INTRODUCTION

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THE VIABILITY OF SKELETAL MUSCLE depends on the presence of a functional microvascular bed that provides adequate supply of oxygen and nutrients to, and removal of waste products from, the tissue. Important insights into muscle microcirculatory function in health and disease have been achieved with the use of intravital microscopy techniques that necessitate surgical exteriorization of the muscle. In this regard, the rat spinotrapezius preparation first described by Gray in 1973 (9) represents one principal model in which to evaluate physiological and pathophysiological phenomena within the microcirculation. For example, the rat spinotrapezius has been pivotal in our understanding of the effects of muscle structure-function relationships and smooth muscle physiology (16, 19, 33) and the role of nitric oxide and calcium in the microcirculation in health (24, 41). In addition, the spinotrapezius muscle preparation has provided insights into the pathophysiological microcirculatory consequences of chronic diseases such as type-1 diabetes (14, 35), hypertension (30), and chronic heart failure (6, 15). The tacit assumption in all of those studies has been that surgical intervention does not impact microcirculatory function either under control conditions or in response to experimental or pathological stimuli. However, it has been demonstrated that arterial and arteriolar pressures are reduced in the cremaster muscle after an exteriorization procedure that interrupts the distal blood supply emanating from the deferential artery (5).

To evaluate the effect of muscle exteriorization on the functional integrity of the microcirculation, the experimental methodology employed must be suitable for evaluation of not only blood flow (O₂ delivery) but also the balance between O₂ delivery and utilization in both the intact and exteriorized preparations. Moreover, in addition to steady-state conditions, the dynamic response of the microcirculation should be evaluated. Consequently, a novel combination of phosphorescence quenching and microsphere techniques was utilized across a range of different experimental conditions, i.e., metabolic stimulation with 2,4-dinitrophenol (DNP), electrically stimulated muscle contractions, and inspired hypoxia and hyperoxia, designed to evaluate the effect of surgical intervention on integrated static and dynamic microcirculatory behaviors within the rat spinotrapezius muscle.

	METHODS

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Animals. We used 4- to 5-mo-old female Sprague-Dawley rats (n = 33, 215-265 g) in these investigations. At this body mass, the spinotrapezius muscle has minimal overlying fascia and thus maintains optimal optical clarity for intravital microscopy.

Spinotrapezius model. The rat spinotrapezius muscle lies in the mid-dorsal region, where it originates in the lower thoracic and upper lumbar region and inserts onto the spine of the scapula. The muscle was prepared surgically with the rat under pentobarbital sodium anesthesia (40 mg/kg ip) with the use of previously described methods (9, 33). Exteriorization was always performed on the left (L) muscle. Although removal of the fascia does improve visibility of the fibers and microcirculation, it causes separation of the fibers and dilatation of microvessels (22, 27). Therefore, exteriorization was performed with minimal fascial disturbance designed to minimize tissue damage (i.e., with the exception of the distal feed artery, all of the vascular and nervous connections remained intact) and alterations of myocyte-to-vessel orientation (22) as well as any associated microcirculatory consequences (9, 27). Briefly, the rat was positioned on a circulation-heated (38°C) Lucite platform, and the spinotrapezius muscle was superfused continuously with the use of a microcirculator (model U3-7A, Julabo, Schwarzwald, Germany) with a Krebs-Henseleit bicarbonate-buffered solution equilibrated with 95% N₂-5% CO₂ (42). The exposed dorsal surface of the spinotrapezius muscle was protected with Saran Wrap (Dow Chemical, Indianapolis, IN), whereas the muscle was sutured with 6.0 silk at five equidistant positions around the caudal periphery to a thin wire horseshoe manifold (40). Thereafter, all exposed surfaces were either covered with Saran Wrap or bathed in the Krebs-Henseleit solution to ~1 mm in depth. The manifold was attached with a swivel to a muscle-stretching apparatus that permitted precise, systematic, and uniform length changes of the entire spinotrapezius muscle along its principal fiber longitudinal axis. This facilitates setting muscle sarcomere length at 2.7 µm, the approximate resting length, thus controlling for length-induced blood flow alterations (33).

Experimental design and conditions. The rats were assigned at random to one of four groups to determine the effects of surgical exteriorization on the spinotrapezius. Group 1 consisted of blood flow measurements under control conditions. In these studies, blood flow was measured in the anesthetized animal, and the L spinotrapezius muscle was then exteriorized and the R muscle exposed before a second blood flow measurement (n = 4) was made. In group 2, the blood flow response to increased metabolic activity induced by metabolic blockade was measured. Blood flow was measured after exposure in the intact R muscle and exteriorization in the L muscle, and both muscles were then superfused with 30 mM DNP before the second blood flow measurement (n = 6). Group 3 determined the response of microvascular PO₂ to electrical stimulation-induced muscle contractions (see Electrical stimulation). Intact and exteriorized muscles were stimulated in random order, and microvascular PO₂ was measured at rest and after 2-3 min stimulation when the microvascular PO₂ had stabilized (n = 6). In group 4, the dynamic response of microvascular PO₂ to hyperoxic (100%) and hypoxic (10%) switches of inspired O₂ was measured. Measurements of microvascular PO₂ were made in intact and exteriorized muscles in random order across the transitions to altered inspired O₂ (n = 7). During each of the experimental conditions, MAP was monitored via the carotid artery (model 200, DigiMed BPA, Louisville, KY).

Blood flow. Tissue blood flows were determined with the use of the radionucleotide-tagged microsphere technique that has been adapted for use in the exercising rat as described originally by Armstrong and Laughlin (1) and modified for use in our laboratories (29). Briefly, polyethylene catheters (PE-10 connected to PE-50) were placed into the right carotid artery and caudal artery. The right carotid artery catheter was advanced toward the heart and secured in a position just inside the aortic arch. This was accomplished by advancing the catheter toward the left ventricle while the arterial pressure waveform was being monitored. When the catheter reached the aortic valve, the pressure waveform became distorted. The catheter was retracted ~2-3 mm and secured in place. The caudal artery catheter was advanced toward the bifurcation of the descending aorta and secured in place. The right carotid catheter was connected to a pressure transducer, and the caudal artery catheter was connected to a 1-ml syringe placed in a withdrawal pump (model 907, Harvard Apparatus, South Natick, MA). Blood withdrawal from the tail artery catheter (microspheres reference sample) was initiated at a rate of 0.25 ml/min. At the same time, arterial blood pressure and heart rate (HR) were recorded from the carotid artery catheter. After 30 s of blood withdrawal, the carotid artery catheter was disconnected from the pressure transducer, and ~250,000 radioactive microspheres were slowly infused into the aortic arch. The microspheres (⁴⁶Sc, ⁸⁵Sr, and ¹¹³Sn) used in the present study were 15 ± 3 µm in diameter as specified by the manufacturer (NEN Research Products, DuPont, Boston, MA). These isotopes were infused in random order under the different experimental conditions in which blood flows were measured (i.e., presurgery, postexteriorization, and post-DNP superfusion). The microspheres were suspended in normal saline containing 0.01% Tween 80 with a specific activity ranging from 7 to 15 mCi/g. Before each infusion, the microspheres were thoroughly mixed and agitated by sonication to prevent clumping. The microspheres were injected into the ascending aorta in a volume of ~0.10 ml over 5-10 s. Blood withdrawal from the caudal artery catheter was maintained for 30 s after the microsphere injection to ensure that all microspheres had been cleared from the withdrawal catheter. The radioactivity of the tissues was determined on a Cobra II Auto-Gamma Spectrometer (Packard Instruments, Downers Grove, IL) set to record the peak energy activity of each isotope for 2 min. The radioactivity of the tissues was then analyzed by computer, taking into account the cross-talk fraction between the different isotopes. Blood flow (expressed as ml · min¹ · 100 g¹ of tissue) to the whole right and left spinotrapezius muscles, solei, and kidneys was calculated by the reference sample method as described by Ishise et al. (13). Adequate mixing of the microspheres was verified for each injection by demonstrating <15% difference in blood flows to the right and left kidneys and/or solei.

Phosphorescence quenching theory. The oxygen dependence of phosphorescence is described by the Stern-Volmer equation (37)
where t_o and t are the phosphorescence lifetimes in the absence of oxygen and at an oxygen pressure microvascular PO₂. The quenching constant (k_Q) is a second-order rate constant related to the frequency of collisions between O₂ and the excited triplet state of the porphyrin and the probability of energy transfer when collisions occur. PO₂ is calculated as
where t_o and t are expressed in microseconds and k_Q in mmHg¹ · s¹. At 38°C and pH 7.4, k_Q = 409 mmHg¹ · s¹ and t_o = 601 µs (26).

Measurement. Initially, 15 mg/kg of the phosphorescent probe palladium-mesotetra(4-carboxyphenyl) porphyrin dendrimer (R2) was infused via arterial cannula. Oxyphor R2 binds tightly to albumin. Specifically, Lo et al. (25) have demonstrated that R2 is essentially completely bound to albumin in solution at a concentration of 0.5% albumin. The concentration of albumin in rat serum is >3 g/dl (i.e., >6-fold that is necessary for complete binding; see Ref. 36). In addition, R2 at a pH of 7.4 possesses a net-negative charge of approximately 14 mV. Both of these properties help to restrict R2 to the intravascular compartment. Microvascular PO₂ was determined by using a PMOD 1000 Frequency Domain Phosphorimeter (Oxygen Enterprises, Philadelphia, PA) with the common end of the bifurcated light guide placed ~2-4 mm above the medial region of the spinotrapezius (i.e., superficial to dorsal surface). This location on the spinotrapezius muscle is the same as that used during intravital microscopy and is ~8-10 mm from the distal (detached) proximity. The excitation light (524 nm) is focused on an ~2-mm diameter circle of exposed or exteriorized muscle surface and samples blood within the microvasculature up to 500 µm deep. The value of microvascular PO₂ principally reflects capillary blood, because this compartment constitutes the majority of intramuscular blood volume (34). The phosphorescence signal (700 nm) was averaged for a 200-ms interval for each microvascular PO₂ measurement, and the measurements were repeated at 2-s intervals.

Electrical stimulation. Stainless steel plate electrodes (2.5-mm diameter) were attached to the muscle proximal to the motor point (cathode) and across the caudal end (anode) close to the spinal attachment to elicit indirect bipolar muscle contractions (31). The muscle was stimulated to contract at 1 Hz for 3 min (5-8 V, 250-µs pulse duration) with the use of a stimulator (model S88, Grass Instruments, Quincy, MA). This stimulation protocol has been demonstrated to increase blood flow two- to threefold within the spinotrapezius muscle (Behnke, Kindig, Musch, and Poole; unpublished results).

Statistical analyses. Data are presented as means ± SE. The differences pre- and postsurgery and between intact and exteriorized muscles under each experimental condition were evaluated with the use of a two-way ANOVA with repeated measures design and paired t-tests as appropriate. Statistical significance was accepted at the P  0.05 level.

	RESULTS

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Groups 1 and 2: Surgical exteriorization and DNP effects on blood flow. Surgical preparation did not alter whole muscle blood flow significantly within either the exteriorized or intact muscles (Fig. 1). Metabolic stimulation with DNP superfusion elevated muscle blood flow over twofold in the intact (19 ± 7 to 42 ± 14 ml · min¹ · 100 g¹, P < 0.05) and exteriorized (16 ± 4 to 35 ± 9 ml · min¹ · 100 g¹, P < 0.05) muscles. The magnitude of this increase was not different between intact and exteriorized muscles (Fig. 2).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Right (R) and left (L) spinotrapezius muscle blood flow pre- and postsurgical preparation. R muscle was exposed with minimal surgery. L muscle was exteriorized after disrupting distal blood supply. Values are means ± SE, n = 4. ns, No significant difference in R vs. L blood flow or change in blood flow pre- vs. postsurgery for either R or L muscles.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   R intact (I) exposed and L (exteriorized, EX) spinotrapezius muscle blood flow under baseline conditions (left) and during superfusion with 30 mM 2,4-dinitrophenol (DNP) (right). All values are means ± SE, n = 6. *P < 0.05, significant increase from baseline to DNP for R and L muscles.

Group 3: Effects of electrically stimulated muscle contractions on microvascular PO₂. Muscle contractions reduced microvascular PO₂ from 30.4 ± 4.3 to 21.8 ± 4.8 mmHg in intact and from 33.2 ± 3.0 to 25.9 ± 2.8 mmHg in exteriorized muscles (Fig. 3). Both conditions showed no difference in microvascular PO₂ between intact and exteriorized muscles.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Spinotrapezius microvascular PO2 (PO2m) at rest (baseline, left) and in the steady state at the end of 2-3 min of 1-Hz contractions (right). All values are means ± SE, n = 6. *P < 0.05, significant decrease in PO2m from baseline to contracting for intact, exposed R, and exteriorized L muscles.

Group 4: Effects of hypoxic and hyperoxic switches on microvascular PO₂. As expected, arterial PO₂ was significantly reduced from 80 ± 3 mmHg breathing room air to 48 ± 5 mmHg on the hypoxic (10% O₂) inspirate. Inspired hyperoxia (100% O₂) elevated arterial PO₂ to 307 ± 57 mmHg. Compared with MAP in normoxia (108 ± 4 mmHg), hypoxia significantly lowered MAP to 96 ± 4 mmHg (P < 0.05), whereas it was unchanged in hyperoxia (110 ± 5 mmHg). Figure 4 demonstrates the magnitude and time course of the microvascular PO₂ changes in response to switches among these inspired O₂ percentages in one exteriorized muscle. As seen in Table 1, neither the magnitude nor the half-time dynamics of the microvascular PO₂ response after each switch were altered significantly in exteriorized compared with intact muscles.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Dynamic response of spinotrapezius (PO2m) across switches in inspired gas from left to right: 21% O2 (room air), 100, 21, and 10% O2 for a representative exteriorized muscle.

	DISCUSSION

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The principal methods for assessing the viability of an exteriorized muscle for microcirculatory investigation include the appearance of vigorous arteriolar flow and arteriolar vasodilation or vasoconstriction during chemical (e.g., adenosine, epinephrine) or oxygen (hypoxic, hyperoxic) challenge. Such assessment may be subjective and is almost never quantitative. Moreover, the inability to observe anything but superficial microcirculatory flow (reflectance microscopy) in the nonexteriorized preparation complicates direct observation of arteriolar vasoaction in intact muscle and thus comparison between intact and exteriorized preparations is not feasible. Thus the present investigation utilized methods and conditions suitable for assessing integrated microcirculatory function in both intact and exteriorized preparations. In no instance was there any indication of a systematic alteration in the basal blood flow (whole muscle) or microvascular PO₂ or the magnitude and/or kinetic response of these variables to metabolic stimulation and change of muscle influent PO₂ postsurgery. The present investigation therefore provides no evidence that the surgical manipulations requisite for exteriorizing the spinotrapezius before intravital transmission microscopy systematically impair microvascular integrity or responses across the range of conditions evaluated herein.

Surgical exteriorization. Skeletal muscle oxygen consumption (O₂) increases up to two orders of magnitude from rest to maximal exercise (7, 20, 21, 32). This extraordinary increase necessitates a substantial elevation of muscle O₂-diffusing capacity in concert with elevated convective O₂ delivery. Theoretical models of O₂ transport and diffusion within muscle consider that one major determinant of muscle O₂-diffusing capacity is the number of red blood cells (RBC) found along the surface of a myocyte at a given time (8, 10). Thus at first it seems reasonable that at exercise onset more capillaries are recruited and the hematocrit within each capillary is elevated (17), thereby increasing RBC number along the fiber surface. However, it is now evident that, at least in some muscles, the majority of capillaries may be "recruited" at rest (4, 12, 15, 33, 42), which limits the opportunity for additional capillary recruitment during exercise. Thus the increased muscle O₂-diffusing capacity from rest to exercise cannot rely greatly on the recruitment of additional capillaries or capillary units per se. Rather, it is likely determined by factors related to augmentation of RBC flow within previously RBC-perfused capillaries (2) or intracellular events (11). The present investigation demonstrates that surgical exteriorization does not elevate muscle blood flow nor alter microvascular PO₂ at rest. These findings support the notion that the high percentage of capillaries supporting RBC flow reported in the spinotrapezius preparation (12, 14, 16, 33) is not an artifact secondary to elevated blood flow resulting from muscle manipulation or damage.

Metabolic stimulation by DNP. Muscle metabolic stimulation by DNP infusion elevates muscle O₂ approximately threefold (3), and DNP superfusion of the exteriorized hamster cremaster muscle increases arteriolar diameter and RBC velocity (39). There is evidence that this occurs via venular-arteriolar diffusion of vasoactive metabolites (38) rather than by a direct DNP-induced relaxation of arteriolar smooth muscle (3). In the present investigation, blood flow increased to 2.2-fold resting levels in both intact and exteriorized muscles, demonstrating that the net effect of DNP on the control of muscle blood flow was unaltered by surgical exteriorization.

Electrically stimulated muscle contractions. Oxygen delivery (O₂) to contracting myocytes is elevated by augmenting muscle blood flow and fractional O₂ extraction. The precise regulators of the increased muscle blood flow are likely contingent on multiple factors such as the intensity, frequency, and duration of the contractions as well as the type of muscle (architecture, fiber type composition, oxidative capacity) and presiding experimental conditions. Given this, arterial driving pressure, muscle pump activity, endothelial cell shear stress, buildup of vasoactive metabolites, and reduced intramuscular PO₂ are each thought to play a role in augmenting muscle blood flow during exercise. In addition, fractional O₂ extraction increases as a hyperbolic function of muscle O₂ (32). During exercise, blood flow (and O₂) and O₂ extraction are regulated tightly such that O₂ delivery increases in proportion to muscle O₂. The measurements of microvascular PO₂ made in the present investigation assess the O₂-to-O₂ relation at rest and during exercise. Thus, whereas neither O₂ nor O₂ themselves were measured, the finding of an unchanged microvascular PO₂ between intact and exteriorized muscles at rest and the magnitude of the fall during contractions is indicative that, irrespective of the exact control mechanisms, the matching of O₂ to O₂ was not altered by surgical exteriorization.

Hypoxic and hyperoxic switches. Altered local PO₂ exerts a profound vasodilatory (hypoxia) or vasoconstrictory (hyperoxia) response (4, 23, 28). Because acute changes in inspired O₂ do not appear to alter pulmonary or tissue O₂ appreciably at rest or during submaximal exercise (17), any subsequent alteration of microvascular PO₂ will depend on the effect of the inspired O₂ on arterial PO₂ (and O₂ content), MAP, and muscle vascular conductance, i.e., arteriolar smooth muscle tone (either modulated locally or neurally in response to central effects of hypo- or hyperoxia). Neither arterial PO₂ nor MAP was significantly different between intact and exteriorized preparations (same rats, order randomized). Hence the similarity of microvascular PO₂, with respect to the magnitude of the absolute change and dynamic profile of microvascular PO₂ across each switch, demonstrate that O₂-mediated arteriolar vasoaction was unaltered by surgical exteriorization.