Functional capillary density in skeletal muscle during vasodilation induced by isoprenaline and muscular exercise

MICROVASCULAR

RESEARCH

20,

156-164

(1980)

Functional Capillary Density in Skeletal Muscle during Vasodilation Induced by lsoprenaline and Muscular Exercise1a2 F. VETTERLEIN AND G. SCHMIDT Institut fir Pharmakologie und Toxikologie der Universitiit Giittingen. Robert-Koch&rape D-3400 Giittingen, Germany

40,

Received May 30, 1978 Functional capillary density in skeletal muscle was studied in an isolated, autoperfused preparation of the abdominal muscles of the rat during different forms of vasodilation. The macromolecule hydroxyethyl starch (MW 450,000), labeled with the fluorochrome lissamine-rhodamine B 200, was intravenously injected. When a certain volume of blood had passed the muscle the tissue was fixed by snap freezing. In histological sections those capillaries which had been perfused by the dye could be visualized in the fluorescence microscope. Increase in total muscular blood flow, measured by arterial drop counting, was induced by intraarterial infusion of isoprenaline and by muscular work (control 30.0 2 2.6, isoprenaline 48.2 + 4.4, postcontraction hyperemia 44.2 f 7.4 ml/min x 1OOg).During hyperemia the functional capillary density (stained capillaries per muscle fiber) was affected in a different way: 0.81 + 0.02 control, 0.71 f 0.03 isoprenaline, and 0.93 f 0.04 postcontraction hyperemia. The data support the view that a rise in total blood flow is not necessarily associated with an increase in functional capillary density. INTRODUCTION

Various physiological experiments point to the existence of a nonuniform blood flow in skeletal muscle during certain forms of vasodilation. This conclusion was drawn from measurements of the integrated capillary pressure (Schroeder, 1966), the clearance rate of 24Na or 1311(Hyman et al., 1959; Renkin and Rosell, 1962; Renkin et al., 1966), and oxygen consumption in the muscle (e.g., Rose11and UvnHs, 1962; Wright and Sonnenschein, 1965; Bolme and Gagnon, 1972; Vetterlein and Schmidt, 1972). These experiments revealed discrepancies between total blood flow and extraction function of the muscle. Various attempts have been made to demonstrate arteriovenous anastomoses in the muscle in which an augmented blood flow might produce heterogeneities in the tissue perfusion. Hammersen (1970), however, a detailed review on the problem of shunts in skeletal muscle stated that the number of such pathways within the skeletal muscle is too small to account for effective bypassing of the nutrient capillary network and thus induction of such heterogeneous perfusion. Alternative explanations are discussed. A variation in length and degree of 1 Part of this investigation was presented at the Meeting of the Gesellschaft fur Mikrozirkulation, October 1977, Miinchen, Germany. * Supported by the Deutsche Forschungsgemeinschaft, SFB 89-Kardiologie, Gottingen. 156 0026i86u8(Y0501~-09$02.00/0 Copyright @ 1980 by Academic Press, Inc. All ri@s of reproduction in any form reserved. Printed in U.S.A.

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branching (Renkin ef al., 1966), different flow velocities in the capillaries (Gaehtgens ef al., 1976), a minor exchange function of vessels supplying only the muscular connective tissue (Barlow, 1959), or a shift in the local perfusion from superficial to deeper layers of the muscle (Vetterlein and Schmidt, 1975) might induce the above-mentioned discrepancies. The present experiments were performed in order to continue the study of changes in capillary perfusion pattern in the muscle during different forms of vasodilation. As a method the blood plasma should be stained intravitally. Histological preparations of the whole cross section of the muscle should then show only those capillaries which had been perfused by stained blood during the period of dye application. METHODS The experiments were performed on male Wistar rats weighing loo-130 g. The animals were anesthetized with 1.4 g/kg urethane ip and subsequent inhalation of a gas mixture of 20% oxygen and 80% nitrous oxide. This additional anesthetic agent was used because otherwise pseudoaffective pain reactions were still observed. For investigation of the muscular perfusion pattern part of the abdominal wall was worked out as an isolated, autoperfused preparation. In detail the following method was employed: During continuous moistening with Ringer’s solution of 37°C the skin was separated from the abdominal wall and the muscles dissected in the feeding area of the deep circumflex iliac vessels, leaving only those structures connected with the body (Fig. 1). Bleeding at the margin of the muscle was stopped by use of histoacrylic cement (Histoacryl). Near the origin of the feeding vessels the vasa pudenda superior externa and some other small side branches of the commune iliac and femoral vessels were ligated. To prevent the blood from clotting during the following procedure heparin (25 mg/kg) was injected via the right femoral vein. This dose had to be repeated every 30 min because as early as 45 min after a single injection thrombi were observed within the tubes of the autoperfusion system. For measurement of blood flow and withdrawal of blood samples an anastomosis was inserted between the left carotid and the femoral artery. A drop chamber with a photoelectric system was interposed in this system. The perfusion pressure was measured in a side branch of the arterial shunt with a Statham pressure transducer, type P 23 Db. This value was continuously recorded on a direct writing system (Beckman Dynograph R). A further tube connected the jugular and femoral veins. After insertion of these cannuli the iliac vessels were ligated proximal to the deep circumflex artery and vein, respectively. Thus all blood entering or leaving the muscle flowed through the tube system. Continuous measurements of total blood flow were possible by drop counting. A registration was performed with an impulse counter (Hewlett-Packard 5214 L) and a printer (Hewlett-Packard thermalprinter 5150 A). Additional volumetric measurements could be performed by directing whole venous blood via a side branch into a gauged capillary glass tube of 50 ~1 and measuring the filling time. All blood which

VETTERLEIN

158

AND SCHMIDT

posifion rguinal

of Me liganmt

femoral

orkry

medial lcmwal circumflrx ves5els

supuiw plxhndnl

sxtsmd vesrcrt

abdominal

w@

deep circumflex iliac wssels

3. FIG. 1. Isolation of the abdominal wall of rat I. Preparation of muscles and vessels.

had been withdrawn or otherwise lost was replaced by donor blood. The anatomosis, too, was filled with blood prior to insertion. Vasodilation was induced with isoprenaline (0.1 pg/kg x min) which was infused at the rate of 10 pl/min into the arterial shunt proximal to the drop chamber. In control experiments only Ringer’s solution was infused. In a further series metabolic vasodilation was induced by direct stimulation of the muscle with 3-5 V,OS msec, and 20 impulses/set for 4 min. For demonstration of the capillaries which had been perfused within a certain time, a fluorescent dye coupled to a macromolecule was injected. Lissaminerhodamine B 200 (RB 200), conjugated to hydroxyethyl starch (MW 450,000), proved to be the best suited substance. This material was observed to migrate into the extravascular space more slowly than two other macromolecules, albumin and globulin. The first signs of extravasation were detected about 3 min after the injection (albumin after about 1 min; globulin, 2 min). The excitation-emission characteristics of RB 200 did not interfere with tissue autofluorescence in the microscopical preparations. In labeling the hydroxyethyl starch with RB 200 the macromolecule first had to be activated by BrCN and then conjugated to a diamine (octamethylendiamine). With this modification the starch could be coupled to the sulfonyl chloride of RB 200 according to the method described by Nairn (1976). The conjugate was purified from unreacted fluorescent material by Sephadex gel filtration and concentrated by pressure dialysis. The dye solution, 5 ml/kg, corresponding to 20 mg/kg, was injected into the right femoral vein. The fixation was performed when

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MUSCLE

410 ~1 blood, corresponding to 20 drops, had passed the anastomosis. Thus the muscle had been perfused with 200 ~1 dye-marked blood since the dead space of the arterial shunt amounted to 210 ~1. Depending on the perfusion rate the muscle was exposed to the dye for 30-45 sec. For fixation the heating devices, which maintained a temperature of 37°C in the isolated tissue, were rapidly removed and the muscle was fixed by compression with the freezing clamp. This instrument was constructed in the form of a pair of tongs. At the end of each branch l-cm-thick aluminum plates were fixed. These plates were adjusted parallel to each other and corresponded in size to the isolated muscle (3 x 4 cm). By clamping the tissue the muscle in its total size could be covered from both sides with the cooled (liquid nitrogen) plates. In the experiments in which the muscle was electrically stimulated 5 set before performing the fixation the electrodes were removed. During this time the total blood flow did not change its steady-state level. To achieve as little mechanical disturbance as possible during this step, the muscle with its heating isolations was held ready in an upright position during the whole experiment (Fig. 2). The frozen muscle was transferred to 60% alcohol at -20°C for freezing substitution and remained there for 24 hr. The following dehydration, using increasing concentrations of alcohol, was performed at room temperature. The tissue was embedded in paraffin and lo-pm sections were cut perpendicular to the main feeding vessels. The slices were deparaffined in xylene and mounted in Entellan. In the incident light fluorescence microscope (Zeiss) those capillaries which contained the dye could well be recognized by their bright fluorescence, using a

c

tight barrier

deep circumflex iliac vessels camman iliac vesse/s

FIG. 2. Isolation of the abdominal wall of rat. II. Autoperfusion system.

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546-nm primary and 590~nm secondary filter system (Fig. 3). For visualization of the muscular fibers a 436/515 nm filter combination was used. For characterization of the area for evaluation in the microscopical preparations the muscle slices were subdivided into five parts (Fig. 4). Microphotographs were taken within the interior and exterior oblique muscle of the central fifth and the two lateral parts. In the transverse muscle no counts could be made because in this part the muscular fibers were sectioned longitudinally throughout. In the photos, representing an area of 0.36 x 0.53 mm, the number of capillaries per muscle fiber was counted. Values of capillaries square millimeter were not determined because of errors caused by shrinkage of the tissue and the inevitability of oblique sections of muscular fibers. RESULTS Only those experiments were taken into the evaluation in which the perfusion rate of the muscle showed constant values during a period of at least 15 min. Under this condition in 27 experiments the blood flow amounted to 28.1 + 1.37 (X + SE) mYmin x 1OOgat rest, determined by venous outflow measurement. During intraarterial infusion of isoprenaline blood flow increased within half a minute to a maximum and then remained at steady state; 3 min after the beginning of the infusion the flow rate amounted to 48.2 + 4.4 ml/min x 1OOg(28.1 f 1.8 at rest, n = 11). During muscular exercise the flow increased from 24.7 + 1.9 to 44.2 + 7.4 ml/min x 1OOg(n = 6). In the control group receiving an infusion of Ringer’s

FIG. 3. Cross section of the internal oblique muscle of the rat with RB-200-marked vessels. Fluorescence excitation and emission, 546/5!Xl and 436/515 nm, respectively; control experiment.

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Iin!./ext. oblique

161

MUSCLE

muscle

FIG. 4. Cross section of the isolated muscle, localization of the areas in which the photomicrographs were taken (x).

solution, the corresponding values amounted to 30.2 + 2.8 and 30.0 + 2.6, respectively (n = 10). One minute after the last withdrawal of blood for measurement of flow rate the fluorescent dye was injected. The application did not elicit changes in blood pressure or heart rate. The evaluation of these experiments showed the following results. In the isoprenaline-treated group a lower functional capillary density was found than during control conditions. This effect was to be seen in all parts of the muscle in which the number of dye-containing capillaries per muscular fiber was determined (Table 1). In contrast to these changes muscular exercise induced opposite effects. Here, the values obtained were greater than those of the controls and those of the isoprenaline-treated group (Table 1). In the two groups in which hyperemia was induced (isoprenaline and muscular exercise) differences in the capillary density of the central parts proved statistically significant (P < 0.05). Regarding these diverse results, the question arose as to whether the changes in capillary density might be caused by variations of the intercapillary distances within the evaluated area. In scanning the microscopic preparations it could be TABLE 1 STAINED CAPILLARIES PER MUSCLE FIBER IN THE ABDOMINAL WALL OF THE RAT DURING CONTROL CONDITIONS AND DURING VASODILATION INDUCED BY ISOPRENALINE AND MUSCULAR EXERCISE

1

Segment M. obliquus

2

3

ext.

int.

ext.

int.

ext.

int.

Control (Ringer’s solution) n = 10

0.89 50.09

0.82 kO.05

0.84 +0.07

0.77 20.04

0.76 -0.04

0.76 r0.04

Isoprenaline (0.1 &kg x min) n = 11

0.72 kO.07

0.69 -to. 10

0.71” 20.06

0.74b +0.04

0.67 20.05

0.70 kO.06

Postcontraction hyperemia n=6

0.86 20.05

0.93 +0.07

0.91c 20.05

I.076 kO.07

1.00 r0.13

0.81 kO.07

a < c,

b-cd

(P < 0.05)

Note. Measurement in the medial (I), lateral (3), and intermediate (2) fifth of the cross section (see Fig. 4). Each value is the g 2 SE.

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observed that there was a considerable variation in the intercapillary distances varying with the diameter of the surrounded muscular fiber. Those capillaries neighboring on the smaller fibers were a shorter distance from each other than those lying near the larger fibers (Fig. 3). A selective influence on capillary density in such distinct areas could not be found, however, in muscular preparations in which vasodilation had been induced. DISCUSSION The present experiments were aimed at providing information on changes in the perfusion pattern within the skeletal muscle during different forms of vasodilation. Previous experiments had shown that in the muscle discrepancies may occur in the behavior of micro- and macroflow in that an augmentation in total blood flow is paralleled by capillary flow reductions in marginal parts of the organ (Vetterlein and Schmidt, 1975). These observations referred only to changes in a small area of the muscular surface, while alterations of the capillary perfusion in the interior of the muscle could not be identified. For this reason in the present experiments a special muscular preparation was evaluated, in which the capillary perfusion pattern could be estimated in the whole cross section of a muscle by staining the plasma and rapidly fixing the tissue. An essential part of the experimental procedure was the attainment of a sudden, complete stoppage of the circulation at a defined time. In preliminary experiments different approaches had been used in this respect. An arrest of the muscular circulation by clamping the feeding vessels or application of low temperature allowed great fluctuations of the blood in the capillaries to occur before reaching the final fixation. Mechanical compression of the tissue proved to be best suited in this respect. Though arterial and venous blood was expressed the capillary blood was rapidly fixed in its position. Therefore mechanical compression combined with application of low temperatures in the form of a freezing clamp (Wollenberger et al., 1960) was applied. Special care was taken to achieve a homogeneous distribution of the intraarterially infused isoprenaline in the blood. For this reason the drug was applied upstream a drop chamber in which system turbulences induced a homogeneous mixture of the two phases. In the abdominal wall preparation a relatively high resting flow rate was found. The data obtained correspond best to those values found in other muscles with predominantly tonic function like the musculus soleus. This type of muscle is perfused at a much higher level than phasic ones at rest (Folkow and Halicka, 1968). A further factor that may have contributed to the high flow rate in the abdominal muscles is the fact that the muscle was denervated and therefore the vascular resistance was reduced (lit. in Hudlicka, 1973). The relatively small changes in functional capillary density that were found during vasodilation may be due to the fact that short-lasting changes in capillary perfusion pattern could not be identified because of the relatively long time of dye exposure (30-45 set). The values obtained under these conditions revealed lower

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counts during isoprenaline vasodilation than during the metabolic one despite equal increases in total blood flow. Different effects on the capillary circulation have been discussed as a cause of diverging results in measurements of exchange function. During vasodilation, induced by isoprenaline, acetylcholine, or activation of cholinergic vasodilator nerves, the decrease in maximal force (Hirvonen et al., 1964), oxygen consumption (Rose11and Uvnls, 1962; Vetterlein and Schmidt, 1972), integrated capillary pressure (Schroeder, 1966), and the lack of changes in clearance rates (Renkin and Rosell, 1962; Hyman et al., 1959) have been attributed to reductions in capillary surface area. The observation of a decrease in capillary flow velocity up to a total stasis during an isoprenaline-induced vasodilation further favors this conception (Vetterlein and Schmidt, 1975). On the other hand, muscular exercise or inhibition of the sympathetic vasoconstrictor tone induced increases in oxygen uptake (Kramer et al., 1939; Wright and Sonnenschein, 1965; Rose11and Uvnh, 1962), tissue clearance (Hyman et al., 1959; Renkin and Resell, 1%2; Renkin et al., 1%6), and integrated capillary pressure (Schroeder, 1961). These changes were discussed as being elicited by a rise in available capillary surface area. The present results obtained in abdominal wall muscles further favor the hypothesis of circulatory effects which induce these changes. Reduction of the capillary density would tend to diminish the exchange between blood and tissue; the enhanced value would contribute to an improvement of this function (e.g., Grunewald and Sowa, 1977). While attempting to compare the present data with those in other reports, no investigations could be found in which the functional capillary density of the skeletal muscle had been studied in a way similar to that in the present study during isoprenaline-induced vasodilation. On the other hand there are many approaches in which these changes have been investigated during muscular exercise (lit. in Hudlicka, 1973). The results are generally in agreement with the present ones in that increases in capillary counts were found by all observers. The magnitude of the change range observed is, however, contradictory and varies from 2- to 30-fold. Different methods and diverging intensities of muscular exercise may account for these discrepancies (e.g., Krogh, 1919; Perry, 1930; Martin et al., 1932; Gray et al., 1967). In those studies in which a dye was used to stain the capillaries an additional factor may be considered. Since the capillary blood flow in the muscle is discontinuous (e.g., Cardon et al., 1970) the number of dye-marked capillaries depends on the time this substance is exposed to the vascular system. This factor varied considerably in the diverse studies and would have contributed to the discrepancies in the intensity of increase in capillary counts during muscular exercise. REFERENCES BARLOW, T. E., HAIGH, A. L., AND WALDER, D. N. (1959). Dual circulation in skeletal muscle. J. Physiol. (London) 149, 18-19. BOLME, P., AND GAGNON, D. J. (1972). The effects of vasodilating drugs and vasoconstrictor stimulation on oxygen uptake in skeletal muscle. Eur. J. Pharmacol. 20, 300-307. CARDON, S. Z., OESTERMEYER,C. F., AND BLOCH, E. H. (1970). Effect of oxygen on cyclic red blood cell flow in unanesthetized mammalian striated muscle as determined by microscopy. Microvasc. Res. 2, 67-76.

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FOLKOW, B., AND HALICKA, H. D. (1968). A comparison between “red” and “white” muscle with respect to blood supply, capillary surface area and oxygen uptake during rest and exercise. Mjcrovasc.

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GAEHTGENS,P., BENNER, K. U., AND SCHICKENDANTZ,S. (1976). Nutritive and non-nutritive blood flow in canine skeletal muscle after partial microembolization. P’uegers Arch. 361, 183-189. GRAY, S. D., CARLSSON,E., AND STAUB, N. C. (1967). Site of increased,vascular resistance during isometric muscle contraction. Amer. .Z. Physiol. 213, 683-689. GRUNEWALD, W. A., AND SOWA, W. (1977). Capillary structures and 0, supply to tissue. Rev. Physiol.

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RENKIN, E. M., HUDLICKA, O., AND SHEHAN, R. (1966). Influence of metabolic vasodilation in skeletal muscle on blood-tissue diffusion. Amer. J. Physiol. 211, 87-98. ROSELL,S., AND UVN&, B. (1962). Vasomotor nerve activity and oxygen uptake in skeletal muscle of the anesthetized cat. Acta Physiol. &and. 54, 209-222. SCHROEDER,W. (1961). Der physiologische Nachweis arteriovendser Kurzschliisse in der Skelettmuskulatur. Pfuegers Arch. 213, 281-287. SCHROEDER,W. (1966). Nutritive und nicht-nutritive Skelettmuskeldurchblutung. Basic Res. Cardiol. 49, 36-49. VETTERLEIN, F., AND SCHMIDT, G. (1972). Changes in oxygen consumption in the hindlimb of the cat during various forms of vasodilation. Naunyn-Schmiedeberg’s Arch. Pharmacol. 275, 263-275. VETTERLEIN, F., AND SCHMIDT, G. (1975). Effects of vasodilating agents on the microcirculation in marginal parts of the skeletal muscle. Arch. Znt. Pharmacodyn. 213, 4-16. WOLLENBERGER, A., RISTAN, O., AND SCHOFFA, G. (1960). Eine einfache Technik der extrem schnellen Abkiihlung grijl3erer Gewebestlcke. Pfluegers Arch. 270, 399-412. WRIGHT, D. L., AND SONNENSCHEIN, R. R. (1965). Relations among activity, blood flow, and vascular state in skeletal muscle. Amer. .Z. Physiol. 208, 782-789.