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Background: Microvascular surgery for the reconstruction of complex defects involves an ischemic period, which may cause flap failure as the result of ischemia/reperfusion injury. We assessed the microvascular consequences of rat cremaster muscle transplantation after prolonged periods of cold storage in HTK-Bretschneider solution (HTK)., Materials and Methods: Cremaster muscle transplantations were performed immediately or after 8 or 24 h of cold storage (4 degrees C) in HTK or saline. Intravital microscopy was used to quantify capillary perfusion and venular leukocyte-endothelium interactions following transplantation., Results: The transplantation procedure itself resulted in 50-65 min of ischemia. After direct transplantation, capillary perfusion was 90% of control. Transplantation after 8 h of cold storage in either HTK or saline did not deteriorate capillary perfusion. When the tissue was stored for 24 h, HTK was superior to saline in preserving capillary perfusion (HTK: 76-83% of control, saline: 30%). Immediate transplantation induced a small increase in leukocyte adhesion. Prolonged cold storage in either fluid resulted in reduced flow velocities (qualitative observations) and edema formation, which hampered quantification of leukocyte-endothelium interactions., Conclusions: Even after 8 or 24 h of cold storage in HTK, transplantation of rat cremaster muscle was successful with good capillary perfusion. Capillary perfusion was better preserved in HTK than in saline.

Background: Our aim was to investigate the potential of the preservation solution Celsior to protect rat cremaster muscle microcirculation during ischemia and reperfusion, and to compare its effects with those of HTK (histidine-tryptophan-ketoglutarate-Bretschneider solution). Because of its anti-oxidant contents, we expected Celsior to be more protective than HTK., Materials and Methods: Capillary perfusion and leukocyte-endothelium interactions were examined in rat cremaster muscle using intravital microscopy. After perfusion with Celsior or HTK (4 degrees C), the cremaster was subjected to 4 or 6 h of warm (33-34 degrees C) ischemia and 2 h of reperfusion. Measurements were performed prior to perfusion and/or ischemia, and 0, 1, and 2 h after restoration of flow., Results: Without Celsior or HTK, capillary perfusion transiently decreased to 50% of baseline after 4 h of ischemia; it remained low (45%) after 6 h of ischemia. Whereas HTK had no significant influence, Celsior deteriorated capillary perfusion: it remained low after 4 h of ischemia (39-48%) and decreased even further after 6 h of ischemia (18-8%). Both preservation solutions similarly reduced the increase in leukocyte-endothelium interactions after ischemia., Conclusions: Preischemic tissue perfusion with Celsior had an adverse effect on capillary perfusion in rat cremaster muscle after 4 and 6 h of ischemia, whereas HTK did not significantly influence this parameter. Both preservation solutions similarly prevented the increase in leukocyte-endothelium interactions after ischemia. These data suggest that HTK is more suited as a preservation solution for muscular tissue than Celsior, especially when the known protective effects of HTK on muscle function are taken into account.

The aim of this work was to develop a model to study the microcirculation and relative levels of intracellular free calcium in the myometrium of pregnant rats. On Day 21 of gestation a lobe of uterus was prepared free, flipped over, and mounted in a superfusion chamber leaving the radix and thereby the innervation and circulation intact. RBC velocity and arteriolar diameters were determined by means of intravital video microscopy before and after stimulation (norepinephrine). To study intracellular free calcium changes, the fluorescent dye Indo-1 AM was added to the superfusate in the chamber. Fluorescence images were recorded and ratios of the images collected at 400 and 506 nm were calculated and changes thereof were assumed to represent intracellular free calcium changes. RBC velocity and arteriolar diameter did not change for at least 1 h, while the response to norepinephrine was similar at the beginning of the experiment and after 120 min. In four separate interventions, the uterus was challenged with 5 x 10(-4) IU/ml oxytocin, 4.5 mM calcium, 5 x 10(-4) IU/ml oxytocin with 4.5 mM calcium, and 5 microM ionomycin, resulting in an increase of the 400/506 nm ratio of 27, 31, 76, and 103%, respectively, representing a relative increase in intracellular free calcium. This novel in vivo model is suitable for monitoring intracellular free calcium changes and to record RBC velocities and blood vessel diameters in the myometrium of pregnant rats., (Copyright 2000 Academic Press.)

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.

Photoactivation of intravascular dyes with high doses of light is a technique used clinically to treat tumors. This procedure results in arteriolar constriction, mast cell degranulation, platelet thrombus formation, and, ultimately, microvascular stasis. In vivo microscopy was utilized in the current study to examine if the endothelial release of prostaglandins and nitric oxide could participate in the microvascular effects of photoactivation. Diameter changes and thrombus formation of arterioles and venules of the cremaster muscle of male Sprague-Dawley rats were quantitated during continuous light activation of intravascular fluorescein isothiocyanate conjugated to bovine serum albumin. Vasoconstriction and thrombus development were assessed separately, using the relationships between the width of the red blood cell column, the inner wall diameter, and the thickness of the plasma layer. Venular photoactivation resulted in thrombus growth which reached 30% of the maximum size by 16.8 +/- 3.71 min and a subsequent growth rate of 6.2 +/- 1.64 microns/min. In arterioles, 30% thrombus growth occurred at 14.0 +/- 2.02 min with a growth rate of 3.0 +/- 0.57 microns/min. Continuous arteriolar photoactivation led to a vasoconstriction of 34.4 +/- 6.87% of the initial vessel diameter. Thirty percent of the maximal constriction occurred after 10.6 +/- 1.26 min of photoactivation. Constriction proceeded at a rate of 3.8 +/- 1.32 microns/min. Topically applied mefenamic acid (a cyclooxygenase inhibitor) and Nw-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) each enhanced both the arteriolar and the venular thrombus growth due to photoactivation. Photoactivation-induced arteriolar constriction was augmented by L-NAME and inhibited by mefenamic acid. These data suggest that the photoactivation of intravascular dyes is accompanied both by the release of nitric oxide, which inhibits thrombus development and arteriolar constriction, and by the release of cyclooxygenase products, which inhibit thrombus growth and induce vasoconstriction. Rats treated with busulfan to induce thrombocytopenia exhibited a 90% decrease in circulating platelets. In these animals, photoactivation caused significantly delayed thrombus growth in arterioles and venules, while arteriolar constriction remained unaltered, suggesting that the vasoconstrictor prostanoid is not of platelet origin.

Spontaneous arteriolar vasomotion and its relation to arterial pressure reduction was studied in the rabbit tenuissimus muscle using intravital microscopy. Vasomotion was observed in all transverse arterioles and their first-order side branches. Vasomotion frequency ranged from 5 to 32 cycles per minute (median: 25 cpm). The relative vasomotion amplitude in transverse arterioles varied from 0.06 to 0.44 (median: 0.22). Vasomotion was generally of the on-off type in first-order side branches. A gradual reduction in arterial pressure as achieved by aortic occlusion resulted in an abrupt disappearance of vasomotion. Vasomotion disappeared between 19 and 59 mm Hg (median: 30.4 mm Hg). No differences were found between the pressures at which vasomotion ceased in transverse arterioles and first-order side branches. With a further reduction of arterial pressure transverse arterioles attained a maximal diameter of 98-265% (median: 119%) of the maximal diameter during vasomotion. After release of occlusion vasomotion reappeared much earlier in the first-order side branches than in their feeding transverse arterioles. It is concluded that although vasomotion influences capillary perfusion, it does not play a major role in the adaptation of vascular resistance following a reduction in arterial pressure.

In unanesthetized bats direct microvascular pressure measurements were made in the tensor plagiopatagii muscle without surgical intervention. Pressure recordings showed great variability due to vasomotion and other physiological stimuli, but were in the same order of magnitude as described in muscle of other species in the anesthetized state. A substantial part of the pressure gradient is dissipated at the level of the small arterioles. Arterial pressure was changed by depressurizing a box containing the body of the bat, while the muscle was under atmospheric condition. In all larger arterioles and in 12 of 19 smaller arterioles a linear relation existed between microvascular pressure and box pressure. Of 19 small arterioles, 7 exhibited a clear biphasic regulatory response to a decrease in arterial pressure. The data are discussed in relation to different models of regulation.