped-flow capillaries (from 0.9 +/- 0.2 to 3.3 +/- 0.4 capillaries/mm); the spatial distribution of perfused capillaries was also 72% more heterogeneous. Mean intercapillary distance (ICD) increased 30% (from 25.7 +/- 0.8 to 33.5 +/- 1.6 microns), and the proportion of capillary pairs with intercapillary distances > 33.8 microns (the 75th percentile of ICDSHAM) was greater with sepsis. Mean capillary RBC velocity increased 17% in CLP rats (391 vs 333 microns/s). Laser Doppler flowmetry was used to assess the functional hyperemic response of the extensor digitorum longus muscle before and after a period of maximal twitch contraction designed to increase oxygen demand. RBC flux was 36% lower in the CLP rats at rest. After contraction, RBC flux increased in both SHAM and CLP rats; however, the relative increase was less in the CLP group. We concluded that sepsis affects the ability of the skeletal muscle microcirculation to appropriately distribute RBC flux and to respond to increases in oxygen need.
Animals↗
Evidence for sensing and integration of biological signals by the capillary network.
The aim of this study was to explore the phenomenon first described by Dietrich (Microvasc. Res. 38: 125-135, 1989) in which a local application of norepinephrine (NE) on a capillary can temporarily reduce flow via constriction of the feeding arteriole. Our objectives were to show that this phenomenon of remote response is not limited to vasoconstriction, can be elicited by materials other than NE, shows stimulus-strength dependency, and can be integrated within the capillary network. We used an intravital micropharmacological approach to iontophoretically apply (in mM concentrations in the pipette) NE, acetylcholine (ACh), 5'-N-ethylcarboxamidoadenosine (NECA, adenosine analogue), K+, and H+ on capillaries of the frog sartorius muscle in situ. Responses were measured in terms of changes in velocity of red blood cells (VRBC) in capillaries or in terms of changes in arteriolar diameter. ACh (3 mM) caused significant increases in diameter (from 34 to 37 microns) and in VRBC (from 250 to 340 microns/s, i.e., 36%). NE (3 mM) reduced VRBC by 16%. The magnitude of ACh and NE velocity responses increased with increasing pipette concentration and with increasing iontophoretic current. The ACh response was blocked by a local pretreatment with atropine. NECA, K+, and H+ caused 20-40% increases in VRBC. Dual application of NE on two capillaries fed by the same arteriole resulted in a greater VRBC reduction than for single NE application. Dual application of NE and ACh significantly attenuated the ACh response.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylcholine↗
Ischemia-reperfusion induced microvascular dysfunction in skeletal muscle: application of intravital video microscopy.
Video microscopy of red cell flow in capillaries at the surface of skeletal muscle provided the opportunity to quantitate ischemia-reperfusion (I-R) induced microcirculatory changes, in vivo. Extensor Digitorum Longus (EDL) muscles of 22 male Wistar rats (300-400 g), anesthetized with sodium pentobarbital (Somnotol, 65 mg kg,-1 IP), were used to measure the number of perfused capillaries (CDper: mm-1) crossing lines drawn perpendicular to the muscle axis, and red blood cell velocity (VRBC: mm/s) within individual capillaries from controls (n = 6), and after 2 hr (n = 4), 3 hr (n = 4), and 4 hr (n = 5) of no-flow ischemia with the muscle temperature maintained at its normal value of 32 degrees C. Ischemia was induced by tightening a tourniquet placed around the limb above the EDL muscle. Measurements were made after 30, 60, and 90 min of reperfusion. To test the usefulness of this skeletal muscle model for evaluating proposed interventions in I-R, the effect of hypothermia (24 degrees C) on the microcirculation following 4 hr ischemia (n = 3) was measured. Edema formation was estimated from the wet/dry weight ratio of the ischemic and contralateral control EDL muscles. Capillary perfusion at the surface of the control muscles was remarkably stable over the 5 hr period studied, while significant changes occurred following the ischemic periods. Significantly lower CDper was measured 30 min following all periods of normothermic ischemia. However, unlike the 2 and 4 hr ischemic periods 3 hr normothermic ischemia resulted in a progressive decline in CDper throughout the reperfusion period. VRBC showed evidence of a hyperemic response following 2 hr normothermic ischemia (control: 0.12 mm/s +/- 0.19 compared to 0.26 mm/s +/- 0.03 following 90 min reperfusion; mean +/- sem). However, no such hyperemia was measured following either 3 or 4 hr normothermic ischemia (i.e., 3 hr control: 0.24 mm/s +/- 0.01 compared to 0.07 mm s +/- 0.003 following 90 min reperfusion). In fact, VRBC was essentially zero 90 min following 4 hr normothermic ischemia (0.01 mm/s +/- 0.01). However, when the muscle was allowed to cool to 24 degrees C during 4 hr ischemia no significant change in either VRBC or CDper was measured compared to pre-ischemic controls. Evidence of edema was found after 3 and 4 hr normothermic ischemia. This study establishes a skeletal muscle model of I-R, which may be useful in testing hypotheses regarding mechanisms of I-R injury, and effectiveness of proposed treatments of I-R.
Animals↗
Microvascular flow response to localized application of norepinephrine on capillaries in rat and frog skeletal muscle.
Recently, Dietrich (1989, Microvasc. Res. 38, 125-135) demonstrated that a local application of a minute amount of norepinephrine (NE, 5.5 mM, 0.01-88 pmole) on a capillary in rat mesentery can elicit constriction of the feeding arteriole 0.5-1.0 mm away. This constriction can reduce or even stop blood flow in capillaries supplied by the arteriole. The main objective here was to show that the phenomenon of reduced flow occurs not only in the rat mesentery but also in other tissues and species. We chose to study the rat tibialis anterior and frog sartorius muscles. Using the same intravital video-microscopic approach as in the mesentery, strong NE stimuli (3 mM) were applied iontophoretically 48 times to 19 capillaries in 10 rats anesthetized with pentobarbital. They resulted in significant reductions (average: 80%) of the red blood cell velocity (VRBC) in capillaries. The onset of these reductions (i.e., 10% decrease from control) occurred within 3-52 sec (average: 20.9 sec) from the time of NE application. Reductions lasted 6.0 min. The same stimuli were applied 42 times to 15 capillaries in 6 frogs anesthetized with urethane. The average VRBC reduction was 86%. The onset occurred within 30.6 sec while the reduction lasted 6.6 min. Under the same conditions, arteriolar diameters in the sartorius muscle decreased significantly from 28.5 to 22.5 microns (n = 8). We also used local microinjection of small droplets of NE (30 mM) to 13 capillaries in 7 frogs. This resulted in a significant VRBC reduction of 64% with an onset time of 44.2 sec and a reduction duration of 17.2 min. Weak NE stimuli (3 microM) applied iontophoretically to 10 capillaries in 5 frogs resulted in marginal, but significant, VRBC reductions (9%). The present study demonstrates that the phenomenon of reduced flow after local application of NE may be a general phenomenon as it occurs also in skeletal muscle in both rat and frog. Our accompanying paper addresses the hypothesis that the phenomenon reflects communication of a NE-induced signal along the capillary.
Animals↗
Capillary as a communicating medium in the microvasculature.
The preceding study (Dietrich and Tyml, 1992. Microvasc. Res. 43) demonstrated that a local application of norepinephrine (NE) on a capillary in a skeletal muscle produces a temporary reduction in blood flow within this capillary. The reduction is mediated via constriction of the supplying arteriole. The objective of the present study was to address the mechanism by which the local NE stimulus is propagated from the capillary to the arteriole. Using intravital video microscopy we measured red blood cell velocity in capillaries, and diameter of supplying arterioles, in the sartorius muscle in anesthetized frogs. Velocity responses were measured following iontophoretic application of NE (3 mM in the pipette) on the capillary, with or without pretreatment with 0.9 mM tetrodotoxin (nerve-specific sodium channel blocker), 30 mM lidocaine (nonspecific sodium channel blocker), and 30 mM yohimbine (alpha 2-receptor blocker). Diameter responses were measured before and after capillary damage introduced by microcautery. Tetrodotoxin did not block the NE-induced velocity reduction (i.e., from 0.2 to 0.07 mm/sec), while lidocaine attenuated it. Yohimbine blocked it only when applied on the same site as NE. Capillary damage abolished the NE-induced arteriolar constriction (i.e., from 27.8 to 21.5 microns). We conclude that the observed responses were not due to (1) direct diffusion of NE from the capillary to the arteriole, (2) conduction along adrenergic nerves, or (3) venous-arteriolar diffusional cross-talk. We interpret our data to indicate that the capillary itself could function as a communicating medium.
Animals↗