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(Circulation Research. 1996;79:551-559.)
© 1996 American Heart Association, Inc.

Articles

Muscle Length Directs Sympathetic Nerve Activity and Vasomotor Tone in Resistance Vessels of Hamster Retractor

Donald G. Welsh, Steven S. Segal

the John B. Pierce Laboratory and the Departments of Cellular and Molecular Physiology & Epidemiology and Public Health, Yale University School of Medicine, New Haven, Conn.

Correspondence to Steven S. Segal, PhD, The John B. Pierce Laboratory, Yale University School of Medicine, 290 Congress Ave, New Haven, CT 06519.

	Abstract

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Increased resistance to blood flow with muscle extension has been explained by the deformation of vessels within the muscle. In the present study, we developed a novel preparation of the hamster retractor muscle to investigate whether passive changes in skeletal muscle length elicit active vasomotor responses through a range of motion (85% to 130% of in vivo length; sarcomere length, 2.69±0.02 to 4.05±0.01 µm) encompassing the classic length-tension relationship. Arterioles (diameter, 32±3 µm) and feed arteries (diameter, 75±4 µm) were observed to progressively constrict (by 8±1 and 17±2 µm, respectively) with muscle lengthening, reducing blood flow by >50%; reciprocal changes occurred with passive shortening. Sodium nitroprusside (10 µmol/L) dilated vessels (to 47±2 and 98±4 µm, respectively) and abolished vasomotor responses to changing muscle length. The coordination of vasomotor responses between arterioles and feed arteries maintained wall shear rate (control, 1764±200 s^-1) and perfusion pressure (60±5 mm Hg) into the arteriolar network. Tetrodotoxin (TTX, 1 µmol/L), phentolamine (1 µmol/L), prazosin (0.1 µmol/L), or 6-hydroxydopamine (1 mmol/L) inhibited vasoconstrictor responses, indicating that action potentials initiated by muscle lengthening give rise to norepinephrine release from sympathetic nerves. As shown with glyoxylic acid staining, sympathetic nerves formed a plexus encompassing arterioles and feed arteries. To test for a reflexive response initiated by intramuscular mechanoreceptors, TTX was applied with micropipettes to proximal segments of feed arteries, thereby neurally "isolating" the muscle from the hamster. Whereas lengthening-induced vasoconstriction persisted in arterioles and in feed artery segments distal to TTX, there was no vasomotor response central to the block. We conclude that passive lengthening stimulates the activity of periarteriolar sympathetic nerves; this activity propagates antidromically along nerve fibers into the feed arteries. These findings identify a mechanotransduction sequence by which the length of skeletal muscle actively governs vasomotor tone and the supply of oxygen to muscle fibers.

Key Words: microcirculation • perivascular sympathetic nerves • mechanotransduction • arterioles • feed arteries

	Introduction

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During exercise, skeletal muscle undergoes rhythmic changes in length and tension, giving rise to mechanical perturbations within the tissue that intermittently reduce muscle blood flow.^{1 2 3 4} Two explanations have evolved to explain the reduction in flow, including (1) passive reductions in vessel diameter due to compression and axial stretch^{3 5 6} and (2) kinking and pinching of vessels due to shear forces generated between muscle fibers and fiber bundles.^{7 8} Such reasoning has implied that the resistance vasculature remains passive when subjected to the mechanical forces produced by muscle fibers. The possibility that microvessels actually respond to changes in muscle length, and thereby actively control the corresponding changes in flow, has not been investigated. Indeed, it is apparent that active vasomotor responses could be initiated via local changes in transmural pressure^{9 10} or luminal perfusion.^{11 12}

In the present study, we tested the hypothesis that passive changes in muscle length elicit active vasomotor responses in the microcirculation. Experiments were performed using a novel preparation of the hamster retractor muscle^{5 13} that enables precise control of muscle length while concomitantly evaluating microvascular hemodynamics. Our findings reveal a unique mechanotransduction sequence in which the length of skeletal muscle fibers actively regulates the caliber of intramuscular arterioles and extraparenchymal resistance arteries. These active responses, which are independent of changes in pressure or luminal perfusion, are governed locally (ie, independent of the central nervous system) through the release of norepinephrine from perivascular nerves. Furthermore, we show that active vasoconstriction accounts for the majority of the effect of muscle lengthening on microvascular perfusion.

	Materials and Methods

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Animal Care and Preliminary Surgery
All procedures were approved by the Institutional Animal Care and Use Committee of the John B. Pierce Laboratory. Male golden hamsters (n=50, 80 to 115 g, Charles River, Kingston, NY) were maintained at 24°C on a 14 h/10 h (light/dark) cycle and were provided rodent chow and water ad libitum. Hamsters were anesthetized with pentobarbital sodium (60 mg/kg IP) and tracheotomized to ensure airway patency. The right carotid artery was cannulated (PE 50) for monitoring arterial pressure (Cobe CDX III transducer coupled to a WPI Transbridge amplifier). A second cannula (PE 50) was secured in the left femoral vein to replace fluids and maintain anesthesia during experiments (10 mg pentobarbital/mL isotonic saline, infused at 410 µL/h). Esophageal temperature was maintained at 37°C to 38°C using heat conducted from a copper plate positioned underneath the hamster.

Retractor Preparation
An incision (6 to 8 cm) was made over the right retractor muscle, which retains the cheek pouch against the body wall.¹³ The exposed tissue was superfused continuously with a bicarbonate-buffered PSS (mmol/L: NaCl 131.9, KCl 4.7, CaCl₂ 2.0, MgSO₄ 1.2, and NaHCO₃ 18; Sigma) equilibrated with 95% N₂/5% CO₂ (pH 7.4, 34°C to 35°C). Connective tissue covering the dorsal surface of the muscle was removed using a stereo microscope, and artificial tendons were secured to each end.⁵ The length of muscle between the tendons was measured with Starrett digital calipers (resolution±0.01 mm). This value was taken as 100% of in vivo muscle length (and of fiber length); sarcomere length was measured as described.⁵ The muscle was then severed from its origin and insertion and reflected to expose the ventral surface. Motor nerve bundles were excised to eliminate associated neural pathways and to resolve feed arteries, and superficial connective tissue was removed.

Upon completion of surgery (duration, 2 to 3 hours), the hamster was positioned on a custom-built transparent acrylic platform (Fig 1) for viewing the retractor muscle and its vascular supply. Each end of the muscle was connected to a micrometer drive using silk suture (4-0); this enabled precise control of muscle length without displacing feed arteries. Fresh PSS superfused the muscle continuously (4 to 5 mL/min) via a superfusion line; effluent was removed using aspiration. Upon completion of experimental procedures, an overdose of pentobarbital was delivered through the venous cannula. The muscle was then excised between tendons and weighed; cross-sectional area was calculated¹⁴ for normalizing isometric tension.

View larger version (36K): [in this window] [in a new window]   Figure 1. A diagram of the hamster retractor preparation. The anesthetized hamster was placed on its side on a transparent Plexiglas viewing stage. The retractor muscle was exteriorized, reflected ventrally, and attached to micrometers with a force transducer mounted in series. PSS was superfused over the tissue at 4 to 5 mL/min and aspirated via wicks placed at the periphery. Below, An exploded diagram of the microvascular network in the hamster retractor muscle (adapted from Reference 5). Typically, two feed arteries (FA) with paired collecting veins (CV) give rise to the microcirculation. Arterioles (1A, 2A, 3A, and 4A) and venules (1V, 2V, 3V, and 4V) are oriented parallel, perpendicular, and at intermediate angles to the muscle axis.

Video Microscopy
The preparation was transferred to the stage of an intravital microscope (Zeiss ACM) and equilibrated for 60 minutes. Feed arteries and arterioles (second- and third-order branches) were observed using bright-field microscopy with Kohler illumination (Zeiss ACH/APL condenser; numerical aperture, 0.32) and a Leitz L25 objective (numerical aperture, 0.35). All of the vessels studied demonstrated brisk and reversible dilation in response to sodium nitroprusside (10 µmol/L) delivered through the superfusion line. The optical image was coupled to a CCD video camera (Hamamatsu C2400) and monitor (Sony PVM 1343 MD); total magnification on the monitor face was x1140.

Hemodynamic Measurements and Calculations
All data were acquired at 100 Hz using a MacLab system (AD Instruments) coupled to a Macintosh IIVX computer; muscle lengths were studied in random order across preparations. Internal vessel diameter was measured with a video caliper (resolution, 1 µm). Vrbc was monitored continuously with an optical Doppler velocimeter^{15 16} ; effective slit width was 9 to 10 µm. Mean red blood cell velocity (Vm) was calculated as Vrbc/1.6. WSR was calculated as 8Vm/D, where D is internal vessel diameter; blood flow was calculated as [(D²/4)Vm].¹⁶

Microvascular perfusion pressure was recorded using servo-null micropressure¹⁷ (model 5A, Instruments for Physiology and Medicine). Borosilicate glass capillaries (Corning No. 7740; outer diameter, 1.2 mm) were pulled (Sutter P-87) and sharpened (Sutter BV-10) at 15° to produce micropipettes with internal tip diameters of 1 µm. Micropipettes were backfilled with degassed NaCl (2 mol/L) and mounted in a Leitz (model M) micromanipulator. Tangential wall tension (T) was calculated as the product of transmural pressure (P) and vessel radius (r) according to the law of Laplace (T=Pr).

A load cell (0 to 100 g; resolution, <100 mg; A.L. Design) mounted on a micrometer drive recorded tension developed by the retractor (Fig 1); platinum electrodes placed at either end of the muscle were used for field stimulation. In seven preparations, passive and maximal tetanic (0.1 millisecond, 50 V at 100 Hz) tension were measured to determine the functional range of the retractor muscle. Muscle lengths ranging from 85% and 130% of in vivo length encompassed the plateau and the ascending and descending limbs of the active length-tension curve (see "Results"); this corresponds to the operational range of skeletal muscle.^{18 19} All subsequent experiments were performed using resting muscles in order to investigate the effect of changing muscle length (and passive tension) on the resistance vasculature independent of vasoactive substances produced by muscle fiber contraction.⁸

Experiment 1: Do Resistance Vessels Respond Actively to Changing Muscle Length?
We tested whether vasoconstriction or vasodilation accompanies changes in muscle length. Diameter and Vrbc were monitored concomitantly in arterioles and feed arteries at 85%, 100%, 115%, and 130% of in vivo muscle length under control conditions and during nitroprusside superfusion to eliminate vasomotor reactivity.

Experiment 2: Do Blood Flow Reductions Initiate Vasoconstriction in Feed Arteries?
We tested whether changes in WSR (an index of luminal shear stress) accompany lengthening or shortening of the retractor muscle. To ascertain whether feed arteries respond to changes in luminal shear stress, blood flow was reduced by gently lowering glass microoccluders (diameter, 25 to 30 µm) onto feed arteries 2 mm upstream or downstream from the measurement site while concomitantly measuring diameter and Vrbc. Proximal and distal occlusions were performed to account for possible changes in transmural pressure (ie, myogenic responses) interacting with the effects of luminal shear stress.²⁰

Experiment 3: Do Increases in Intravascular Pressure Initiate Length-Induced Vasoconstriction in Feed Arteries?
It is possible that a myogenic response in the proximal feed arteries could be triggered by changes in microvascular resistance within the muscle.²¹ Therefore, feed artery pressure and diameter were monitored at 85%, 100%, 115%, and 130% of in vivo muscle length. Micropressure was measured just proximal to the insertion of the feed artery into the muscle; with systemic pressure known, these values reflect vascular resistance external to the muscle and provide a direct index of the pressure that is driving flow into the arteriolar network within the muscle.²²

Experiment 4: Does Muscle Length Direct Neural Activation to Induce Vasoconstriction?
The presence of perivascular sympathetic innervation was evaluated by staining preparations with glyoxylic acid.^{23 24} Diameter and Vrbc were monitored in arterioles and feed arteries at 85% and 130% of in vivo muscle length under control conditions and during superfusion with TTX (1 µmol/L), phentolamine (1 µmol/L), or prazosin (0.1 µmol/L) to inhibit action potentials, ₁- and ₂-adrenoceptors, or ₁-adrenoceptors, respectively. To produce chemical sympathectomy, 6-OHD (1 mmol/L) was applied for 10 minutes followed by a 2-hour recovery.²⁵ The efficacy of pharmacological blockade and 6-OHD treatment was evaluated by PNS (1 millisecond, 100 V at 32 Hz) with a glass microelectrode positioned on a feed artery or arteriole, 2 to 3 mm from the measurement site.^{25 26}

Experiment 5: Does the Increase in Sympathetic Nerve Activity Arise From an Afferent-Efferent Reflex?
With motor nerves excised, the neural connections remaining between the hamster and the retractor muscle are the perivascular nerves that run along the feed arteries (see "Results"). Thus, a protocol was developed to neurally "isolate" the retractor muscle from the hamster. Arterioles and feed arteries were observed at 85% and 130% of in vivo muscle length under control conditions and with TTX (10 µmol/L) perfused (60 µL/min) from micropipettes (internal diameter, 30 µm) onto feed artery segments located 3 mm proximal to the muscle. Feed arteries were observed at the origin of TTX treatment and at a downstream site located between the muscle and the treated segment; arteriolar diameter was monitored as described above. The efficacy of this local TTX blockade was tested by applying PNS to the distal end of the feed artery.

Statistics
Summary data are reported as mean±SE. Diameter, blood flow, WSR, and micropressure were analyzed with respect to muscle length using linear regression and repeated measures ANOVA (experiments 1 through 3). When significant F ratios were obtained, post hoc analyses were performed using comparison of least-square means. Paired t tests were used to compare vessel diameters (control versus nitroprusside superfusion) at a given muscle length and to evaluate the effect of TTX, phentolamine, prazosin, and 6-OHD (experiments 4 and 5). Comparisons were considered statistically significant at P.05. When multiple t tests were performed, a family-wide significance level was maintained by dividing this P value by the number of comparisons.

	Results

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Mean arterial pressure at the beginning and end of experiments (duration, 3 to 4 hours) was 89±4 and 87±1 mm Hg; corresponding values for heart rate were 381±4 and 389±9 bpm.

Vasomotor Responses of Resistance Vessels to Changing Muscle Length (Experiment 1)
As the length of the retractor muscle increased, the diameter of arterioles and of feed arteries decreased (Fig 2; r=.66 and .81, respectively). Vasomotor responses to each change in length were complete within 10 seconds and sustained (eg, >10 minutes) at each position. Lengthening-induced reductions in arteriolar diameter were independent of branch order and orientation to muscle fibers⁵ ; therefore, results were pooled. Superfusing the retractor muscle with nitroprusside dilated arterioles and feed arteries (to 47±2 and 98±4 µm, respectively) and abolished length-induced vasomotor responses. As seen previously,⁵ muscles typically "buckled" at lengths <80%, and blood flow stopped at >130%, of in vivo muscle length.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of muscle length (percentage of in vivo resting length) on tension development and microvessel diameter. A, Active tension (maximal tetanic) and passive tension (n=7) are shown. B and C, Diameters of arterioles (B, n=40) and feed arteries (C, n=13) decreased as muscle length increased (r=.66 and .81, respectively); nitroprusside eliminated this effect. Data are expressed as the change in diameter (µm) relative to 85% in vivo muscle length. Diameters at 85% were as follows: arterioles, 32±3 µm (control) and 47±2 µm (nitroprusside); feed arteries, 75±4 µm (control) and 98±4 µm (nitroprusside). Sarcomere lengths (n=10) were as follows: at 85%, 2.69±0.02 µm; at 100%, 3.10±0.01 µm; at 115%, 3.54±0.01 µm; and at 130%, 4.05±0.01 µm. *P.05 vs values at 85%. #P.05 for control vs nitroprusside (10 µmol/L).

Blood Flow, WSR, and Luminal Shear Stress at Different Muscle Lengths (Experiment 2)
As shown in Fig 3, stepwise increases in muscle length progressively reduced feed artery blood flow by 50%. The corresponding reductions in diameter and Vrbc maintained a constant WSR in feed arteries, suggesting that vasomotor responses were associated with the regulation of luminal shear stress.¹¹ However, when flow was reduced by 25% and 50% (the amount observed in Fig 3) for up to 5 minutes, feed artery diameter remained unchanged despite the proportionate fall in WSR (Table) and luminal shear stress. These same vessels constricted with muscle lengthening and dilated with nitroprusside (as above).

View larger version (12K): [in this window] [in a new window]   Figure 3. Effect of muscle length (percentage of in vivo resting length) on feed artery hemodynamics. Whereas blood flow decreased as muscle length increased (A, n=13), WSR (B, n=13), and microvascular perfusion pressure (C, n=7) were unchanged. *P.05 vs values at 85%. In panel B, Vrbc was as follows: 85%, 26.7±3.6 mm/s; 100%, 27.2±3.8 mm/s; 115%, 26.3±3.3 mm/s; and 130%, 23.2±2.8 mm/s. For experiments in panel C, carotid arterial pressure was 91±5 mm Hg at 85% and did not change with muscle length.

View this table: [in this window] [in a new window]   Table 1. Effect of Blood Flow and WSR on Feed Artery Diameter

Microvascular Perfusion Pressure at Different Muscle Lengths (Experiment 3)
Confirming results from experiments 1 and 2, muscle lengthening reduced feed artery diameter from 76±6 µm (85%) to 62±5 µm (130% in vivo length). Despite the significant reduction in vessel diameter, perfusion pressure at the entrance to the arteriolar network was unchanged throughout the range of motion (Fig 3C). Tangential wall tension in feed arteries decreased (P<.05) from 300±33 dyne/cm (85%) to 255±34 dyne/cm (130%) during muscle lengthening. Pressure in the veins exiting from the muscle averaged 10 mm Hg across muscle lengths (n=3).

Sympathetic Nerve Activation and Deactivation With Muscle Lengthening and Shortening (Experiment 4)
Lengthening the retractor reduced diameter and blood flow throughout the resistance network under control conditions (Fig 4 and above); TTX or phentolamine superfusion inhibited these responses to muscle lengthening. In control experiments performed at rest (at 85% of in vivo length), PNS reduced arteriolar and feed artery diameter by 27±2 µm (n=8) and 21±2 µm (n=5), respectively; superfusion with either TTX or phentolamine abolished these responses, confirming norepinephrine release from perivascular sympathetic nerves (Fig 5). In a similar manner, prazosin inhibited both lengthening- and PNS-induced vasoconstriction (diameter change: feed arteries [n=6], 36±3 µm [control] and 6±1 µm [prazosin]; arterioles [n=12], 21±1 µm [control] and 4±1 µm [prazosin]). Additional controls verified that TTX superfusion did not interfere with constriction of feed arteries (resting diameter, 68±7 µm; response, 45±6 µm; n=5) or arterioles (rest, 44±4 µm; response, 34±3 µm; n=10) to 0.1 µmol/L norepinephrine. After treatment with 6-OHD, the responses of feed arteries (n=4) and arterioles (n=7) to both muscle lengthening and PNS were abolished without a significant effect on resting diameters (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 4. Effect of muscle lengthening (from 85% to 130% of in vivo resting length) on microvessel diameter (A) and blood flow (B) under control conditions and with 1 µmol/L TTX, 1 µmol/L phentolamine, or 0.1 µmol/L prazosin in the superfusate. Data are expressed as the change from 85% to 130%; numbers in parentheses indicate the number of vessels studied. Diameters at 85% were as follows: feed arteries, 67±5 µm (control), 68±7 µm (TTX), 66±6 µm (phentolamine), and 85±6 µm (nitroprusside); arterioles, 46±4 µm (control), 44±4 µm (TTX), 47±5 µm (phentolamine), and 68±5 µm (nitroprusside). Blood flows at 85% were as follows: feed arteries, 60±16 nL/s (control), 59±19 nL/s (TTX), 57±22 nL/s (phentolamine), and 146±28 nL/s (nitroprusside); arterioles, 20±8 nL/s (control), 15±6 nL/s (TTX), 20±9 nL/s (phentolamine), and 63±15 nL/s (nitroprusside). Values for prazosin experiments were similar to those reported for phentolamine. *P.05 compared with control.

View larger version (108K): [in this window] [in a new window]   Figure 5. Glyoxylic acid reaction for catecholamines illustrates rich perivascular innervation for arterioles (A) and feed arteries (B) of the retractor muscle. In panel A, note lack of innervation of adjacent venule (edges highlighted by arrows); in panel B, note boundaries of endothelial cells visible through vessel wall; adjacent vein had no reaction with glyoxylic acid. Preparations were excised, incubated,24 and processed for observation using fluorescence microscopy (395- to 440-nm band-pass excitation, 470-nm long-pass emission; Zeiss filter set No. 487705); epi-illumination was from a 75-W xenon lamp. Preparations were imaged using a Nikon CSN Plan Apo x60/1.25 objective. Photomicrographs were obtained using Kodak Ektachrome 160T film in a Contax 167MT camera mounted on a Zeiss No. 456029 phototube. Bar=20 µm.

Changes in Sympathetic Nerve Activity Arise From Within the Muscle (Experiment 5)
Consistent with preceding experiments, muscle lengthening reduced feed artery and arteriolar diameter under control conditions (Fig 6). Lengthening-induced constriction in feed artery segments distal to the site of TTX perfusion was unaffected, as were the responses of intramuscular arterioles. In contrast, vasomotor responses were abolished at or above the site of TTX perfusion. In control experiments (n=4), PNS constricted arterioles (by 18±2 µm) and feed arteries (by 26±2 and 32±4 µm at the origin and distal sites, respectively). Discrete TTX perfusion blocked responses to PNS only at or above the treated site; identical results were obtained in the paired feed artery (Fig 6, inset) that was simultaneously perfused with TTX micropipettes.

View larger version (19K): [in this window] [in a new window]   Figure 6. Effect of muscle lengthening (from 85% to 130% of in vivo resting length) on diameter of arterioles and feed arteries under control conditions and with 10 µmol/L TTX applied with micropipettes to proximal segments of feed arteries. Data are expressed as the change from 85% to 130%; numbers in parentheses indicate the number of vessels studied. Diameters at 85% were as follows: feed arteries, 86±11 µm (control), 90±11 µm (TTX), and 112±11 µm (nitroprusside); arterioles, 57±8 µm (control), 56±7 µm (TTX), and 74±8 µm (nitroprusside). *P.05 vs control. Top, Illustration (not to scale) of discrete TTX application with micropipettes. Tetrodotoxin (10 µmol/L, 60 µL/min) was perfused onto two feed artery segments 3 mm proximal to the retractor muscle; both vessels were treated simultaneously. Boxes indicate sites at which diameters were measured: Origin refers to feed artery segment treated with TTX; Distal refers to feed artery below the region of TTX treatment. The direction of superfusate flow (Distal to Origin) minimized exposing the distal feed artery or arterioles to TTX. Venous networks have been eliminated for clarity; dashed lines represent the site at which feed arteries enter the retractor muscle.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The increased resistance to blood flow with muscle lengthening was first reported early this century² and has since been confirmed in a variety of preparations.^{3 4 27 28 29} This increase in resistance has been attributed to the compression,^{27 29} axial stretch,^{3 6} and kinking^{3 5 7 30} of vessels within muscle. These conclusions have been based on measures of total organ blood flow and have assumed that the vasculature remains passive. In the present study, we have used intravital microscopy to investigate whether specific resistance vessels are responsive to muscle length within a physiological range of motion,¹⁸ as defined by the classic length-tension relationship of skeletal muscle.¹⁹ Our findings reveal that passive changes in muscle length elicit active vasomotor responses throughout the resistance network, both within and external to the tissue. Increasing muscle length resulted in vasoconstriction and a reduction in blood flow; passive shortening produced vasodilation and elevated flow. Pharmacological interventions indicate that length-induced vasomotor responses are triggered by norepinephrine release from perivascular sympathetic nerves in a manner that is independent of the central nervous system. Thus, our findings point to a unique mechanotransduction sequence between skeletal muscle fibers, periarteriolar sympathetic nerves, and resistance vessels: action potentials triggered intramuscularly are propagated antidromically along the sympathetic nerve fibers, initiating coordinated vasomotor activity throughout the resistance network. This "ascending vasoconstriction" results in the maintenance of WSR in feed arteries and perfusion pressure to the microcirculation.

Resistance Vessels Are Sensitive to Muscle Fiber Length (Experiment 1)
Throughout a functionally defined range of motion, a reciprocal relationship was observed between muscle length and the diameter of arterioles as well as feed arteries (Fig 2). These effects were highly reproducible and sustained across muscle lengths; such active responses counter the view that blood vessels remain passive during changes in muscle length. In fact, the abolition of vasomotor responses with nitroprusside confirmed that length-induced changes in vessel diameter were not simply the result of mechanical deformation. Whereas the effects of muscle fiber contraction on vasomotor tone were not evaluated, it seems likely that the release of metabolic vasodilators could override the vasoconstriction reported in the present study, particularly for intramuscular arterioles.

Vasomotor responses of arterioles were independent of branch order or orientation to muscle fibers,⁵ indicating that vessels subjected either to axial stretch or compression were similarly responsive to excursions in muscle length. Because the resistance network is contiguous, it is apparent that responses originating at one location (eg, in vessels subjected to axial stretch) can spread into interconnected branches.^{31 32} This was readily demonstrated by observing that feed arteries responded in concert with arterioles yet were physically unperturbed during muscle extension or shortening. Indeed, this correspondence in vasomotor activity between parent and daughter branches²¹ led us to question how the changes in feed artery diameter were coordinated with those of arterioles.

Blood Flow Reductions Do Not Induce Feed Artery Constriction (Experiment 2)
Lengthening-induced constriction of feed arteries corresponded with reductions in feed artery blood flow (Fig 3A). Moreover, WSR, an index of the tugging force exerted by blood flow on the endothelial cell surface, remained constant throughout the retractor's range of motion (Fig 3B). This finding suggested that an increase in downstream (ie, arteriolar) resistance with muscle extension may have induced feed artery constriction by diminishing the release of vasodilators from endothelial cells.^{11 12} To directly test for such an effect, feed artery blood flow was systematically reduced (by the amount observed during muscle lengthening [Fig 3A]) with microoccluders positioned either upstream or downstream from the measurement site. Although feed arteries dilated with nitroprusside and constricted with muscle lengthening (data not shown), these vessels were surprisingly unresponsive to even a 50% reduction in WSR (Table). The lack of a causal relationship between luminal flow (or WSR) and the diameter of feed arteries demonstrates that a flow-dependent mechanism^{11 12} does not mediate feed artery responses to changing muscle length.

Microvascular Perfusion Pressure Remains Constant During Vasomotor Responses to Changing Muscle Length (Experiment 3)
Arteriolar constriction during muscle lengthening could increase the pressure in feed arteries and thereby trigger a myogenic vasoconstriction.^{9 10} Therefore, in addition to the microocclusion studies performed in experiment 2, we tested for a myogenic response by simultaneously measuring feed artery pressure and diameter just proximal to the microcirculation at each muscle length studied. The constancy of feed artery pressure during vasoconstriction (Fig 3C) resulted in corresponding reduction in tangential wall tension, which is inconsistent with the myogenic response. Although we were unable to resolve wall thickness, its increase with vasoconstriction¹⁰ would have reduced tangential wall stress (=Pr/wall thickness) even more than calculated for wall tension. Nevertheless, the constriction of feed arteries in concert with arterioles effectively maintained a constant microvascular perfusion pressure (Fig 3C). Analogous to observations in soleus and extensor digitorum longus muscles of the rat,²² one third or more of the pressure drop occurred external to the retractor muscle. Thus, irrespective of muscle length, vascular resistance proximal to arterioles contributed 37% to 38% of the total resistance to blood flow at rest.

Muscle Length Directs Sympathetic Nerve Activity (Experiment 4)
Arterioles and feed arteries of the retractor muscle are densely innervated by perivascular sympathetic nerves (Fig 5). Efferent sympathetic nerve traffic increases via central command centers in the brain stem³³ and via afferent input from mechanoreceptors and chemoreceptors in skeletal muscle.^{34 35} Whereas performing our experiments in resting muscle minimized the likelihood of chemoreceptor activation, the length-induced changes in feed artery and arteriolar diameters may have been neurally mediated. We tested this possibility by evaluating diameter responses to changing muscle length in the absence and presence of TTX, a specific inhibitor of the fast voltage-sensitive sodium channels that underlie action potentials. We found that TTX abolished lengthening-induced vasoconstriction and did so without affecting resting vessel diameter. Control experiments verified that TTX reversibly blocked vasoconstriction during PNS. Nevertheless, in the presence of TTX, microvessels retained the ability to constrict and dilate when superfused with norepinephrine and nitroprusside, respectively. When combined with previous reports that TTX did not alter intercellular conduction,^{31 32} resting potential, or excitatory potentials,^{36 37} our data support the specificity of TTX and demonstrate that action potential generation is required to elicit the release of norepinephrine from perivascular nerves. Our findings thereby contrast with a direct effect of muscle stretch on promoting neurotransmitter release from the nerve terminal.³⁸

The demonstration (using glyoxylic acid) of catecholamines in perivascular nerves (Fig 5) together with the blockade of PNS-induced vasoconstriction with TTX led us to test whether norepinephrine was the neurotransmitter that mediated the vasomotor responses to changing muscle length. Analogous to the effect of TTX, phentolamine inhibited the constrictions of both feed arteries and arterioles (Fig 5A). Lengthening-induced vasomotor responses were also blocked by prazosin, a selective ₁-adrenoceptor antagonist, and by chemical sympathectomy.²⁵ These results indicate that norepinephrine released from perivascular nerve fibers mediates lengthening-induced vasoconstriction via ₁-adrenoceptors.

Sympathetic Nerves Are Activated Within the Muscle (Experiment 5)
There are two apparent mechanisms by which muscle length could effect perivascular sympathetic nerve activity in skeletal muscle. First, mechanosensitive afferents³⁴ activated by changes in passive tension (Fig 2A) could reflexively activate perivascular efferent fibers. Alternatively, muscle lengthening could directly trigger action potentials in the perivascular nerve fibers. To distinguish between these pathways for signal transduction, a localized neural blockade was implemented. Because the motor nerve bundles were excised during surgical preparation, the neural connection remaining between the hamster and the retractor muscle was along the feed arteries (Fig 5). Thus, TTX was applied with micropipettes to proximal segments of feed arteries (Fig 6, inset), effectively isolating the retractor muscle from the central nervous system (verified with PNS as described in "Results"). This local treatment with TTX blocked lengthening-induced vasoconstriction at or above the treated segment, yet vasomotor responses in arterioles and distal segments of feed arteries were maintained (Fig 6).

These experiments reveal that muscle lengthening activates perivascular sympathetic nerve fibers within the retractor muscle, giving rise to antidromic propagation of action potentials into feed arteries, resulting in norepinephrine release and vasoconstriction. This finding contrasts sharply with the established orthodromic activation of sympathetic nerves via outflow from higher brain centers and paravertebral ganglia.^{33 35 39} It should also be recognized that because TTX blocked vasoconstriction at and beyond the treated segment, the present findings argue against cell-to-cell conduction^{31 32 40 41} or venous-arterial diffusion²¹ of norepinephrine as the mechanism of "ascending" vasoconstriction.

Summary and Conclusions
The present findings provide the first evidence that intramuscular and extraparenchymal resistance vessels actively respond to mechanical forces within muscle, independent of muscle fiber activation or the release of vasoactive metabolites. Passive extension of the retractor muscle reduced feed artery and arteriolar blood flow by 50% to 60%. Treatment with TTX, phentolamine, or prazosin eliminated approximately two thirds of this effect, indicating that the increase in vascular resistance arises primarily from the activation of sympathetic nerves; the remaining approximately one third of the effect is explained by compression and kinking of microvessels within the muscle, as proposed in earlier studies.^{3 5 7} The coordination of feed artery and arteriolar responses during muscle lengthening maintained WSR in feed arteries and perfusion pressure to the microcirculation. In contrast to the cell-to-cell conduction of vasomotor responses via gap junctions,^{40 41} the responses identified in the present study are mediated by propagation along perivascular nerves.

The mechanism by which changes in muscle length influence the firing in perivascular nerve fibers is unclear. It is plausible that muscle length activates stretch-sensitive ion channels in these fibers, causing depolarization and the generation of action potentials. Such an effect could be mediated by integrin binding to the extracellular matrix³⁸ and warrants further study. Alternatively, muscle extension may increase the release of a paracrine substance that gives rise to action potentials in perivascular nerves, perhaps via a local axon reflex. However, this explanation remains speculative until such a mechanism is identified in skeletal muscle. Nevertheless, our findings define a unique mechanotransduction sequence by which physiological changes in the length of skeletal muscle are intrinsically coupled to the neural regulation of peripheral resistance and the supply of oxygen to muscle fibers.

	Selected Abbreviations and Acronyms

 

6-OHD = 6-hydroxydopamine PNS = perivascular nerve stimulation PSS = physiological saline solution TTX = tetrodotoxin Vrbc = centerline red blood cell velocity WSR = wall shear rate

	Acknowledgments

 
This study was supported by a postdoctoral fellowship (Dr Welsh) from the Connecticut Affiliate of the American Heart Association (AHA) and a Grant-In-Aid from the AHA (National Center) and was performed during the tenure of an Established Investigatorship Award (Dr Segal) from AHA and Genentec, Inc. Special thanks are extended to Drs D.T. Kurjiaka and A.J. Fuglevand for valuable discussions, to B.D. Walker and M.S. Fritz for excellent technical assistance, to S.E. Brett Welsh for artwork, to Dr C. Desjardins for a critical review of this work, and to Micro Video Instruments for use of the objective used to acquire the images in Fig 5.

Received February 26, 1996; accepted May 15, 1996.

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