Krrrv FRONEK AND BENJAMIN W. ZWEIFACH University of California, San Diego, AMES-Bioengineering, M-005. La Jolla, California 92093 Received September 17, I976 Blood flow and microvascular pressure distribution were determined in arterioles of the tenuissimus muscle by use of intravital microscopy and the radioisotope-labeledmicrospheres technique. The tlow rates were found to range from 3.8 up to 11.7 mm/set in arterioles and precapillaries with diameters of from 50 to 9 m. An idealized construction of segmental dichotomization was developed on the basis of volume flow distribution. It was found that the terminal arterioles divide into five precapillaries. Microvascular pressure showed a sharp drop in this region. The distribution of microspheres was used to compare relative blood flow in dissected and undissected paired muscles on the right and left sides. The dissection associated with the intravital preparation, and the ensuing 2-hr exposure of the tenuissimus muscle, did not influence appreciably the blood flow in this skeletal muscle. It is concluded that the tenuissimus muscle preparation is well suited for microvascular studies.
INTRODUCTION The supply of blood and its distribution in a striated skeletal muscle has, until recently, been studied almost exclusively on an input-output basis. With the development of quantitative microvascular techniques, it has become possible to approach the microcirculation of skeletal muscle directly and to determine basic microcirculatory parameters. M. tenuissimus has the characteristic attributes of a skeletal type of striated muscle, since it contains a mixture of white and red fibers (Eriksson and Myrhage, 1971). The muscle is straplike and consists of several thin layers of muscle fibers, so that it is well suited for direct microscopy. Blood flow in individual surface capillaries of the tenuissimus muscle in the cat has been described by Eriksson and Myrhage (1972), and in the rabbit by Tuma et al . (1975). This approach is not suitable for determining the total blood flow to the whole muscle. Rutili and Arfors (1976) using the radiolabeled-microsphere technique measured total flow in the tenuissimus muscle of the rabbit, and concluded that its flow rates compare well with the blood supply to other skeletal muscles. The tenuissimus muscle in the cat is more delicate, and transillumination facilitates flow measurement on vessels as large as 100 pm. In a recent report, Fronek and Zweifach (1975) described the pressure distribution in the successive microvessel segmentsof the tenuissimus muscle in the cat, in conjunction with micropressure changesresulting from vasodilator perturbations. As a further extension, it is the purpose of the present study to delineate the blood flow distribution in the hierarchy of microvesselsfrom the large feeding arterioles to the true capillaries. Furthermore, the physiologic state of m. tenuissimus under exteriorized conditions was tested in a whole organ study using the radioisotope-labeled-microspheretechnique. I This research was supported by USPHS Grant HL- 10881. 181
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Experiments were performed on 16 cats of average weight, 1.2-1.8 kg, anesthetized with ketamine 20 mg/kg in combination with chloralose 50-70 mg/kg. The systemic arterial pressure was measuredby a catheter placed in the contralateral femoral artery. The cat was encased in a supporting plastic mold and the m. tenuissimus was dissected and placed under the microscope in the samemanner as described previously (Fronek and Zweifach, 1975). Special care was taken to maintain the preparation under constant physiological temperature. The limb in which the m. tenuissimus was dissected was wrapped with heating tape. A second heating tape was wrapped around the light pipe. A thermistor sensing the temperature underneath the free edge of the tenuissimus muscle servedto activate a temperature regulating circuit. Magnifications of 300 to 400 were obtained from UM Leitz objectives having a working distance of several centimeters. By shifting the focus, microvessels at different depths and of different diameters were available for observation. The servo-nulling method was used for microvascular pressure measurements (Wiederhielm et al., 1964; Intaglietta et al., 1970), and an electrooptical technique of image splitting was utilized for vessel diameter determination (Baez, 1966; Intaglietta and Tompkins, 1973). Microvascular blood flow velocities were monitored by continuous recording of the optical density changesproduced by the blood flow stream in the microvessel. The actual velocity was measured as a time delay between two sensing sites positioned at a known distance apart, and the signals were continuously recorded by on-line crosscorrelation (Intaglietta et al., 1970). Before measurements were made, the surgically exposed area was covered with a plastic sheet (Saran Wrap) for 20-30 min. Microvascular pressure measurement was made through a circular opening in the plastic sheet,covered with mineral oil. Whole organ flow studies with radioisotope-labeled microsphereswere performed on eight cats. The protocol of the experiments was basically the sameas that used by other investigators (Neutze et al., 1968; Roth et al., 1970; Foreman et al., 1976; Marcus et al., 1976). The day before the experiment, the cats were prepared in the following way: They were anesthetized with pentobarbital, 30 mg/kg, intubated, and artilically ventilated. A PE 190 tubing filled with heparin was placed via open chest into the left atrium of the heart. The other end of the tubing, attached to a stopcock, was exteriorized through a skin tunnel in the interscapular region, and was used as an injection site. On the following day, microspheres were administered as follows: carbon microspheres (supplied by 3M Co.) of average size (15 + 5 m) labeled with 85Sr,14rCe, and 51Cr were used. The specific activity (cpm) and the number of particles were established for each shipment. A well-mixed sample of O-4-0.5 ml was taken into a heparinized Hamilton syringe and the exact amount was determined by weight and corrected for specific gravity. Just prior to injection, the sample was diluted up to 4 ml with dextran. After the injection, the syringe and catheter were flushed with an additional 4 ml of dextran. Residual activity in the syringe and catheter was measured and subtracted from the initial activity of the injected sample. The injection was performed slowly, to avoid possible transient changes in heart rate which may occur with rapid injection rates. Each injected sample contained 311 x lo6 microspheres.The 85Sr-labeledmicrospheres were injected shortly after the cat was anesthetized.The right
m. tenuissimus was then dissected, exposed, and covered with the plastic sheet. The temperature was maintained at 37’ and the preparation was left undisturbed under these conditions for an hour, at which point spheres labeled with 141Cewere administered. Two hours later, sphereslabeled with *‘Cr were injected. Cats were sacrificed with an overdose of pentobarbital and the following tissue samples were taken: (1) both mm. tenuissimi, (2) section from adjacent mm. biceps femoris, and (3) both kidneys. Each sample was blotted, weighed, and placed in plastic counting vials, and the disintegration of single isotopes was counted in the respective specific-spectrum windows: **Sr in 440-640 keV, 141Cein 65-200 keV, and *‘Cr in 260-390 keV. From the injected activity and the activity of the given sample normalized for 100 g, the percentage of total flow to the given region was determined and the ratio between the dissected and intact mm. tenuissimi (left :right) was calculated. RESULTS Blood velocity under steady-state conditions was measured in microvessels ranging from precapillaries (9 m) up to arterioles (50 w in diameter) by positioning sensing diodes along the centerfine of the vessel. Velocity us Volumetricjlow Studies of flow-velocity profiles across different-sized tubes indicate that the ratio of centerline velocity of the erythrocytes to the mean erythrocyte velocity plus plasma is 1.6. This correction factor was determined by Baker and Wayland (1974) for tubes from 80 to 23 ,um. Lipowsky (1975) further demonstrated the validity of this factor down to tubes of 17 ,um in diameter. For tubes smaller than 17 q, expe&nentai data for centerline erythrocytes velocity and the relationship to the mean velocity of the whole blood are not yet available. For this reason, we preferred to present the results as non-corrected flow velocities. The flow velocities expressedin this way range from 3.8 mm/set in the smallest arterioles to 11.7 mm/set in the 50-m vessels. Table 1 summarizes such hemodynamic data. Volume flow was calculated as a product of flow velocity and the vessel cross-section area, assuming a circular cross section for the arteriolar microvessels. TABLE
MICROVASCIJLAR FLOW AND PRESSURE DISTRILWION IN M. TENUISSIMUS Vessel diameter ti)
9 21.0 40.7 50.0
Yk 0.3 + 0.4 f 1.0 + 2.8
Volume flow (ml x lo-J/min)
2.0 17.2 89.0 312.5
f + f +
0.15 1.6 10.9 50.0
3.8 9.4 11.5 11.7
(mm Hd + + + +
1.4 1.4 1.7
38 + 5.3 70 f 4.0 88 f 6.2 96 f 3.8
Vascularity and Blood Flow Data on Aow distribution in successive segments of the microcirculation make it possible to calculate the number of vessels at each of these cross sections of the
network. The branching pattern of the minute blood vesselsis not uniform and, ideally, one should differentiate between true dichotomy, parallel branching, and arcading arterioles. This is not always possible in the vascular network of the skeletal muscle, mainly due to the three-dimensional alignment of the vessels.As shown schematically in Fig. 1, the relative distribution of blood flow in successivebranches of the arterioles is directly related to the number of branches at each of these segments.Arterioles with an average diameter of 50 pm and volume flow of 3.1 x lo-) ml/min subdivide into five smaller vessels.Assuming that the flow is distributed in a comparable manner down to the 16 pm level, someeight precapillaries are formed with an average diameter of 9 pm. SCHEMATlC DERWED
OF ARTERIOLAR FROM VOLUME
BRANCHING FLOW VALUES
ARTERIOLE g x lo-’
I - 4Gd ml/min
PRECAPILLARY 2 * 10-9 mllmin
TRUE CAPtLLARIES 6.0 x lo-6 rnlhni”
, e, 5,,,
FIG. 1. Ratio of flows in successive dichotomies of small blood vessels provides an estimate of the number of branches at successivecross sections of the microvascular bed. It assumesthat all of the parent vessel flow is distributed to these vessels.
The reduction in volume flow and the corresponding drop in microvascular pressure are greatest between the 21- and 9-pm vessel groups. Precapillary vasomotion was consistently observed, and was associated with fluctuations in flow velocity and diameter of about g-10%. Pressure-Flow Relationships Figure 2 presents the flow data relative to microvascular pressure. On the average, both flow velocity and microvascular pressuresdecreasewith the degree of branching. Blood flow velocities and arteriolar diameter tend to fluctuate more with vasomotion than does microvascular pressure. The sharp drop in pressure noted in arterioles between 21 and 9 pm is associatedwith a drop in flow velocity from 9.4 to 3.8 mm/set. Microsphere Measurement of Flow Distribution The microsphere technique was used to substantiate the viability of our microvascular striated muscle model. Data presented here include only those experiments in which it was found that the left : right flow ratio for the kidney was close to 1.0. It is our experience that a flow ratio of 1.0 for the kidney is good evidence of adequate mixing. When Y3r was administered before any dissection was started, the muscle distribution ratio consistently indicated a somewhat higher overall flow in the left limb. This result
PRESSURE AND VELOCITY DISTRIBUTION IN M.TENUISSIMUS MICROVESSELS
FIG. 2. Pressure (A) and flow velocity (a) in arterioles of different diameters. Contours of the curves t the standard errors of the mean are shown.
was independent of the position of the animal, and was seenin both muscleswhich were studied, m. tenuissimus and m. biceps femoris (Fig. 3). 14iCewas injected after the m. tenuissimus had been dissected and exposed for an hour. The slight fall in the ratio can be interpreted either as an increase in flow on the dissectedside, or a decreaseon the undissected side. The change was not statistically significant. Measurements made 2 hr later show that the flow had increased in both muscles (Fig. 3), and had returned to essentially initial values. M.TENUISSI MUS
M. BICEPS FEMORIS
Ih 2h 14&e 51Cr
FIG. 3. The radioactivity (cpm/l g) measured in samples from both hind limbs is exp& as left: rightside ratio. C, control period; lh and 2h, 1 and 2 hr after dissection and exposure of the right m tenuissimus. The isotope with which the microspheres was labeled is indicated.
DISCUSSION New information and evidence have become available with the development of intravital microscopic techniques for quantitative measurement of pressure and flow. It is possibly to carry out continuous measurements of pressure in vessels down to the capillaries to determine the diameter of selectedvesselsand to monitor the blood dew
velocities in standardized preparations. The tissue in exteriorized preparations had to be handled in a special way to make data acquisition possible. A fundamental question revolves around the extent to which physiological conditions have been affectedby such handling. Can organ hemodynamics of the undisturbed muscle be duplicated under microscopic conditions? By their very nature, microvascular studies on skeletal muscle are more open to criticism than, for instance, studies on capillaries on the skin nailfold. It is therefore significant when we compare our data with that obtained by other authors (Table 2) TABLE 2 Fmw VEUXITY RATE IN THE MKROVASCULATURE
Johnson and Wayland Johnson Gaehtgens et al.
Intaglietta et al. Burton and Johnson Gentry and Johnson Eriksson and Myrhage Richardson et al. Zweifach and Lipowsky Intaglietta et al.
1972 1972 1972 1971 1975 1975
M. sartorius M. pectoralis M. tenuissimus Omentum Mesentery M. tenuissimus Omenturn Pia Nailfold Brain Brain Cremaster
Rosenblum Butti et al. Koo and Cheng
Cat Cat Frog Cat Cat Cat Cat Rabbit Mice Human Rat
Rosenblum Prewitt and Johnson
Size of microvesselsa old Capillary Capillary Capillary <30 >30 20 Capillary Capillary Capillary 20-80 7-25 Capillary Capillary Arterioles Capillary 78.9 55.0 13-46 20
Velocity bdsec) 0.0-5.2 0.5-1.0 upto 1.7 8.65 k 1.79 5.65 f. 1.04 10.0 0.38 0.33 + 0.19 0.0-1.5 0.1-2.38 0.7-12.00 up to 10 ID-l.5 1.88-6.0 0.3-2.35 12.4 14.7 2.0-26 2.36 + 0.29
a Range from arterioles to capillaries.
that we find general agreement even though the data represent different tissues and species. Various investigators have presenteddifferent values for the number of capillaries per unit area in skeletal muscle as determined by histological techniques. In general, earlier estimates of capillary density are higher than reported in more recent studies (Phyley and Groom, 1975). A capillary density of 1000 capillaries/mm2 of skeletal muscle appearsto be a realistic estimate. Intravital microscopy indicates that all of the capillaries do not contain an active flow at any given time. Furthermore, different groups of capillaries show first an open-flow and then a no-flow state at variable intervals. When a vasodilatory agent is introduced, all of the capillaries gradually assumean active flow, and capillary fiow intermittency of this type disappears.
Under rt28tiagcondition8, therefore, the arteriolar vol~c flow is dii only population. Our direct in u&o measurementsof flow show to a fractionofthe that only about 30-35% of the capilk& are being actively pehmd. If we as8umcthat a capillary density of 1000/mmz corresponds to the vasodilated state, the calculated flow per capillary under steady-stateconditions would be 6.0 x 10e6ml/mm. The volume flow for the smallest vesselsin the present experiments for which such measurements were ma& was, on the average, 2 x 10e5 ml/min per vessel. In these studies, the 9.0 + 1.0-m precapilhuies were the smallest vesselsin which we measured flow. The diameter of the true capillaries in skeletal muscle of the cat is, on the average, 5 to 6 m. In most instances, each of the 9- to 10-q precapillaries further subdivides into three to five capillaries. Direct contact with ambient air was avoided by covering the muscle with a plastic sheet (Saran Wrap) which has been shown to be impermeable to gases.Microvascular pressureswere measured under a layer of mineral oil. In this way, it was not necessary to use a suffusion solution, and the viability of the preparation was improved from several aspects: Tendency for the muscle to swell, which may otherwise occur alter several hours, was minimized and, more importantly, the free exchange of gaseswith ambient air was avoided (Duling and Beme, 1970; Prewitt and Johnson, 1976). The standard microsphere technique used to evaluate organ flow is most accurate when there are at least 400 particles of each isotope in each tissue sample. A number of difficulties are encountered in adapting this method for small tissue samples (the total weight of the m. tenuissimus is only 90-120 mg), because administration of larger numbers of spheres leads to disturbances of cardiovascular function and tissue perksion is impeded. For these reasons, we preferred to use a smaller number of particles per m. tenuissimus, and not to formulate any quantitative conclusions regarding absolute volume flow based on this technique. The data were then compared with the results obtained from m. biceps femoris, where the critical number of particles was easily exceeded. As previously indicated, the objective of these experiments was to assessthe influence of the dissection and exposure of the tenuissimus muscle necessary for intravital microscopy. A comparison was therefore made of the relative blood flow distribution in the dissected and undissected sides by determining the ratio of left-to-right radioactivity. Such L : R ratios were somewhat greater than 1.O,indicating a higher perfusion rate in the left limb muscle. The effect of the specific gravity of the radiolabeled microspheres was excluded by initial experiments, where’the position of the cat was alternated for each injection. Rutili and Arfors (1976) carried out a similar study on rabbit tenuissimus muscle using Sephadex microspheres with a low tendency to sediment. When a left-to-right ratio is calculated from their data there is a similar, statistically insignificant, trend for a higher blood supply to the left hind limb muscle. The phenomenon is more noticeable in the ratio derived from flow values for the tenuissimus muscle than from the values for the biceps femoris. One can only speculate concerning the basis for such a difference. When the initial perfusion ratio of the tenuissimus muscle is compared with that observed 1 to 2 hr alter dissection, the blood flow conditions in both muscles were similar. Inasmuch as one tenuissimus muscle was dissected and exposedwhile the other was left undisturbed, we concluded that the type of dissection and exposure used does not significantly interfere with the normal operational status of the muscle.
FRONEK AND ZWEIFACH
It is obvious that blood flow velocities and flow rates of the type obtained for different-sized microvesselsin the exteriorized muscle preparations can serve as a frame of reference for studies designed to analyze the hemodynamics and fluid transfer in the terminal vascuiature.
ACKNOWLEDGMENTS The authors wish to acknowledge the skillful assistance of Steve Kovalcheck, John Firrell, William Brown, and Sy Kmtz throughout the experiment, and of Mrs. Peme Whaley in the preparation of the manuscript.
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