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Perfusion of skeletal muscle varies considerably during rest, exercise, or when arteries are occluded. The extent that a muscle can adapt to changes in flow demand is often expressed as the ratio of the highest inducible flow and control flow, the microvascular blood flow reserve capacity (MBFRC). However, perfusion of the nutritive capillaries of skeletal muscle may not only be improved by the increase in blood flow proportional to the increase in arterial flow, but also by diverting originally shunted flow towards the muscle proper. Consequently, MBFRC is not a good measure of capillary flow reserve, unless the assessed flow in both conditions is purely nutritive in nature. Therefore, in critical conditions, flow measurements in large vessels are not appropriate to assess MBFRC. In muscle, capillaries are compliant, i.e., with varying transmural pressure capillary diameter varies. During high perfusion states, when capillary transmural pressure is increased, capillary compliance results in increased capillary diameter and, hence, in reduced resistance and increased exchange surface area. This results in improved perfusion and enlarged capillary exchange surface area. In low perfusion states, capillary diameter is reduced. This augments the detrimental effects of the low perfusion status. Operative restoration of perfusion pressure not only increases the driving force for perfusion, but also leads to (passive) dilatation of the capillary bed and an extra reduction in resistance to flow, and, hence, a disproportional increase in flow.

Accumulation of intracellular free calcium (Ca2+i) may play an essential role in the ischemia/reperfusion injury of skeletal muscle. Although it has been shown that Ca2+i levels significantly increase during ischemia/reperfusion, it is still a matter of debate whether Ca2+i increases during ischemia alone. It was the aim of this study to monitor the in vivo Ca2+i levels in the rat spinotrapezius muscle during ischemia of varying duration and reperfusion, using a ratiometric fluorescence technique, and to investigate the relationship between the postischemic flow patterns and Ca2+i, if any. The muscle was loaded with Indo-1/AM and imaged by a cooled digital camera. Pre- and postischemic tissue perfusion was assessed by means of an analogue camera. Our results show that short-term ischemia (5, 15 and 30 min) and subsequent reperfusion (60 min) does not alter Ca2+i homeostasis and that tissue perfusion promptly recovers after the insult. One or two hours of ischemia resulted in changes in Ca2+i levels, varying from preparation to preparation; increases in some and no changes in others. In these preparations three distinct flow patterns - normal, compromised and no-reflow - could be distinguished during the 60-min reperfusion. Our main conclusion is that in skeletal muscle Ca2+i levels may increase, the increase probably depending on the muscle fiber type exposed.

The understanding of the regulation of the free cytosolic [Ca2+] ([Ca2+]i) in skeletal muscle is hampered by the lack of techniques for quantifying free [Ca2+]i in muscle fibres in situ. We describe a model for studying the dynamics of free [Ca2+]i in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles of the rat in vivo using caffeine superfusion to induce changes in free [Ca2+]i. We assumed that differences in sensitivity between the two muscle types for this substance reflect differences in intracellular Ca2+ handling in the fibres of which these muscles consist. The Indo-1 ratiometric method, using intravital microscopy with incident light, was adapted to measure free [Ca2+]i in vivo. Fluorescence images were collected by means of a digital camera. Caffeine superfusion at 37 degrees C for 2 min, at concentrations of 1, 2, 5, 10 or 20 mmol/l, induced a concentration-dependent increase in free [Ca2+]i and revealed differences in caffeine sensitivity between the muscle types, with the SOL being more sensitive. In a separate set of experiments the contracture threshold, as assessed by topical application of caffeine, was determined in both muscle types. EDL had a higher threshold for developing contracture than SOL. These finding are in agreement with previous in vitro studies. We may conclude that the dynamics of free [Ca2+]i can be assessed reliably in intact mammalian muscle in vivo.

Intracellular free Ca2+ concentration ([Ca2+]i) plays an essential role in physiological regulatory processes and common pathological conditions. Better understanding of these phenomena is still hampered by problems encountered in the quantitative assessment of [Ca2+]i changes, especially in blood-perfused organs. This study demonstrates that the ratiometric fluorescence technique can be adapted for quantitative in vivo [Ca2+]i determinations. The rat spinotrapezius muscle was topically loaded with indo 1-AM and imaged by a cooled digital camera. Ratio images were calculated in small regions (100 micrometers x 100 micrometers) practically devoid of large vessels in the resting state, after 30 min of ischemia, 20 min of reperfusion, or ionomycin or manganate treatments. When we assumed an average [Ca2+]i of 100 nM in the resting blood-perfused muscle, ischemia increased [Ca2+]i to approximately 200 nM. During reperfusion [Ca2+]i decreased to approximately 140 nM. Ionomycin induced an increase in [Ca2+]i to well above 750 nM. Manganate reduced Ca2+-dependent fluorescence to virtually zero. Our main conclusion is that changes in [Ca2+]i can be monitored and quantitatively determined in vivo.

Loose ligation of a sciatic nerve in rats (chronic constriction injury; CCI) provokes sensory, autonomic, and motor disturbances like those observed in humans with partial peripheral nerve injury. So far, it is unknown whether these motor disturbances result from (mechanical) allodynia or from damage to the motor neuron. These considerations prompted us to assess, in CCI rats, the density of motor axons in both the ligated sciatic nerve and the ipsilateral femoral nerve. To this end, we determined the number of cholinesterase positive fibres. It has been demonstrated previously that muscle fibre type density may be used as a measure of motor denervation and/or hypokinesia. Therefore, the myofibrillar ATPase reaction was employed to assess fibre type density in biopsies obtained from the lateral gastrocnemius muscle (innervated by sciatic nerve) and rectus femoris muscle (innervated by femoral nerve). We observed axonal degeneration of motor fibres within the loosely ligated sciatic nerve, both at an intermediate (day 21) and at a late stage (day 90) after nerve injury. The reduction in the number of motor nerve fibres was more pronounced distal to the site of the ligatures than proximal. A (less pronounced) reduction of motor fibres was observed in the ipsilateral (non-ligated) femoral nerve. In line with these findings, we observed altered fibre type densities in muscle tissue innervated by the ligated sciatic nerve as well as the non-ligated femoral nerve indicative of motor denervation rather than hypokinesia. The findings of this study suggest that the motor disorder induced by partial nerve injury involves degeneration of motor nerve fibres not only within the primarily affected nerve but also within adjacent large peripheral nerves. This spread outside the territory of the primarily affected nerve suggests degeneration of motor neurons at the level of the central nervous system.

Mechanical interaction between tissue stress and blood perfusion in skeletal muscles plays an important role in blood flow impediment during sustained contraction. The exact mechanism of this interaction is not clear, and experimental investigation of this mechanism is difficult. We developed a finite-element model of the mechanical behavior of blood-perfused muscle tissue, which accounts for mechanical blood-tissue interaction in maximally vasodilated vasculature. Verification of the model was performed by comparing finite-element results of blood pressure and flow with experimental measurements in a muscle that is subject to well-controlled mechanical loading conditions. In addition, we performed simulations of blood perfusion during tetanic, isometric contraction and maximal vasodilation in a simplified, two-dimensional finite-element model of a rat calf muscle. A vascular waterfall in the venous compartment was identified as the main cause for blood flow impediment both in the experiment and in the finite-element simulations. The validated finite-element model offers possibilities for detailed analysis of blood perfusion in three-dimensional muscle models under complicated loading conditions.

The observed positive correlation between cessation of red blood cell flow in capillaries at low perfusion pressures and the oxygen tension (PO2) in the superfusion solution may be due to oxygen-dependent arteriolar constriction. To test this hypothesis, we investigated capillary flow cessation during aortic occlusion and concomitant changes in diameters of terminal arterioles and capillaries in normal and vasodilated vascular beds of rabbit tenuissimus muscle (n = 15) by means of video intravital microscopy. In the vasodilated bed, arteriolar tone was eliminated by local application of 10(-4) M adenosine (ADO). The PO2 in the superfusate was varied locally, i.e., in the solution between objective lens and muscle surface. At a local PO2 of 40 mm Hg without ADO, flow ceased in about 50% of the capillaries during aortic occlusion while the arterioles dilated to 118% of control (median; p < 0.001). Addition of ADO led to an increase in arteriolar and capillary diameter to 220% (median; p < 0.001) and 121% (median; p < 0.05), respectively. Under ADO, the incidence of capillary flow cessation was reduced (p < 0.05) to about 20%. Elevation of the local PO2 from 40 to 100 mm Hg in the presence of ADO did not lead to a significant change in the incidence of flow cessation, nor to changes in arteriolar or capillary diameter. In the presence of ADO, median arteriolar and capillary diameters during aortic occlusion were 96% (p < 0.001) and 7% (p < 0.05) larger than their control diameters without ADO, respectively. In summary, it is suggested that the incidence of flow cessation may depend on both the arteriolar and the capillary diameter. Of these two factors, capillary diameter may be the most important one, because its changes affect the interaction between red blood cells and the vessel wall in the narrow capillaries, and, hence, the resistance to flow. In the presence of ADO, at elevated local PO2 levels flow cessation still occurs in about 20-30% of the capillaries, suggesting that arteriolar contraction is only in part responsible for the incidence of flow cessation.

The effects of locally applied adenosine (ADO) and/or femoral artery pressure reduction (induced by complete aorta occlusion) on capillary diameter were investigated in the tenuissimus muscle of anesthetized rabbits. Capillaries were visualized by means of intravital video microscopy. Diameters were measured using an image shearing device. During control femoral artery pressure (median: 83 mm Hg) and without ADO, median capillary diameter was 4.3 microns (range: 3.2-5.3 microns; 27 capillaries in 7 animals). Complete aorta occlusion (median femoral artery pressure: 18 mm Hg) resulted in a reduction of capillary diameter to 3.9 microns (2.7-4.7 microns; p < 0.0001). Subsequent reactive hyperemia resulted in an increase in diameter to 5.2 microns (3.7-6.0 microns; p < 0.0001). Locally applied ADO (10(-4) M) probably led to complete vasodilation of the arterioles, because their diameters did not further increase during reactive hyperemia after complete occlusion. ADO (10(-4) M) induced an increase of control capillary diameter to 5.5 microns (4.1-6.4 microns; median relative increase: 27%; p < 0.0001), resulting in a decrease of capillary resistance by 61%. In the presence of ADO, aorta occlusion resulted in a capillary diameter decrease to 4.7 microns (3.4-6.1 microns); p < 0.0001). Subsequent reactive hyperemia resulted in an increase to maximally 5.6 microns (4.3-6.4 microns; p < 0.0001). This diameter was approximately the same as the control diameter during ADO. During occlusion in the presence of ADO, capillary diameter was significantly larger (11%; p < 0.0001) than during control without ADO. The capillary diameter changes induced by the various interventions were mainly passive, i.e., proportional to capillary transmural pressure changes. However, capillary diameter was larger during aortal occlusion in the presence of ADO than during control femoral artery pressures without ADO, even though capillary pressure was probably higher in the latter case. It is proposed that the prolonged increase in transmural capillary pressure due to ADO may induce changes in capillary wall configuration, leading to larger diameters.

Perfusion of capillaries was investigated in the tenuissimus muscle of young anesthetized New Zealand White rabbits during control conditions, complete aorta occlusion, and reactive hyperemia at various local oxygen tensions. Capillaries were visualized with bright-field microscopy. The number of capillaries perfused under experimental conditions was compared with that during control conditions. Capillary diameter was measured to assess whether the interventions caused changes in luminal diameter. During control conditions at a local pO2 of about 20 mm Hg, capillary perfusion fluctuates; instantaneous capillary density is smaller than anatomical capillary density. When the aorta is (partially) occluded, capillary perfusion becomes continuous and instantaneous capillary density equals anatomical capillary density. The latter is also observed during the early phase of reactive hyperemia, prior to the reappearance of flowmotion. Capillary diameter is not invariant during these interventions, but decreases by 8% during occlusion and increases by 12% during reactive hyperemia. The concomitant change in perimeter and cross-sectional area should be factored in with functional capillary density, when tissue exchange surface area or volume flow are considered. When during control conditions, the muscle becomes locally (under the microscope lens) exposed to a higher oxygen tension, capillary diameter does not change. However, the relative number of capillaries perfused at complete aorta occlusion is unity at low local oxygen, and diminishes with increasing local oxygen to become 0 at an oxygen tension of about 70 mm Hg. In preparations in which capillary diameter is not invariant under the experimental conditions, functional capillary density can only be used to compare the number of perfused capillaries with the number of capillaries anatomically present. Capillary diameter has to be factored in when tissue perfusion or exchange surface area are considered.

When perfusion pressure is reduced, red blood cell flow in the capillaries of skeletal muscle ceases at a positive pressure difference across the vascular bed, while arterioles dilate and venules are not constricted. This flow cessation (i.e., cessation of red blood cell flow) and luminal diameter changes in capillaries following femoral arterial pressure reduction were investigated in the rabbit tenuissimus muscle in situ (n = 42) using intravital video microscopy. Arterial pressure was reduced by occlusion of the aorta distal to the renal arteries. During the experiments, leg and muscle were placed in a sealed box. The muscle was exposed to low PO2 by leading a gas mixture deprived of O2 through the box. Locally at the muscle surface, i.e., under the microscope objective, PO2 was varied by varying the PO2 in the superfusion solution. In all experiments, the remainder of the muscle was kept at low (< 20 mm Hg) PO2. The incidence of flow cessation was virtually zero at low local (< 20 mm Hg) PO2 and became almost 100% at local values above 70 mm Hg. Initial equivalent capillary diameters were 3.1-5.8 microm (median 4.0 microm) and did not correlate with local O2 tension. During aorta occlusion, capillary diameters significantly (P < 0.0001) decreased by a median value of 8% at all local PO2 values; in 14 out of 54 capillaries local diameter became less than 2.8 microm. The extent of diameter reduction did not correlate with PO2. In the 14 capillaries in which the diameter became less than 2.8 microm flow cessation occurred in only four cases. The minimal diameter reached was always at the site of an endothelial nucleus. The capillary diameter reductions are probably due to passive recoil. In the 48 capillaries in which flow ceased, only in four cases did a red blood cell stop at the site of the nucleus. We conclude that capillary diameter reductions (local and generalized) lead to a considerable increase in capillary resistance which contributes to the occurrence of flow cessation but cannot solely explain it.

Capillary diameter changes were studied in the tenuissimus muscle of 29 urethan-anesthetized New Zealand White rabbits. Capillaries were visualized with transillumination bright-field microscopy (saltwater lens, x 50; resolution approximately 0.3 microns). Median capillary diameter during the control period was 4.4 microns (range 3.2-6.9 microns). Complete aortic occlusion resulted in a reduction of median femoral arterial pressure to 17 mmHg (range 4-22 mmHg). During 2 min of occlusion, capillary diameter decreased by 6%, with greater change on the arteriolar side of the capillary than on the venular side. During reactive hyperemia after release of the occluder, capillary diameter maximally increased by 12% compared with the control period, with a larger response at the arteriolar end of the capillary than at the venular end. Median capillary resistance was estimated to increase by 27% during occlusion and to decrease by 36% during peak reactive hyperemia. The observed diameter changes are compatible with the idea that capillaries change their diameter relative to changes in transmural pressure.