perfused capillary surface area in postural and locomotor skeletal muscle

MICROVASCULAR RESEARCH 24, 142-157 (1982)

Perfused Capillary Surface Area in Postural and Locomotor Skeletal Muscle’

PAUL F. MCDONAGH,~ ROBERT W. GORE, AND SARAH D. GRAY*

Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724; and *Department of Human Physiology, School of Medicine, University of California,

Davis, California 95616

Received September II, 1981

A measure was made of the perfused capillary surface area per gram of tissue (S,) in postural, anterior latissimus dorsi (ALD), and locomotor, posterior latissimus dorsi (PLD), skeletal muscles in chickens. The animals were anesthetized with L.A. Thesia and the ALD and PLD muscles were prepared for observation using a modification of the method developed by R. E. Klabunde and P. C. Johnson (1977, Amer. J. Physiol. 232, H41 I-417). S, was determined from the relationship S r = TdLO,, where d = capillary diameter, L = capillary length, and Or = number of perfused capillaries per gram. Capillary lengths and diameters were measured directly through the microscope. Estimates of Or were measured in two ways: by a flow method (OF), and by an histochemical method (OF). It was observed that the average capillary diameter in PLD was the same as in ALD muscles, but the average capillary length in PLD was two times longer than in ALD muscles. Both the flow and histochemical methods yielded values of Or that gave similar values of S, for PLD muscles (SF = 24.7 + 20.0 cm’ig, S,” = 13.7 * 14.4 cm’ig), but different values for ALD muscles (Sp = 9.8 f 6.8 cm’lg, S p = 42 8 ? 19.1 cm’lg). The difference between SF and S,” for ALD muscles could be explained by the more irregular and longer perfusion path observed in postural versus locomotor muscle. The flow method appears to underestimate S, in capillary beds with irregular perfusion paths. The results indicate that only a small fraction of the total capillary bed was perfused in resting skeletal muscle and that S,” for ALD was approximately three times larger than SF for PLD.

INTRODUCTION

The fiber composition of most skeletal muscle is mixed, but muscles that are used primarily for posture are tonically active and are composed mostly of slow oxidative (SO) and fast oxidative, glycolytic (FOG) fibers. On the other hand, muscles that are used primarily for locomotion are phasically active and composed mostly of fast glycolytic (FG) and FOG fibers. Some of the apparent interactions between the cardiovascular system and postural and locomotor muscle functions have been investigated previously (Smith and Giovacchini 1956;

’ Supported by NIH Grants HL-13437, HL-17421, HL-17998, HL-07249, and a Grant-in-Aid from the Arizona Heart Association. A portion of this work was presented at the 1980 Microcirculatory Society Meeting, Anaheim, California.

2 Present address: Department of Cardiothoracic Surgery, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven, Conn. 06510.

142

0026-2862/82/050142- 16$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.

PERFUSED CAPILLARY SURFACE AREA IN MUSCLE 143

Folkow and Halicka, 1968; Hudlicka, 1969; Gray, 1971; Klabunde and Johnson, 1977). Folkow and Halicka (1968) found in cats that both blood flow and the capillary filtration coefficient (CFC) of postural soleus muscle were twice the flow and CFC of locomotor gastrocnemius muscle. They assumed that hydraulic conductivity (L,) was the same for all capillaries, so they could use CFC as a comparative measure of perfused capillary surface area per gram of tissue (S,). They concluded that at rest the soleus had twice the perfused capillary surface area of gastrocnemius muscle. Recent studies, however, have raised questions about the validity of the notion that blood flow and Sr are greater in highly oxidative skeletal muscles. For example, Bockman et al. (1980) found that flow to the resting oxidative soleus muscle of cats was not greater than blood flow to the more glycolytic gracilis muscle. Maxwell et al. (1980) found no consistent relationship between the total capillary density of skeletal muscles and their oxidative capacity or blood flow. In an earlier paper, Maxwell et al. (1977) reported that cat soleus and gastrocnemius muscles had similar oxidative ca- pacities. Klabunde and Johnson (1977) found that reactive hyperemia was similar in postural and locomotor muscle. McDonagh and Gore (1978) reported that the hydraulic conductivities (L,) of postural and locomotor muscles were not equal, as Folkow and Halicka had assumed. Thus, CFC, which is the product of L, and Sr, may not be an acceptable comparative measure of Sr in these two types of skeletal muscle. In order to compare our earlier single capillary L, data with whole organ CFC results, and to correlate muscle metabolism with vascularity, a measure of the perfused capillary surface area of postural and locomotor muscle was needed. No data for perfused capillary surface area in ALD and PLD muscles were available in the literature, so the following study was performed to determine Sf in postural and locomotor skeletal muscle.

THEORY

The functional capillary surface area (Sf) is the total capillary surface area of the perfused capillaries per gram of tissue. In this context the term “perfused” means that fluid is moving in the vessel. The fluid does not need to contain red cells. The value of Sr may be much less than the total anatomical capillary surface area (S,) because only a fraction of the total capillary population is normally perfused at any one time (Gray et al., 1982; Honig et al., 1970; Lindblom et al., 1980; Lalone and Johnson, 1979). The perfused capillary surface area per gram of tissue (Sr) can be defined mathematically as

where

Sf = Sc yf Nvlp, [II

S, = perfused capillary surface area per capillary. yf = number of perfused capillaries in a capillary-equivalent volume (V). V is

defined as a volume of tissue having unit cross-sectional area and thick- ness equal to one capillary length.

N, = the number of capillary-equivalent volumes per unit volume. N, equals unity length divided by capillary length.

P = tissue density.

144 MC DONAGH, GORE, AND GRAY

The perfused capillary surface area can be measured if representative values of all terms on the right-hand side of Eq. [l] can be determined. The perfused capillary surface area of a capillary (S,) is defined as

S, = ndL, PI

where d = internal capillary diameter and L = anatomical capillary length. To determine S,, capillary diameters and lengths can be measured from in vivo microvascular preparations (Eriksson and Myrhage, 1974; Gore, 1974; Honig et al., 1977; Klitzman and Duling, 1979) or from Microfil casts of the vascular bed being studied (Bassingthwaighthe et al., 1974; Plyley and Groom, 1975; Plyley et al., 1976). The number of perfused capillaries in a capillary equivalent volume (rr) can be measured by several techniques similar to those employed by Gray and Renkin (1977), Klitzman (1979), and LaLone and Johnson (1979) to determine the number of perfused capillaries per gram (03. The relationship between yf and Bf is

Of = yf NV/P. [31

For example, for a unit volume 1 mm on a side and a capillary length of 0.5 mm, of will be 2 times yJp.

In the present study, Sf was determined in two different ways. One method combined measurements of total muscle blood flow with single-capillary flow. We shall call this the “Flow Method” for determining perfused capillary surface area per gram (Sp). The second method involved determining yf from histologic sections of india ink-perfused muscles. We shall call this the “Histologic Method” for determining the perfused capillary surface area per gram (SF). The rationales of these two methods are described below along with their underlying assumptions.

Determination of Sp (Flow Method)

The number of perfused capillaries in a capillary-equivalent volume (r?) can be calculated from the average of single-capillary and total organ blood flow measurements. Consider a unit volume of tissue 1 mm on a side. If we know the blood flow per unit weight to the muscle (Q-r) and the capillary length, then the blood flow to a capillary equivalent volume (QV) is

Qv = QTP/N~. [41

Equation [4] states that the blood flow per capillary-equivalent volume is equal to the product of blood flow per unit weight (QT) and the tissue density (p, unit mass/unit volume) divided by the number of capillary-equivalent volumes per unit volume (N,). The blood flow per perfused capillary (Qc> can be determined from single-capillary measurements as

Qc = nd2 v,/4h, [51

where

v, = the average red cell capillary velocity. A = Coefficient relating the capillary red cell velocity (v,) to the capillary bulk

fluid velocity (vb). That is, A = v&.


The number of perfused capillaries in a capillary equivalent volume (yp) can be calculated from

Y? = Qv/Q C’ WI

The rationale underlying Eq. [6] is similar to the one recently used by Klitzman and Duling (1979) for a red cell mass balance in the cremaster muscle. Several conditions are necessary to employ Eq. [6] to estimate yf. The capillary dimensions (d and L) must be independent of flow. The coefficient A must be known for the vascular bed under study. The capillary dimensions and velocities must be representative of the whole muscle. Substituting Eq. [2] and [4]-[6] into Eq. [I] gives, for the flow method,

SF = 4WId)(Q~lv,). [71

The principal condition necessary to combine Eq. [6] with Eq. [l] is that the average length of a flow path in the capillary-equivalent volume (V) is equal to one capillary length. A flow path is defined here as the total distance traveled by a particle of blood from one cross-sectional face of V to the opposite cross- sectional face of V. Additionally, Eq. [7] requires that all the blood must flow through capillaries, so there can be no shunts. From our direct observations of microvascular flow in muscle, we have not observed any significant arteriovenous anastomoses or thoroughfare channels. Other workers (Eriksson and Myrhage, 1974; Klitzman and Duling, 1979) have not observed anatomical shunts in skeletal muscle. Thus, the “no-shunt” requirement appears to be well satisfied in skeletal muscle.

Determination of SfH (Histologic Method) The number of perfused capillaries in a capillary-equivalent volume (yf) can

also be determined from perfused capillary density measurements. This technique is similar to that employed by Bassingthwaighte et al. (1974) to estimate the total capillary surface area (ST) in the left ventricle of the dog; by Myrhage and Hudlicka (1976), to estimate ST in the rat extensor hallucis proprius muscle; and by Gray and Renkin (1978) to estimate ST in rabbit gastrocnemius and soleus muscles. To evaluate the perfused capillary density, an intravascular marker, such as an india ink solution, is infused upstream from the arterial input to the muscle. During the ink infusion, the muscle is frozen in situ and a biopsy is taken. The biopsy is sectioned transverse to muscle fibers, and the numbers of ink-filled capillaries per cross-sectional area (4,“) are counted. The number of perfused capillaries per capillary equivalent volume (yfH) can be calculated as

Yf” = @. Bl

For example, considering a unit tissue volume of l-mm’ cross section and 1 mm thick, if the perfused capillary density was found to be 300 capillaries/mm2 and the average capillary length was found to be 0.5 mm, then yf” = 300 caps/V. The number of perfused capillaries per unit volume (mm3) is 2 x 300 = 600 caps/mm3. The perfused capillary surface area per gram of tissue (SF) can be calculated by combining Eqs. [l], [2], and [8] to give

S,” = rd & N,/p. [91


Note that the vascular length term L cancels out explicitly but remains in Eq. [9] implicitly because L is required to determine N,.

Tissue Preparation The skeletal muscle model employed in these studies was modified from the

anterior latissimus dorsi (ALD) and the posterior latissimus dorsi (PLD) avian muscle preparation developed by Klabunde and Johnson (1977). Since our single capillary hydraulic conductivity measurements were made in these muscles (McDonagh and Gore, 1978), perfused surface area determinations were also made using this preparation. The ALD is a postural muscle composed entirely of oxidative fibers, whereas the locomotor PLD is composed entirely of glycolytic fibers (Gray et al., 1982). Five- to eight-week-old white leghorn chickens were anesthetized with L.A. Thesia3 (3.5 cc/kg im with maintenance doses of lo-15% of initial dose). A branch of the left brachial artery was cannulated for systemic blood pressure measurement. Body temperature was monitored and maintained at 40”. The muscle stage was kept at 39” and a specially designed tissue chamber was used to contain the silicone oil covering the muscles. This preparation allowed direct viewing of the microcirculations of both the ALD and PLD muscles. The blood and neural supplies to these muscles were left intact. After preparing the muscles, the animal stage was mounted under a Leitz intravital microscope. Capillary fields were brought into focus and the microscope image was viewed through a video camera and a TV monitor.

Determination of Se To compute functional capillary surface area per gram from blood flow data

(SF), an average value of all the variables on the right-hand side of Eq. [7] was required. Capillary lengths (L) were determined directly using a Nikon filar micrometer eyepiece. In this study the capillary length was measured from the end of a terminal arteriole to the beginning of a collecting venule. Capillary internal diameters (d) were measured from videotapes of capillary fields (Sony AV-3650 videotape recorder). In stop-frame videotape playback a calibrated video micrometer described by Gore (1974) was used to measure capillary diameter. Red blood cell velocity in capillaries (v,) was determined from frame- by-frame analysis of the displacement of a red blood cell with time in the capillary. For this muscle preparation the red cells essentially filled the capillary. There was little apparent plasma space separating the red cell from the capillary en- dothelium. Approximately 10 red cells were analyzed in each capillary and a mean red cell velocity calculated for that capillary. The specimen-to-monitor magnification was 1059 x , thus, velocities up to 3 mm/set could be reliably measured. That is, in our system, a red cell moving at 3 mrn/sec took three or four video fields to cross the monitor face. A value of X = 1.3 was used in Eq. [5] to relate capillary red cell velocity to bulk fluid velocity. The A factor was estimated from known values of capillary hematocrit (McDonagh and Gore, 1978) and measures of capillary plasma and red cell velocities by Gaehtgens et al. (1976) and Albrecht et al. (1979).

’ L.A. Thesia is a commercially available anesthetic with pentobarbital and chloral hydrate as its active ingredients. It is very similar to Equi Thesin, which is also a common avian anesthetic.


Total blood flow (Q?) to the ALD and PLD muscles was determined using 15 pm microspheres (Strontium-85, 3M Corp.) and the reference organ technique (Heymann et al., 1977). For this series of experiments, a catheter was placed in the right brachial artery and connected to a Harvard withdrawal pump. A second catheter was inserted into the right carotid artery and connected to a Statham blood-pressure transducer. The catheter was advanced toward the heart while observing the pressure trace. The catheter was secured, after its position in the left ventricle was confirmed by observation of a typical left ventricular pressure waveform. The location of the left ventricular catheter was later confirmed at autopsy. For each microsphere injection, the reference pump withdrawal (0.196 ml/min) was begun 1 min prior to injection. A l-ml microsphere solution (approximately 4.5 x lo5 spheres in saline with Tween 80) was injected into the left heart via the left ventricular catheter. The microsphere injection took approximately 20 set and was followed by a l-ml flush of heparinized saline (30 set). The reference pump withdrawal was continued for 3 min following injection. The animal was immediately sacrificed with sodium pentobarbital and both the left and right ALD and PLD muscles were removed, blotted, and placed in preweighed test tubes. The reference organ blood (approximately 1.2 ml) was placed in three test tubes along with three flushes of the reference blood syringe. Additionally, contralateral muscles supplied by branches of both the thoracic and abdominal aorta were sampled to check the bilateral distribution of the spheres. The tissue samples, reference organ tubes, and three samples of the injectate were counted for Strontium-85 (window = 410 keV to x) to 2% error (10,000 counts) with a Searle gamma counter.

Measurement of SfH To determine the functional capillary surface area from histologic data (SF),

measurement of functional capillary density (4,“) was required to calculate the number of perfused capillaries per capillary-equivalent volume (Eq. [S]). For this series of experiments chickens were anesthetized with L.A. Thesia. The right common carotid artery was cannulated and a dialyzed, filtered, india ink solution, described by Gray and Renkin (1978), was infused at systemic pressure for 90-120 sec. The muscles were then frozen in situ with isopentane cooled in liquid nitrogen, removed, and sectioned (12 pm) in a cryostat. Muscle cross sections were stained with eosin as a background for the india ink-filled capillaries. Microscopic fields (1.6-3.6 mm*) were photographed and capillary counts were made of fields approximately 0.4 mm* in area. The measure of &’ was combined with the measures of d and L to calculate S,” using Eq. [9]. The value of tissue density used was 1.06 g/cm3 (Gore and Whalen, 1968; Close, 1972). Details of the method of measuring the capillary density from the histologic sections are given in Gray and Renkin (1978). Details of the method as applied to the ALD and PLD muscles have been described by McDonagh et al. (1980) and by Gray et al. (1982).

Data Analysis The results are expressed as mean *SD. Comparisons were made using the

Student t test for independent samples and a paired t test when appropriate. A propagation-of-errors analysis was made on the computed values of SF and S,” using the approach described by Young (1962).


RESULTS

The capillary dimensions and hemodynamic data measured in the ALD and PLD muscles are summarized in Table 1. The capillary diameters (7-8 Frn) were larger than mammalian values (4-5 pm) (Eriksson and Myrhage, 1974; Klitzman and Duling, 1979; Plyley et al., 1976) but the average diameter of capillaries in ALD muscles (7.3 + 1.1 km) was not significantly different from those in PLD muscles (7.9 + 1.5 pm) (P = 0.09). The capillary lengths were similar to those reported for mammals (Eriksson and Myrhage, 1974; Honig et al., 1977; Myrhage and Hudlicka, 1976; Smaje et al., 1970), but the ALD capillaries (625 -+ 260 pm) were significantly shorter than the PLD capillaries (1288 ? 548 pm) (P < 0.001). The average red blood cell velocities (ALD = 0.51 -+ 0.22 mmlsec, PLD = 0.34 ? 0.21 mm/set) were not significantly different (P = 0.07). The mean red cell velocities determined in our study are nearly identical to those reported by Klabunde and Johnson (1977) (ALD = 0.56 mm/s, PLD = 0.34 mm/s), who used the dual-slit photometric technique (Wayland and Johnson, 1967). However, they found that the ALD and PLD velocities were significantly different (P < 0.001). The difference in significance between our results (P = 0.07) and Kla- bunde and Johnson’s results (P < 0.05) may be due to our smaller sample size. The microsphere experiments yielded values for resting muscle blood flow in chickens (==6 ml/min/lOO g) that were similar to mammalian resting muscle flow rates. No statistical difference in blood flow was found in any of the left and right muscle pairs studied (ALD, PLD, pectoralis, and gastrocnemius). Also, no statistical difference was found in flow to the left and right kidneys. Thus, mixing of the microspheres appeared adequate and the ventricular cannulation did not alter blood flow. In 10 of the 14 muscle pairs examined, the resting blood flow through the oxidative ALD muscle was greater than flow through the glycolytic PLD muscle. However, the mean difference was not statistically significant at the 95% confidence level (paired t test). This result is in agreement with Bockman et al. (1980) who found no statistical difference in resting blood flow to cat soleus (oxidative) and gracilis (glycolytic) muscles. Other investigators (Folkow and

TABLE 1 MUSCLE CAPILLARY DIMENSIONS AND BLOOD FLOW MEASUREMENTS FOR DETERMINATION OF PERFUSED

CAPILLARY SURFACE AREAS

Muscle d (w) L km) v,

(mmisec) QT

(mlimin/lOO g) 4,”

(caps/mm’)

ALD 7.3 625 0.51 6.74 21.1” k 260 20.22 I2.06

n 22 22 I 14

PLD 7.9 1288 0.34 5.94 t 1.5 k 548 kO.21 21.46

nb 45 39 30 14

P’ 0.09 <O.OOl 0.07 0.112d

a SD. b n = Number of determinations. Not necessarily equal to number of animals. ‘ Two-tailed probability. d Paired t-test.

198 2112

41

58 2 40

33

<0.025


TABLE 2 FUNCTIONAL CAPILLARY SURFACE AREA FOR ALD AND PLD MUSCLES

OF (x 10-4) 0,” (x 1o-J) SP S,” Muscle (caps& (capsk) (cm’ig) (cm’ig)

ALD 6.9 29.9 9.8 42.8 26.8” ? 19.1

PLD 7.8 4.3 24.7 13.7 220.0 k 14.4

’ SD from propagation-of-errors analysis.

Halicka, 1968; Hudlicka, 1969) have found significantly higher blood flows in highly oxidative muscle than in glycolytic muscle. Also, our blood flow values (ALD = 6.74, PLD = 5.94 ml/min100 g) are lower than the total blood flow values reported by Hudlicka (1969) for the ALD and PLD muscles (14.6 and 10.7 ml/min/lOO g, respectively) using the xenon clearance technique.

Regarding the ink-perfusion studies, we found that the ink-perfused capillary density for the ALD muscle (198 ? 112 caps/mm*) was more than three times greater than the perfused capillary density of the PLD muscle (58 ? 40 caps/ mm*) (P < 0.025).

A value for the number of perfused capillaries per gram of tissue (@) was computed by applying the single capillary and total muscle blood flow data in Table 1 to Eqs. [3] and [6]. Also, the number of ink-perfused capillaries per gram (@) was calculated from the perfused capillary density data (r#$) in Table 1 and Eqs. [3] and [8]. The results are given in Table 2. The flow method (Eqs. [3] and [6]) yielded similar values of @ for both the ALD (6.9 x lo4 perfused caps/ g) and PLD (7.8 x lo4 perfused caps/g) muscles. The result was the same whether the mean Qc was calculated using mean values of d and v, (Eq. [5]) or from the mean of individual calculations of Qc. However, the histologic approach (Eqs. [3] and [S]) yielded almost a sevenfold larger 0,” for the ALD (29.9 x lo4 perfused caps/g) than the PLD (4.3 x lo4 perfused caps/g).

Perfused capillary surface areas (Sr) were then calculated by both methods (Eqs. [7] and [9]) and the results are given in Table 2. From Eq. [7], the perfused surface area (SF) for the ALD was 9.8 t 6.8 cm*/g, and SF for the PLD muscle was 24.7 ?Z 20.0 cm’/g. For the histologic approach, Eq. [9], the value of 0,” for the ALD muscle was almost seven times the value of 13,” for the PLD, but the mean S,” for the ALD (42.8 cm’/g) was only three times greater than the mean S,” for the PLD (13.7 cm*/g). Thus, although the ALD had roughly seven times as many perfused capillaries as the PLD, the ALD surface area was only about three times greater because ALD capillaries are only half as long as PLD capillaries.

DISCUSSION

The purpose of this study was to determine the functional or perfused capillary surface area (Sr) in postural and locomotor skeletal muscles. Sr was measured in two ways, based on two different rationales. The results from both approaches indicate that the perfused capillary surface area of postural muscle is not clearly


different from the Sf of locomotor muscle. The results from the histologic method indicate that the mean value of SF for the ALD (42.8 cm’/g) was three times larger than S,” for the PLD muscle (13.7 cm’/g) despite a sevenfold difference in the perfused capillary densities.

The values of surface area reported here are lower than many reported values (Pappenheimer and Soto-Rivera, 1948; Myrhage and Hudlicka, 1976; Gray and Renkin, 1978). This lack of agreement may be due to differences in animal models as well as due to different methodologies. In our study an avian model was used. The muscle fiber characteristics of the slow, tonic ALD muscle in the chicken are somewhat different than found in slow-twitch mammalian muscles (Ashmore et al., 1978). However, referring to Tables 1 and 3, we found that the values of total blood flow, capillary velocity, capillary density, and capillary dimensions for the avian ALD and PLD muscles, were quite similar to mammalian values. On that basis our capillary surface area results should be similar, not smaller than other reported values. Another possible explanation is that the duration of india ink addition was too short. In our determination of S,“, india ink was added to the blood for 90-120 sec. From a recent report by Renkin et al. (1981), this duration of ink infusion should be more than sufficient to mark perfused capillaries. The principal difference between our results and earlier reports is that our values represent the perfused or functional capillary surface area (Sf) not the total anatomical surface area (S,). Other workers have also found an &/ST of 0.5 or less. Lindbom et al. (1980) recently reported that the perfused capillary density in rabbit tenuissimus muscle is less than half the total capillary density. LaLone and Johnson (1979) reported that only 20% of the capillaries were perfused in the resting cat sartorious muscle. Honig et al. (1970) estimated that only 10% of the capillaries were perfused in the highly glycolytic cat gracilis muscle. Thus, there can be as much as a lo-fold difference between the results for perfused and total capillary surface areas of skeletal muscle.

The agreement between the flow (24.7 2 20.0 cm’/g) and histologic (13.7 & 14.4 cm*/g) measures of Sr for the PLD muscle indicates that for the PLD the requirements and assumptions in both methods were reasonably well satisfied. This was not the case for the ALD muscle (SF = 9.8 and SF = 42.8 cm*/g). The difference in SF and S,” for the ALD implies that the conditions inherent in Eqs. [7] and [9] were not met for this thicker, highly oxidative muscle. The number of perfused capillaries per capillary equivalent volume (-#) was calculated cor- rectly for the ALD by applying the single vessel and total flow data to Eq. [6]. But, in order to combine Eqs. [6] and [l] to give Eq. [7], it is necessary that L equal the length of a flow path. This condition may not have been satisfied for the ALD muscle. This point is illustrated in Fig. 1. The upper panels of Fig. 1 show longitudinal sections of india ink-perfused ALD and PLD muscles. The postural ALD is a completely oxidative muscle with numerous intercapillary anastomoses, while the locomotor PLD muscle is highly glycolytic with few, if any, intercapillary connections. The vasculature of the PLD is a very simple, parallel microcirculation. The PLD is only a few muscle fibers thick and the microcirculation is essentially two dimensional. The PLD capillaries are long and straight, much like the idealized vasculature represented by a Krogh cylinder model. The lower panels of Fig. 1 are simplified schematic representations of the capillary architectures of the ALD and PLD muscles shown in the upper


ALD (TONIC)

PLD 1 PHASIC)

FIG. 1, (Upper) Longitudinal sections of india ink-perfused anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) muscles. (Lower) Schematic representation of typical flow paths taken by red blood cells in the oxidative ALD (left) and glycolytic PLD (right) muscles. The dotted lines represent hypothetical lines of section through the muscles.

panels. The arrows depict a hypothetical path taken by blood passing through a portion of the ALD and PLD capillary beds. Although the paths shown in the bottom panels of Fig. I are only illustrations, we routinely observed these flow patterns in our in viva studies. In the ALD muscle, a red cell will often travel from arteriole to venule in a circuitous manner passing from one capillary to another via intercapillary connections. Often the red cell will move perpendicular to the plane of focus via an intercapillary connection. Thus, in the ALD, the actual path traveled by a red cell from arteriole to venule or through a capillary- equivalent volume (flow path) may be much longer than the anatomical capillary length. The use of the anatomical L in Eq. [7] may cause the ALD SF to be underestimated. In contrast, in the PLD muscle, a red cell travels from arteriole to venule in a straightforward manner along a single capillary. In this case, the actual flow path more likely equals the anatomical capillary length and the conditions inherent in Eq. [7] are better satisfied.

Regarding the histologic method (Eq. [9]), similar flow paths were probably taken by the india ink during perfusion of these muscles. The dotted lines in the lower panels of Fig. I represent hypothetical planes of section through these muscles. From this figure it can be seen that, from histologic analysis, a single path in the ALD muscle would be counted as three perfused capillaries. This overestimate of the number of flow paths would tend to offset the underestimate of path length, and Eq. [9] would give a more reasonable value of Sf for the ALD. In the glycolytic PLD muscle a single path would be counted as one


capillary, and the path length would equal the capillary length. Thus, Eq. [9] would also give a reasonable estimate of Sr for the PLD muscle. In addition, we did not observe any thoroughfare channels or arteriovenous anastomoses in the PLD muscle, which is only a few muscle cell layers thick. Hence, the requirement of no A-V shunts was also satisfied for the PLD muscle. Since we were unable to visually follow the entire flow path of many red cells through the ALD microcirculation, A-V shunting cannot be ruled out in this muscle. Freeze-fixing both muscles in situ helped insure an accurate determination of perfused capillary density.

Assuming that SF for ALD (42.8 cm*/g) is the true perfused capillary surface area then a SF = 9.8 cm’ig for ALD implies that the average flow path was roughly 4.4 times the anatomical capillary length. This value is more than twice the 2.0 tortuosity factor estimated by Renkin et al. (1981) for highly oxidative muscles. Referring to Fig. 1, the number of capillary segments per capillary in the chicken ALD may be quite similar to the 4.4 segments/capillary value reported for the frog sartorius muscle by Plyley et al. (1976). If this is the case, a tortuosity factor for ALD could exceed 2.0. Also ALD capillary flow is not totally uni- directional, which would increase the flow path length. As illustrated in the lower left panel of Fig. 1, we frequently observed red cells flow in opposite directions in adjacent capillary segments. In the case of the PLD muscle, the capillary segment length is equal to the anatomical capillary length and any tortuosity factor is probably close to 1.0.

Table 3 is a summary of the literature of microvascular dimensional and hemodynamic data on skeletal muscles whose muscle fiber composition is known. From the available data both the perfused capillary surface area (Sr) and the total capillary surface area (S,) were calculated. Sr was calculated whenever possible using both Eqs. [7] and 191. ST was always calculated from Eq. [9] using available data on total capillary density. This analysis was performed to compare our results with the literature, to examine the relationship between muscle metabolism and vascularity, and to gain some insight into how intercapillary anastomoses may affect surface area calculations. The first column lists the animal, the muscle evaluated, and the literature references from which parameters were obtained. Columns 2-7 give the hemodynamic and morphologic data used in the computations. These values came from one or more of the references given in column 1. The Sr and ST columns give values of the perfused and total capillary surface areas, respectively, computed from the data in columns 2-7. The method column describes which method (histoiogic, Eq. [9]; flow, Eq. 171) was used to calculate Sr and ST. For Eq. [9], we used values for p = 1.06 g/cm* (Gore and Whalen, 1968; Close, 1972) and A = 1.3 (estimated from Gaehtgens et al. (1976) and Albrecht et al. (1979)) in Eq. [7]. Column 11 describes each muscle in terms of its fiber-type composition according to the nomenclature of Peter et al. (1972). The percentage fiber composition column gives a histochemical description of the muscle in terms of its twitch characteristics (S, slow twitch; F, fast twitch) and its biochemical characteristics (0, oxidative; G, glycolytic). The intercapillary connections column gives a qualitative assessment of the degree of intercapillary anastomoses in that muscle.

Referring to Table 3, it can be seen that the calculated Sr values for chicken ALD and PLD muscles are in the same range as calculated mammalian values.

TABL

E 3

DET

ERM

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8).


The calculated values of ST for ALD and PLD muscles are well within the range of mammalian ST values previously reported in the literature.

Also, from this analysis it can be seen there is no clear relationship between the oxidative capacity of a skeletal muscle and the total capillary surface area (ST). For example, Gray and Renkin (1977) found in the rabbit that the oxidative soleus had twice the total capillary surface area of the more glycolytic gastrocnemius muscle. In the present study we found in the chicken that the ST of the oxidative ALD was only 20% greater than the ST of the glycolytic PLD muscle. The difference between these results may be due to a difference between mammals versus birds. Also, our measured capillary length in the PLD was twice that of the ALD muscle. Gray and Renkin (1978) assumed a value for L = 1000 km in which case a capillary equivalent volume equaled a unit volume (i.e., N, = 1). Maxwell et al. (1980) also found no clear relationship between capillarity and the oxidative capacity of a muscle.

Good agreement was obtained for the perfused capillary surface areas calculated by the histologic and flow methods for the chicken PLD, the rat gracilis, and the cat sartorius muscles. These muscles all have a high proportion of glycolytic muscle fibers and few intercapillary connections. In contrast, poor agreement in Se and SF was found for the chicken ALD and the rat cremaster muscles. These muscles have a high proportion of oxidative muscle fibers and numerous intercapillary connections. For the ALD and cremaster muscles, the flow method (Se) yields a much smaller value of surface area than the histologic method. Thus, as we found for the ALD muscle, it is likely that the cremaster capillary length (615 pm) (Smaje et al., 1970) is less than the average flow path length. Using Eq. [9], there was good agreement between ST calculated (223 cm2/g) for the cremaster muscle versus the 244 cm2/g value reported by Smaje et al. (1970). This analysis suggests that only half of the total capillary surface area is perfused in the oxidative cremaster muscle.

Table 3 also lists the results of surface area calculations for the cat tenuissimus and the rat extensor hallucis proprius (EHP) muscles. For the cat tenuissimus our calculation of total surface area (S.y = 103 cm’ig) agrees well with Erickson and Myrhage’s (1974) value (S = 90 cm’ig) and the Schmidt-Schoebein et al. (1977) value of 101 cm’/g (using their estimate of p = 1.28 g/cm”). Since the tenuissimus is a highly glycolytic muscle with few intercapillary anastomoses, it is likely that the histochemical approach gave an accurate value of ST for this muscle. Thus, it appears that all the capillaries were filled with carbon solution at the time of fixation in the Schmidt-Schoenbein et al. study. We calculated from literature data that the perfused capillary surface area in cat tenuissimus muscle was approximately 10% of the total capillary surface area. A ratio of Sf/ST = 0.1 seems small, but the tenuissimus is a highly glycolytic muscle and this value agrees with our 11% value for the totally glycolytic PLD muscle and the 10% value reported by Honig et al. (1970) for the glycolytic gracilis muscle. From the rat EHP muscle data we calculated a similar value of total surface area (S,“) as that reported by Myrhage and Hudlicka (1976) (170 and 160 cm’ig, respectively). Their approach is conceptually quite similar to the histologic method that we used (Sy).

A technique for calculating the capillary surface area in cardiac muscle has been developed by Bassingthwaighte et al. (1974). Their approach is based on


the rationale that the surface area per unit weight equals the product of the capillary surface area per tissue cross-sectional area times an equivalent cross- sectional area per tissue weight. The difference between the Bassingthwaighte et al. approach and Eq. [9] is that their relationship assumed that NV = 1. This may not be the case because the authors indicate that the mean coronary capillary length in dogs may be much larger than 1000 pm. If so, then NV would be much smaller than unity. Additionally, it is implicitly assumed in their relationship that each capillary counted in cross section equals a perfused path. From recent direct observations of coronary microvascular flow in the rat heart (McDonagh et al., 1981; McDonagh and Niven, 1982) it appears that the coronary capillary perfusion pattern is quite heterogeneous. It is possible that capillary cross-sectional counts in the highly oxidative heart may overestimate the true number of perfused paths.

The large difference in Sf and ST found in this study, and in our review of the literature, may help explain the lack of agreement between the results of single- capillary and whole-organ solute transport studies (Crone and Christensen, 1979). Single capillary studies often report solute permeability coefficients (P) that are an order of magnitude larger than those calculated by dividing whole organ PS values by an assumed surface area (S) (Crone et a/., 1978; Crone and Christensen, 1979). Often the measured PS value is divided by the total capillary surface area, ST. If SP/ST is actually less than 1, then the resulting P value will be smaller than the true solute permeability coefficient. This discrepancy also occurs when whole-organ fluid-exchange studies are compared to single capillary hydraulic conductivity measurements (Gore and McDonagh, 1980).

In summary, we found that the perfused capillary surface area (S,) of postural skeletal muscle was not clearly different from the Sf of locomotor muscle. Thus, different hydraulic conductivities (~5,) may also be an important determinant in the different fluid-exchange capabilities of postural versus locomotor skeletal muscle. We also found in agreement with Bockman et ul. (1980) that blood flow through oxidative muscles is not significantly different than blood flow through glycolytic muscles. With regard to the two methods for determining perfused surface area described in this report, we conclude that the flow method under- estimates surface area of oxidative muscles with numerous intercapillary connections, while the histologic method gives a reasonable estimate of surface area. For glycolytic muscles with few intercapillary connections, both the flow and histologic methods appear to be acceptable for computing &. We found that postural muscle has shorter capillaries and faster red cell flow than locomotor muscle. However, the flow path in the ALD is circuitous, so the transit time through this oxidative muscle may be longer. We also deduced that only a small fraction of the total capillary surface area is perfused at rest, indicating a significant capillary reserve in skeletal muscle. Since surface area is a normally varying physiological parameter, it is felt that this type of measurement is necessary when comparing whole-organ data to the results of single-capillary transport studies. An independent measurement of S,- is also often necessary when whole-organ studies are performed to examine the effect of some intervention on capillary permeability.


ACKNOWLEDGMENTS

We express our thanks to Patricia Ferrer and Laurie Dodd for their excellent technical assistance, and to Kathleen Cocks and Mary Peters for preparing this manuscript.

REFERENCES

1. ALBRECHT, K. H., GAEHTGENS, P., DRIES, A., AND HEUSER, M. (1979). The Fahraeus effect in narrow capillaries (i.d. 3.3 to 11.0 pm). Microvusc. Res. 18, 33-47.

2. ARIANO, M. A., ARMSTRONG, R. B., AND EDGERTON, V. R. (1973). Hindlimb muscle fiber pop- ulations of five mammals. J. Hisfochem. 21(l), 51-55.

3. ASHMORE, C. R., KIKUCHI, R., AND DOERR, L. (1978). Some observations on the innervation patterns of different fiber types of chick muscle. Exp. Neural. 58, 272-284.

4. BASSINGTHWAIGHTE, J. B., YIPINTSOI, T., AND HARVEY, R. B. (1974). Microvasculature of the dog left ventricular myocardium. Microvasc. Res. 7, 229-249.

5. BOCKMAN, E. L., MCKENZIE, J. E., AND FERGUSON. J. L. (1980). Resting blood flow and oxygen consumption in soleus and gracilis muscles of cats. Amer. J. Physiol. 239, H516-524.

6. CLOSE, R. I. (1972). Dynamic properties of mammalian skeletal muscles. Physiol. Rev. 52, 129-197.

7. CRONE, C., JENSEN-FROKJAER, J., FRIEDMAN, J. J., AND CRISTENSEN, 0. (1978). The permeability of single capillaries to potassium ions. J. Gen. Physiol. 71, 195-220.

8. CRONE, C., AND CHRISTENSEN, 0. (1979). Transcapillary transport of small solutes and water. In “International Review of Physiology, Cardiovacular Physiology III,” (A. C. Guyton and D. B. Young, eds.), Vol. 18, Chap. 5. Univ. Park Press, Baltimore.

9. ERIKSSON, E., AND MYRHAGE, R. (1972). Microvascular dimensions and blood flow in skeletal muscle. Acta Physiol. Stand. 86, 211-222.

10. FOLKOW, B., 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. Microvasc. Res. 1, l-14.

11. GAEHTGENS, P., BENNER, K. U.. SCHICKENDANTZ, S., AND ALBRECHT, K. H. (1976). Method for simultaneous determination of red cell and plasma flow velocity in vitro and in vivo. PJ~ugers Arch. 361, 191-195.

12. GORE, R. W., AND WHALEN, W. J. (1968). Relations among tissue PO?, QOz, and resting heat production of frog sartorius muscle. Amer. J. Physiol. 214(2), 277-286.

13. GORE, R. W. (1974). Pressures in cat mesenteric arterioles and capillaries during changes in systemic arterial blood pressure. Circ. Res. 34, 581-591.

14. GORE, R. W. AND MCDONAGH, P. F. (1980). Fluid exchange across single capillaries. Annu. Rev. Physiol. 42, 337-357.

15. GRAY, S. D. (1971). Responsiveness of the terminal vascular bed in fast and slow skeletal muscles to a-adrenergic stimulation. Angiologica 8, 285-296.

16. GRAY, S. D., AND RENKIN, E. M. (1977). Blood supply in relation to cell metabolic type in mixed skeletal muscle. Fed. Proc. 36, 552. [Abstract]

17. GRAY, S. D., AND RENKIN, E. M. (1978). Microvascular supply in relation to fiber metabolic type in mixed skeletal muscle of rabbits. Microvasc. Res. 16, 406-424.

18. GRAY, S. D., MCDONAGH, P. F., AND GORE, R. W. (1982). Comparison of functional and total capillary densities in fast and slow muscles of the chicken. Submitted for publication.

19. HENRICH, H. N., AND HECKE, A. (1978). A gracilis muscle preparation for quantitative microcirculatory studies in the rat. Microvasc. Res. 15, 349-356.

20. HEYMANN, M. A., PAYNE, B. D., HOFFMAN, J. I. E., AND RUDOLPH, A. M. (1977). Blood flow measurements with radionuclide-labeled particles. Progr. Cardiovasc. Dis. 20, 55-79.

21. HONIG, C. R., FRIERSON, J. L., AND PATTERSON, J. L. (1970). Comparison of neural controls of resistance and capillary density in resting muscle. Amer. J. Physiol. 218(4), 937-942.

22. HONIG, C. R., FELDSTEIN, M. L., AND FRIERSON, J. L. (1977). Capillary lengths, anastomoses, and estimated capillary transit times in skeletal muscle. Amer. J. Physiol. 233(l), H122-H129.

23. HUDLICKA, 0. (1969). Resting and postcontraction blood flow in slow and fast muscles of the chick during development. Microvasc. Res. 1, 390-402.

24. JOHNSON, P. C., BURTON, K. S., HENRICH, H., AND HENRICH, LJ., (1976). Effect of occlusion duration reactive hyperemia in sartorius muscle capillaries. Amer. J. Physiol. 230, 715-719.


25. KLABUNDE, R. E., AND JOHNSON, P. C. (1977). Reactive hyperemia in capillaries of red and white skeletal muscle. Amer. J. Physiol. 232(4), H411-417.

26. KLITZMAN, B. (1979). “Microvascular Determinants of Oxygen Delivery in Striated Muscle,” Ph.D. dissertation. University of Virginia, Charlottesville.

27. KLITZMAN, B., AND DULING, B. R. (1979). Microvascular hematocrit and red cell flow in resting and contracting striated muscle. Amer. J. Physiol. 237, H481-H490.

28. LINDBOM, L., TUMA, R. F., AND ARFORS, K. E. (1980). Infuence of oxygen on perfused capillary density and capillary red cell velocity in rabbit skeletal muscle. Microvasc. Res. 19, 197-208.

29. LALONE, B. J., AND JOHNSON, P. C. (1979). Arteriolar-capillary network analysis in resting cat sartorius muscle. Microvasc. Res. 17(3), Part 2, S19. [Abstract]

30. MAXWELL, L. C., BARCLAY, J. K., MOHRMAN. D. E., AND FAULKNER, J. A. (1977). Physiological characteristics of skeletal muscles of dogs and cats. Amer. J. Physiol. 233(l), C14-C18.

31. MAXWELL, L. C., WHITE, T. P., AND FAULKNER, J. A. (1980). Oxidative capacity, blood flow, and capillarity of skeletal muscles. J. Appl. Physiol. 49(4), 627-633.

32. MCDONAGH, P. F., AND GORE, R. W. (1978). Comparison of hydraulic conductivities in single capillaries of red versus white skeletal muscle. Microvasc. Res. 15, 269. [Abstract]

33. MCDONAGH, P. F., GORE, R. W., GRAY, S. D., AND FERRER, P. (1980). Perfused capillary surface area in tonic and phasic skeletal muscle. Microvasc. Res. 20, 119-120.

34. MCDONAGH, P. F., LAKS, H., AND WILLIAMS, S. K. (1981). Direct visualization of transport in the coronary microcirculation-Effect of global ischemia-reperfusion. Microvasc. Res. 21, 250.

35. MCDONAGH, P. F., AND NIVEN, T. (1982). Direct visualization of transcoronary albumin exchange-Effect of perfusate composition. Microvasc. Res. 23, 266.

36. MYRHAGE, R., AND HUDLICKA, 0. (1976). The microvascular bed and capillary surface area in rat extensor hallucis proprius muscle (EHP). Microvasc. Res. 11, 315-323.

37. MYRHAGE, R. (1978). Capillary supply of the muscle fiber population in hindlimb muscles of the cat. Actu Physiol. Scund. 103, 19-30.

38. PAPPENHEIMER, J. R., AND SOTO-RIVERA, A. (1948). Effective osmotic pressure of the plasma proteins and other quantities associated with the capillary circulation in the hindlimbs of cats and dogs. Amer. .I. Physiol. 152, 471-491.

39. PETER, J. B., BARNARD, R. J., EDGERTON, V. R., GILLESPIE, C. A., AND STEMPEL, K. E. (1972). Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemisrry 11, 2627-2633.

40. PLYLEY, M. J., AND GROOM, A. C. (1975). Geometrical distribution of capillaries in mammalian striated muscle. Amer. J. Physiol. 228(5), 1376-1383.

41. PLYLEY, M. J., SUTHERLAND, G. J., AND GROOM, A. C. (1976). Geometry of the capillary network in skeletal muscle. Microvusc. Res. 11, 161-173.

42. PREWITT, R. L., AND JOHNSON, P. C. (1976). The effect of oxygen on arteriolar red cell velocity and capillary density in the rat cremaster muscle. Microvasc. Res. 12, 59-70.

43. RENKIN, E. M., GRAY, S. D., AND DODD, L. R. (1981). Filling of microcirculation in skeletal muscles during timed India ink perfusion. Amer. J. Physiol. 241, 174-186.

44. SCHMID-SCHOENBEIN, G. W., ZWEIFACH, B. W., AND KOVALCHEK, S. (1977). The application of stereological principles to morphometry of the microcirculation in different tissues. Microvusc. Res. 14, 303-317.

45. SMAJE, L., ZWEIFACH, B. W., AND INTAGLIETTA, M. (1970). Micropressures and capillary filtration coefficients in single vessels of the cremaster muscle of the rat. Microvasc. Res. 2, 96-110.

46. SMITH, R. D. AND GIOVACCHINI, R. P. (1956). The vascularity of some red and white muscles of the rabbit. Acru Anut. 28, 342-358.

47. WAYLAND, H., AND JOHNSON, P. C. (1967). Erythrocyte velocity measurement in microvessels by a two-slit photometric method. J. Appl. Physiol. 22, 333-337.

48. YOUNG, H. D. (1962). “Statistical Treatment of Experimental Data,” pp. 96-101. McGraw-Hill, New York.

perfused capillary surface area in postural and locomotor skeletal muscle

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