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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 role of lumbar sympathectomy in the treatment of limb ischemia secondary to arteriosclerosis obliterans has been controversial. Increased temperature and rubor of the skin, which usually follow sympathectomy, have generally been interpreted as indicative of improved nutritive skin blood flow. However, the existence of a (nonnutritive) thermoregulatory level of skin microcirculation makes such an extrapolation questionable. We investigated the total (mainly thermoregulatory) skin blood flow (TSBF) in the hindlimb of 15 male Lewis rats by means of laser Doppler flowmetry and the nutritive skin blood flow (NSBF) by means of capillary microscopy (red blood cell velocity). Transcutaneous oximetry was used to assess skin oxygenation (SO). Measurements were performed before and 2 and 28 days after ligation of the common iliac and iliolumbar artery. Subsequently, either a surgical resection of the sympathetic chain (L2-L6) was performed or a sham operation. Measurements were repeated 2 and 28 days later. For the group of 15 rats as a whole, TSBF (p < 0.05), NSBF (p < 0.05), and SO (p < 0.05) were found to be drastically reduced at day 2 after litigation compared to preligation values. This reduction partially recovered during the following weeks. TSBF (p < 0.05) and NSBF (p < 0.05), however were still reduced at day 28 after ligation compared to preligation values, whereas the SO at this time tended to be lower (p = 0.11). In the sympathectomy group the TSBF was found to be increased at day 2 (p < 0.05) and day 28 (p < 0.05) after sympathectomy, both compared to values obtained at day 28 after ligation. Sympathectomy did not have an effect on NSFB and SO. The sham procedure had no effect on the TSBF, NSBF, or SO. These results indicate that in case of lower limb ischemia, sympathectomy improves skin blood flow at the thermoregulatory but not the nutritive level of skin microcirculation. This may be related to the fact that the thermoregulatory vessels are mainly sympathetically controlled, whereas the nutritive capillaries are mainly controlled by local (nonneural) factors.

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.

Transcutaneous oximetry (TcpO2) performed at 37 and 44 degrees on the dorsum of the foot and capillary microscopy of the nailfold of the big toe were applied to 85 patients with various (including asymptomatic) stages of lower limb ischemia to appreciate the relationship between (disturbances in) capillary perfusion and skin oxygen tension. In mildly diseased patients, capillary perfusion as measured by direct observation, was preserved. In critically ischemic patients in the supine position, red blood cell-perfused capillary density was reduced. Nutritive perfusion was severely reduced and showed an absent reactive hyperemia after a 1-min arterial occlusion. Also, postural vasoconstrictive activity was reduced. TcpO2 measured at 37 degrees was very low already in mildly diseased patients, illustrating the poor oxygen diffusion toward the skin. At 44 degrees, TcpO2 was severely reduced in critically ischemic patients. Reactive hyperemic response and postural vasoconstriction were suppressed, due to local heating of the skin. Measurement of the TcpO2 has limitations in the assessment of nutritive perfusion, as opposed to capillary microscopy, since it is an indirect measure of skin perfusion, not necessarily derived from capillaries only. The obligatory local skin heating impairs physiological studies as to hyperemic reserve capacity or postural constriction mechanisms. Thus, transcutaneous oximetry is a poor method of characterizing pathophysiological mechanisms occurring in skin nutritive microcirculation. However, capillary microscopy and transcutaneous oximetry can give additive information as to the severity of peripheral ischemia. Peak red blood cell velocity during reactive hyperemia using capillary microscopy and the resting TcpO2 at 44 degrees, both measured in the supine position, appeared to be valuable microcirculatory parameters in detecting critical limb ischemia.

TMo date, capillary microscopy, transcutaneous oximetry (tcpO2) and laser Doppler fluxmetry are frequently used in the investigation of skin microcirculation in patients with lower limb ischaemia. The concomitant microcirculatory disturbances may be useful in addition to macrocirculatory parameters to discriminate the different degrees of ischaemic severity. The best ways of application of these methods and the choice of the best parameters to assess ischaemia have been insufficiently investigated. Therefore, skin microcirculation was investigated with the use of these techniques in 130 patients with different stages of lower limb ischaemia, divided according to their ankle-to-brachial pressure index (ABI). Patients were investigated in the sitting and the supine position. Measurements were performed at rest and during reactive hyperaemia following arterial occlusion, and before and after local skin heating. The reactive hyperaemic response using laser Doppler fluxmetry differed in every patient group investigated. Capillary red blood cell velocity was markedly impaired in critically ischaemic patients (ABI < 25%). Transcutaneous oxygen pressure measurements at rest rendered the highest positive predictive value (PV; 87%) to classify patients as having clinically severe ischaemia (Fontaine 3 or 4). Ankle and toe pressure measurements provided a PV value of 78%. Microcirculatory parameters and techniques appear to be useful as an addition to standard macrocirculatory techniques to assess the severity of lower limb ischaemia. This is particularly of importance in patients in whom macrocirculatory parameters are unattainable.

Posturally induced microvascular constriction in the skin of the leg is disturbed in severe ischaemia. It is unknown whether this disturbance is of local or central origin and whether the stage of ischaemia at which this disturbance occurs differs when the nutritive and thermoregulatory flow levels are compared. We investigated the effect of posture on the skin microcirculation in 21 patients with unilateral severe ischaemia. The results were compared with those from the contralateral, asymptomatic leg and with results from 11 age-matched controls. Patients were investigated in supine and sitting positions, using capillary microscopy to measure nutritive flow, and laser Doppler fluxmetry (LDF) to measure thermoregulatory flow, of the big toes. In the supine position, capillary flow and LDF were lower in the diseased than in the asymptomatic and control legs. After changing from the supine to the sitting position, capillary perfusion decreased in all three groups, but was most pronounced in the controls. Laser Doppler flux decreased in the controls, but increased in the diseased legs, suggesting disturbed vasoconstriction mechanisms in the deeper skin microvessels. These findings indicate that in severe limb ischaemia, posturally induced microvascular reactivity is sustained at the nutritive level but not at the thermoregulatory level. This disturbed reactivity is considered a local phenomenon, as it is not observed in the contralateral leg.

Systolic ankle and toe pressure measurements are considered to be the best way of documenting arterial occlusive disease. In the European consensus, chronic critical limb ischaemia is defined as persistent pain with an ankle pressure lower than 50 mmHg. To investigate the possible adjunct value of microcirculatory assessment, capillary microscopy and transcutaneous oximetry were performed in 21 asymptomatic persons (F1), 89 claudicants (F2) and 54 patients with critical limb ischaemia (F3/4). Capillary morphology (diameter, density) and dynamics [red blood cell velocity (RBCV), peak RBCV and time to peak RBCV], as well as transcutaneous oximetry parameters were determined for each Fontaine group and compared with ankle and toe pressure measurements. Despite considerable overlap, ankle and toe pressures were significantly (p less than 0.001) different between F1, F2 and F3/4 patients. Capillary density (p less than 0.05), diameter (p less than 0.05), peak RBCV (p less than 0.05) and time to peak RBCV (p less than 0.01), as well as transcutaneous oximetry parameters (p less than 0.001) were significantly different between all groups and impaired with progression of ischaemia. However, a similar overlap between all groups was observed, except the supine TcpO2 parameter which separated F3/4 patients completely from the other groups. In all patients with critical limb ischaemia, dynamic parameters, such as peak RBCV (p less than 0.01) and time to peak RBCV (p less than 0.001), were significantly lower as compared to non-critically ischaemic patients, irrespective of an ankle pressure below or above a value of 50 mmHg, illustrating the additional value of microcirculatory assessment in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Patients suffering from severe lower limb ischemia may experience pain relief on leg dependency despite the fact that dependency normally results in arteriolar vasoconstriction. To clarify this possible paradox, skin microcirculation of the limb was investigated in 75 patients with different stages of lower limb ischemia and in 12 asymptomatic subjects., Methods and Results: Using nailfold capillary video microscopy, red blood cell-perfused capillary density and diameter and red blood cell velocity were assessed in supine and sitting positions. Capillary density increased by changing from the supine to the sitting position, especially in patients with limb-threatening ischemia (showing a 4.5-fold increase versus a 1.5-fold increase in asymptomatic subjects). In subjects without or with mild ischemia, capillary perfusion was two to four times lower in the sitting than in the supine position. In patients with limb-threatening ischemia, perfusion was strongly reduced, being slightly higher in the sitting position. Patients with relief of pain while sitting did not always have a higher capillary perfusion but did have a higher capillary density in the sitting position., Conclusions: The arteriolar postural vasoconstrictive mechanism at the nutritive level is still intact in subjects without or with mild ischemia but not in patients with severe ischemia. Capillary recruitment rather than disturbed arteriolar vasoconstriction could explain why patients with severe leg ischemia prefer leg dependency.

1. Skin microcirculation was investigated in 12 asymptomatic subjects and 76 patients, grouped according to their ankle-to-brachial systolic blood pressure index, in order to evaluate to what extent posturally induced microvascular constriction is dependent on the stage of leg ischaemia at different local skin temperatures. 2. Skin microcirculation was assessed in the supine and sitting position by using laser Doppler fluxmetry at unheated skin temperature and at 36 degrees C, and transcutaneous oximetry at 37 degrees C and 44 degrees C. 3. Skin perfusion and oxygenation diminished with decreasing ankle-to-brachial systolic blood pressure index. In healthy control subjects, perfusion and oxygenation were reduced when changing from the supine to the sitting position, but were enhanced in patients with severe leg ischaemia (ankle-to-brachial systolic blood pressure less than 30%), indicating disturbed posturally induced vasoconstriction. 4. Increasing the local skin temperature resulted in a higher perfusion and masked the posturally induced vasoconstriction in healthy subjects. In patients with severe leg ischaemia, however, perfusion was unaltered by the temperature increase, apparently because the microvessels were already maximally dilated. The induction of reactive hyperaemia produced no additional increase in perfusion or oxygenation. 5. It is concluded that posturally induced microvascular constriction in the skin is disturbed in patients with severe leg ischaemia (ankle-to-brachial systolic blood pressure index less than 30%). Disturbed microvascular constriction upon dependency was also seen in healthy subjects after local skin heating. This suggests that posturally induced vasoconstriction is mainly regulated by local mechanisms.

To determine whether postoperative oedema could be predicted preoperatively by microcirculatory parameters, we studied nutritive capillary blood flow in 21 patients before and after limb salvage procedures. All patients had severe lower limb ischaemia and underwent femoro-popliteal or femoro-crural bypass surgery. The systolic ankle-branchial arm index and systolic toe pressure were used as macrocirculatory parameters. Intravital capillary microscopy was used to measure red blood cell (RBC) and peak RBC velocity and time to peak RBC velocity after release of a 1 min arterial occlusion in the nailfold of the toe. Transcutaneous pO2 was measured on the dorsum of the foot at rest, during oxygen inhalation and following a release of a 5 min occlusion. After surgery the mean systolic ankle-brachial index and systolic toe pressure and all transcutaneous pO2 parameters improved significantly (P less than 0.001). Mean peak RBC velocity increased from 0.156 mm/s to 0.310 mm/s (P less than 0.05), indicating that the reactive hyperaemic response in the capillary bed had improved. Eleven patients developed postoperative oedema. There were no differences in postoperative macro and microcirculatory parameters between the patients with oedema (n = 11) and those without oedema (n = 10). However, preoperatively RBC velocity and peak RBC velocity were significantly lower (P less than 0.05) and time to peak RBC velocity was significantly longer (P less than 0.01) in patients who developed oedema. These findings show that in patients with severe limb ischaemia vascular surgery improves both macro- and microcirculatory blood flow, but that these patients develop oedema after vascular surgery when microcirculatory blood flow is compromised preoperatively.

Epidural spinal cord electrical stimulation (ESES) was performed on 10 patients with severe limb ischemia due to atherosclerotic disease. Microcirculatory parameters were assessed before and after ESES. Bright field microscopy was used to assess capillary diameters and red blood cell (RBC) velocity in the dorsum of the foot. Fluorescein microscopy was used with intravenously injected sodium fluorescein to study capillary density and sodium fluorescein appearance time in the dorsum of the toe. The systolic ankle/arm pressure ratio and toe pressure measurements were used as macrocirculatory parameters. After ESES, clinical improvement was confirmed by intravital microscopy. Capillary density increased (p less than 0.001), RBC velocity in capillaries already perfused before ESES increased from 0.054 mm/sec to 0.762 mm/sec (p less than 0.001), and sodium fluorescein appearance time decreased from 72 to 45 seconds (p less than 0.001). Capillary diameter did not change significantly so that the increase in RBC velocity may be interpreted as enhanced volume flow. Systolic ankle/arm pressure ratios and digital arterial pressure did not change significantly. The current results show that in patients with severe occlusive arterial disease of the lower limbs, ESES recruits capillaries not perfused in the control situation and enhances skin blood flow, improvements that may explain the beneficial clinical effects of ESES.

Background: The object of this study was to develop an animal model in which changes in microvascular haemodynamics and leucocyte–vessel wall interactions due to acute limb ischaemia–reperfusion (I/R) can be measured in the skin. Furthermore, it was investigated whether these changes are related to local muscle injury. Methods: Male Lewis rats were subjected to unilateral limb ischaemia for 1 h (n = 8) or 2 h (n = 8) by cuff inflation, or to a sham protocol (n = 6). Intravital video microscopic measurements of leucocyte–vessel wall interactions, venular diameter, red blood cell velocity and reduced velocity (which is proportional to wall shear rate) were performed in skin venules before ischaemia and at 0·5, 1, 2, 3 and 4 h after the start of reperfusion. Oedema and leucocyte infiltration of ischaemic/reperfused skeletal muscle were quantified histologically. Results: In skin venules, both 1 and 2 h of ischaemia induced a significant increase in leucocyte rolling (six and five times baseline, respectively; P < 0·05) and adherence during reperfusion (eight and four times baseline; P < 0·05). No significant increase in muscular leucocyte infiltration was detected. After an initial hyperaemic response of 180 per cent of baseline values (P < 0·05), blood flow decreased to about 60 per cent after 4 h of reperfusion in skin venules of both experimental groups. I/R induced tibial muscle oedema, the severity of which depended on the ischaemic interval (wet to dry ratio: control, 4·0; 1 h, 4·5 (P not significant); 2 h, 5·8 (P < 0·05)). Conclusion: A non-invasive animal model was developed that enables investigation of the consequences of acute limb I/R. [ABSTRACT FROM AUTHOR]

Ischemia/reperfusion injury (IRI) is often the final and irreversible factor causing flap failure in microvascular surgery for reconstruction of complex defects. Use of vascularised composite tissue allografts (CTA's) may lead to a reduction of donor site morbidity and functional loss. Of all tissues present in CTA's, muscle is one of the least tolerant to ischemia. The aim of the study was to assess the feasibility of a transplantation of the cremaster in rats and to assess effects of a preservation solution on IRI. IRI leads to decreased capillary. Authors developed a useful model for the observation of the microcirculation in the rat cremaster muscle after transplantation and to assess the effects of Histidine-Tryptophan-Ketoglutarate on IRI. Transplantation of the cremaster did not induce a major change in capillary perfusion.