The role of perfusion washout in limb revascularization procedures

Amputated rat hindlimbs were subjected to either normothermic (26 degrees C) or hypothermic (4 degrees C) ischemia. Experimental limbs had their microcirculation washed out (either before or after the ischemic insult) with a physiologic acellular plasma substitute previously reported to enhance flap survival following extended periods of warm ischemia. Control limbs were not washed out; i.e., stagnant blood remained in these limbs. Following the ischemic interval, amputated limbs were replanted. Monastral blue B, a colloidal pigment capable of labeling leaky blood vessels, was administered systemically to all rats just prior to vascular declamping. Limb biopsies of skin and muscle were harvested 30 minutes following revascularization in order to assess Monastral labeling and, therefore, the functional integrity of the microcirculation. Results confirm that stagnant blood under conditions of warm ischemia is detrimental to the functionality of the microcirculation in both skin (p less than 0.03) and muscle (p less than 0.007). Accordingly, perfusion washout, when performed prior to the ischemic period, enhances limb survival following 6 hours of warm ischemia (p less than 0.01). Hypothermia protects against the detrimental effects of stagnant blood; perfusion offers no benefit if hypothermic conditions prevail. Physiologic mechanisms responsible for these findings are discussed.     

Estimation of age-related changes in the regulation of peripheral blood flow in humans

The age-dependent features in the state of skin microvascular bed has been studied with laser Doppler flowmetry in healthy volunteers of different age groups. To reveal the reaction of skin blood flow in response to short-term ischemia, the occlusive test has been carried out. To estimate the contribution of rhythmic components to blood flow signal, continuous wavelet-transform spectral analysis was used. Age-dependent increase of pulse-wave amplitude and decrease of respiratory wave amplitude reflecting age-dependent changes in functioning of arteriolar and venular links of microvascular bed have been observed at rest. In response to short-term ischemia the age-dependent reduction of reserve resources has been revealed in functioning of arteriolar link of microvascular bed. The reduction of activity of myogenic, neurogenic and endothelial regulation systems have been shown at rest in ageing.     

Age-related differences in the dynamics of the skin blood flow oscillations during postocclusive reactive hyperemia

Age-related changes in peripheral microcirculation were studied using laser Doppler flowmetry in 60 apparently healthy subjects. The response of microcirculation to short-term ischemia was studied using the occlusion test. Changes in the amplitude of the peripheral blood flow oscillations were determined using time-amplitude analysis based on continuous adaptive wavelet filtration. The oscillation amplitude in the frequency range of the heart rate was found to reach the maximum with a delay after the removal of the occlusion, whereas in the range of the respiratory rhythm, no delay was observed. The hyperemic response to short-term ischemia is assumed to develop under the predominant influence of the arterial-arteriolar component, whereas the dynamics of amplitude oscillations in the range of the respiratory rhythm is a result of the devastation of the venular component after removal of occlusion. In response to short-term ischemia, the maximum oscillation amplitudes of myogenic, neurogenic, and endothelial rhythms decreased with age, which demonstrates the restriction of the regulatory control of the peripheral blood flow by the corresponding systems.     

The role of the coronary microcirculation in myocardial recovery from ischemia.

The aim of thrombolysis, angioplasty, and coronary artery bypass surgery is to "reperfuse" ischemic myocardium; however, reperfusion can cause further cardiac damage and compromise the coronary microcirculation. Because nutrient supply and exchange and delivery of pharmacologic agents require a patent microvasculature, the coronary microcirculation plays a major role in myocardial recovery from ischemia. It is known that ischemia-reperfusion can cause an increase in coronary permeability and microvascular plugging (No-reflow). The permeability to macromolecules is increased more than the permeability to smaller molecules. The permeability increase leads to extravasation of plasma proteins and a permeability edema. Furthermore, proteins that normally remain extravascular are now free to wash out the heart. Both microvascular effects, increased coronary permeability and No-reflow, compromise cardiac function. The degree of damage depends on the nature (No-flow versus low-flow) and length of ischemia. Unfortunately, both the increase in coronary permeability and the reduction in perfused capillarity advance with time during early reperfusion. Although the increase in permeability does not require the presence of platelets or leukocytes, it is apparent that the No-reflow response does. Mechanisms that may explain the microvascular responses to ischemia include cell swelling, damage caused by oxygen free radicals, and inflammatory responses that may or may not involve granulocytes. The permeability response may involve a calcium-mediated endothelial contraction because the macromolecular leakage that follows ischemia can be prevented by pretreating hearts with the calcium blocker nisoldipine. Protection of the coronary microcirculation should be included in any attempt to improve treatment of occlusive coronary artery disease.     

A comparative study of reactive hyperemia in human forearm skin and muscle

Reactive hyperemia (RH) in forearm muscle or skin microcirculation has been considered as a surrogate endpoint in clinical studies of cardiovascular disease. We evaluated two potential confounders that might limit such use of RH, namely laterality of measurement and intake of non-steroidal anti-inflammatory drugs (NSAIDS). Twenty-three young non-smoking healthy adults were enrolled. In Experiment 1 (n=16), the RH elicited by 3 min of ischemia was recorded in the muscle (strain gauge plethysmography, hand excluded) and skin (laser Doppler imaging) of both forearms. In Experiment 2 (n=7), RH was determined in the dominant forearm only, one hour following oral acetylsalicylic acid (1 g) or placebo. In Experiment 1, peak RH was identical in both forearms, and so were the corresponding durations of responses. RH lasted significantly less in muscle than in skin (p=0.003), a hitherto unrecognized fact. In the skin, acetylsalicylate reduced duration (43 vs. 57.4 s for placebo, p=0.03), without affecting the peak response. In muscle, duration tended to decrease with acetylsalicylate (21.4 vs. 26.0 s with placebo, p=0.06) and the peak increase in blood flow was blunted (27.2 vs. 32.4 ml/min/100 ml tissue with placebo, p=0.003). We conclude that, when using RH as a surrogate endpoint in studies of cardiovascular disease, a confounding by laterality of measurement need not be feared, but NSAIDS may have an influence, although perhaps not on the peak response in the skin.     

Reliable assessment of skin flap viability using orthogonal polarization imaging

Intraoperative evaluation of skin flap viability has primarily been dependent on clinical judgment. The purpose of this study was to determine whether an orthogonal polarization spectral imaging device could be used to accurately predict viability of random-pattern skin flaps. Orthogonal polarization spectral imaging is a newly developed technique that visualizes the microcirculation using reflected light without the use of fluorescent dyes and allows for noninvasive real-time observation of functional microvascular networks. In Sprague-Dawley rats (n = 24), three types of random skin flaps were designed with unknown zones of viability (n = 8 per group). After flap elevation, the skin flaps were evaluated by both clinical examination and orthogonal polarization spectral imaging. Areas of the flap determined to be nonviable by clinical examination were measured and marked. Orthogonal polarization spectral imaging was subsequently performed, and areas of the skin flap with stasis (i.e., cessation of red blood cell movement) in the dermal microcirculation on orthogonal polarization spectral imaging were measured and marked. The skin flaps were then secured in place. Flaps were evaluated on a daily basis for clinical signs of ischemia and necrosis. On postoperative day 7, the total amount of random skin flap necrosis was measured and recorded. Clinical examination of the random skin flaps significantly underestimated the actual amount of eventual flap necrosis, and as result was a very poor predictor of flap necrosis. By contrast, assessment of microcirculatory stasis using the orthogonal polarization spectral imaging device correlated well with the subsequent development of necrosis in all groups. In the three groups, the average amount of flap necrosis predicted by clinical examination deviated from actual necrosis by approximately 2 to 4 cm. However, the amount that orthogonal polarization spectral imaging differed from actual necrosis was 0.1 to 0.3 cm. Therefore, orthogonal polarization spectral imaging was an excellent predictor of eventual flap necrosis and much more accurate than clinical observation (p < 0.001). Intraoperative evaluation of axial and random pattern flap viability has traditionally been based on clinical examination as no other reliable, convenient test currently exists. The authors demonstrated that an orthogonal polarization spectral imaging device accurately predicts zones of necrosis in random pattern flaps by directly visualizing cessation of microcirculatory flow. Intraoperative stasis in the dermal microcirculation correlated precisely with subsequent flap necrosis. Orthogonal polarization spectral imaging was significantly more accurate than clinical examination, which consistently underestimated flap necrosis. The orthogonal polarization spectral imaging technique may have value in the intraoperative assessment of skin flap perfusion such as that required after skin-sparing mastectomy.     

Microcirculation characteristics in apparently healthy subjects during acute hypoxia and intermittent hypoxic training

The characteristics of the microcirculation in the forearm skin of 29 apparently healthy male volunteers were studied in acute hypoxia and during intermittent hypoxic training (IHT) using computer laser Doppler flowmetry. It was shown that short-term exposure of apparently healthy subjects to simulated acute hypoxia optimized the microcirculation owing to sympathetic innervation and concomitant rearrangement of microvascular tone regulation (activation of skin perfusion and reduction of blood flow through arteriovenular shunts when the neurogenic tone component increases). A second (placebo-controlled) series of experiments showed that long-term hypoxic preconditioning (20 IHT sessions) facilitated fixing of the adaptive dynamic rearrangements aimed at microcirculation improvement. The microvasculature response during acute hypoxia and a course of IHT depends on the initial sensitivity of subjects to simulated hypoxia. Significant perfusion enhancement in the tissues studied and microvascular tone normalization were observed in the subjects that were initially sensitive to hypoxia.     

Effects of thermal factors on the state of microcirculation of the small intestine in acute ischemia

The influence of normo- (38 degrees C), hyper- (42 degrees C) and hypothermia (20 degrees C) on microcirculatory disturbances caused by acute local ischemia of the small intestine was investigated with the help of biomicroscopy as well as morphological methods. Ischemia was modeled by ligation of the intestine look eventrated through the abdominal wall incision of a rat onto the microscope stage for 1 h. It was shown that hyperthermia intensified microcirculatory disorders and stimulated destructive processes in tissues and hypothermia promoting microcirculation and decreasing metabolism and restrained the development of these processes. Important peculiarity of the microvascular response to ischemia, hyper- and hypothermia was revealed: heterogeneity of the reaction of different parts of microvascular bed. Appropriate evaluation of the microcirculation state in such conditions can be obtained taking into account not only the qualitative character of microvascular reaction but also an extent of this reaction manifestation in different parts of microvascular bed.     

硒对自由基损伤的红细胞引起微循环紊乱的保护作用--动物实验及临床研究

为研究硒保护实验动物大脑表面微循环免受自由基损伤红细胞的干扰作用,以及术前服硒对心脏手术病人在体外循环过程中皮肤和肌肉微循环的保护效果,在动物实验中利用荧光标记的图象分析法观察大鼠脑微小动静脉直径,流速和通透性的改变,并在临床观察中用激光多普乐法对服硒组心脏手术病人皮肤和肌肉微循环的变化与对照组进行比较研究。     

Direct evidence of the vasodilator action of carbon dioxide on subcutaneous microvasculature in rats by use of intra-vital video-microscopy

The therapeutic effects of carbon dioxide (CO₂) on cutaneous tissue blood flow in the human have long been well recognized. Although CO₂ has vasodilator action, in-vivo evidence of its action on the microcirculation of the skin, and of its mechanism, has rarely been reported. We studied the direct effects of CO₂ on in-vivo microvasculature and blood flow rate by using an intra-vital video-microscopic system. Brown Norway rats were anesthetized by intraperitoneal administration of alpha-chloralose and urethane. In order to measure inner diameter and red blood cell velocity (Vrbc) for a microvessel, the dorsal skin window was draped on an observation box placed inside a bath. Vrbc was derived from the cross-correlation function of paired segments of dual-window intensity in the video of microvascular images of the subcutaneous tissue. We measured pH in subcutaneous tissue by making a dorsal skin tube. After topical application of CO₂ dissolved in water via the skin of the rat, we observed both vasodilatation and an increase in blood flow of the micro vessels. The pH of subcutaneous tissue also decreased after CO₂ application. The CO₂ reduced the pH of subcutaneous tissue and inhibited vascular smooth muscle contraction, resulting in dilatation of the vasculature of the skin microcirculation.     

Microscopic instrumentation and analysis of laser-tissue interaction in a skin flap model

A dorsal skin flap model for microcirculatory studies has been modified for "in vivo" studies of laser-tissue interaction with microcirculation. An experimental apparatus has been built implementing a laser delivery system, video microscopy during irradiation, and thermal recordings. This model has been used to study irradiation effects on microcirculation using the argon laser (488 and 514.5 nm) and the argon pumped dye laser at 577 nm. The results include: measurements of the optical properties of the model; dosimetry measurements for the production of embolized and stationary coaguli in arterioles and venules; and focal vessel disappearance of venules irradiated with the argon or the argon pumped dye laser at 577 nm; a method to determine light attenuation in the model; a unique method for measurements of blood flow velocity in arterioles and venules and measurements obtained with this method; measurements of transient and steady state temperatures during irradiation and a study of laser induced photorelaxation phenomena in venules.     

An experimental neurovascular island skin flap for the study of the delay phenomenon.

We present an experimental neurovascular island skin flap. It is a consistent, reproducible model which produces a definite pattern of surviving skin flap area versus skin flap necrosis. There is a constant, anatomically definable nerve and vascular supply to the flap. This model permits independent experimental manipulation of the neural, arterial, and venous supply to the skin. It is useful, therefore, for the study of the vascular mechanisms of the skin microcirculation. We also demonstrated that increased flap survival can be produced by a delay involving denervation alone (leaving the vascular supply intact) or by devascularization alone (leaving the nerve supply intact). We conclude that both the adrenergic denervation and the ischemia contribute to the production of the delay phenomenon. We suggest that sustained vasodilation--vascular smooth muscle relaxation--is the vascular mechanism that accounts for the delay phenomenon.     

Transmural difference in coronary arteriolar dilation to adenosine: effect of luminal pressure and K(ATP) channels

Coronary blood flow in the subendocardium is preferentially increased by adenosine but is redistributed to the subepicardium during ischemia in association with coronary pressure reduction. The mechanism for this flow redistribution remains unclear. Since adenosine is released during ischemia, it is possible that the coronary microcirculation exhibits a transmural difference in vasomotor responsiveness to adenosine at various intraluminal pressures. Although the ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in coronary arteriolar dilation to adenosine, its role in the transmural adenosine response remains elusive. To address these issues, pig subepicardial and subendocardial arterioles (60-120 micrometer) were isolated, cannulated, and pressurized to 20, 40, 60, or 80 cmH(2)O without flow for in vitro study. At each of these pressures, vessels developed basal tone and dilated concentration dependently to adenosine and the K(ATP) channel opener pinacidil. Subepicardial and subendocardial arterioles dilated equally to adenosine and pinacidil at 60 and 80 cmH(2)O luminal pressure. At lower luminal pressures (i.e., 20 and 40 cmH(2)O), vasodilation in both vessel types was enhanced. Enhanced vasodilatory responses were not affected by removal of endothelium but were abolished by the K(ATP) channel inhibitor glibenclamide. In a manner similar to reducing pressure, a subthreshold dose of pinacidil potentiated vasodilation to adenosine. In contrast to adenosine, dilation of coronary arterioles to sodium nitroprusside was independent of pressure changes. These results indicate that coronary microvascular dilation to adenosine is enhanced at lower intraluminal pressures by selective activation of smooth muscle K(ATP) channels. Since microvascular pressure has been shown to be consistently lower in the subendocardium than in the subepicardium, it is likely that the inherent pressure gradient in the coronary microcirculation across the ventricular wall may be an important determinant of transmural flow in vivo during resting conditions or under metabolic stress with adenosine release.     

Effects of poorly perfused peripheries on derived transit time parameters of the lower and upper limbs.

A simple and non-intrusive approach termed the pulse transit time ratio (PTTR) has recently been shown to be a potential surrogate of the ankle-brachial index (ABI). PTTR is based on the principle of PTT, which is known to be temperature-sensitive. In this study, 23 healthy adults with normally perfused peripheries and 10 with poorly perfused peripheries were recruited. No significant change in PTTR was observed between those with cold (1.287+/-0.043) and normal (1.290+/-0.027) peripheries (p>0.05). A cold periphery may cause pulse waveform changes and indirectly affect PTT owing to poor skin microcirculation, but may have a limited effect on PTTR, which is useful as an ABI alternative.     

Study of endothelium-dependent blood oscillations in the human skin microcirculatory bed

Cutaneous microcirculation parameters were studied with laser Doppler flowmetry in healthy volunteers. To investigate endothelial-dependent peripheral blood flow oscillations the iontophoresis of 1% acetylcholine solution was carried out. To estimate the contribution of rhythmical components in blood flow signal the continuous wavelet-transform spectral analysis was used. To reveal correlation between microcirculation parameters under study the correlation analysis was used. The microcirculation index was shown to be the factor producing cross-correlation dependences. The only positive significant correlation between the blood flow oscillation amplitude in the range of endothelial activity normalized to mean microcirculation index at rest and maximal microcirculation index during the iontophoresis of acetylcholine was revealed.     

Evidence for non-neurotensin receptor-mediated effects of xenin (1-25)--focus on intestinal microcirculation

Xenin (1-25) has been detected in various locations in mammalians. It has structural similarities with neurotensin and its intestinal effects are claimed to be mediated by neurotensin receptors. It has been shown to influence gastrointestinal motility. The effects of xenin (1-25) on intestinal microvascular perfusion after ischemia/reperfusion have not been investigated yet. Therefore, the superior mesenteric artery was clamped for 40 min in Wistar rats (n=8). Ten minutes prior to reperfusion, intravenous infusion of xenin (1-25) (5 nmol/kg/h) was started. By means of intravital microscopy, microvascular perfusion in the mucosal layer was assessed. Animals (n=8) with and without clamping of the superior mesenteric artery and infusion of the carrier solution served as controls.After ischemia/reperfusion, xenin (1-25) increased the density of perfused microvessels and the capillary red blood cell velocity compared to ischemic controls. Capillary red blood cell velocity was elevated (p<0.05). Xenin (1-25) improved the heterogeneous distribution of mucosal blood flow during reperfusion demonstrated by an increase of both the perfusion index and the percentage of perfused microvessels.We conclude that the effects of xenin (1-25) on intestinal microcirculation are significantly different from those previously described for neurotensin. A more complex effector mechanism must be postulated that may involve other regulatory peptides and receptors.     

Pathomechanism of chronic venous insufficiency and leg ulcer

Uniform view of chronic venous diseases has been formed in the last 3 decades. Chronic venous insufficiency (CVI) is a functional disorder of the venous system of the lower limb. The basis of the pathology is always the venous hypertension caused by valvular insufficiency and reflux with or without venous outflow obstruction. Epifascial, subfascial and transfascial forms of CVI can be distinguished. In the practice these forms are almost always combined. The consistent venous hypertension is the initiating factor in alterations in the microcirculation which leads to skin changes and venous ulceration. The precise mechanism of the development of venous leg ulcer is still uncertain. A recent hypothesis suggests that leukocytes are trapped in the capillaries and attaching to the endothel they become activated and release proteolytic enzymes, free radicals which have destructive effects on lipid membranes, proteins as well as on many connective tissue compounds. The endothelium plays active role in the complex mechanism. Increased expression of tissue metalloproteinases has been observed in the periulcer skin. The presence of perivascular leukocyte infiltration and fibrin cuff is a reflexion of an inflammatory process. The clinical stages of CVI are likely to be the results of a systemic inflammatory response to a period of venous hypertension.     

In situ assessment of the brain microcirculation in mechanically-ventilated rabbits using sidestream dark-field (SDF) imaging

Assessment of the cerebral microcirculation by on-line visualization has been impossible for a long time. Sidestream dark-field (SDF) imaging is a relatively new method allowing direct visualization of cerebral surface layer microcirculation using hand-held probe for direct contact with target tissue. The aim of this study was to elucidate the feasibility of studying the cerebral microcirculation in situ by SDF imaging and to assess the basic cerebral microcirculatory parameters in mechanically ventilated rabbits. Images were obtained using SDF imaging from the surface of the brain via craniotomy. Clear high contrast SDF images were successfully obtained. Total small-vessel density was 14.6+/-1.8 mm/mm(2), total all-vessel density was 17.9+/-1.7 mm/mm(2), DeBacker score was 12.0+/-1.6 mm(-1) and microvascular flow index was 3.0+/-0.0. This method seems to be applicable in animal studies with possibility to use SDF imaging also intraoperatively, providing unique opportunity to study cerebral microcirculation during various experimental and clinical settings.     

Pathophysiology of ischemic skin flaps: differences in xanthine oxidase levels among rats, pigs, and humans

Oxygen-derived free radicals have been implicated in a variety of diseases and pathologic processes, including ischemia reperfusion injury (IRI). Based on experimental work with rat skin-flap models, the enzyme xanthine oxidase (XO) has been proposed as a major source of free radicals responsible for tissue injury and flap necrosis. The presence of this enzyme is variable within different tissues of a specific species and between species. Xanthine oxidase levels in pig and human skin have not previously been reported. The activity of xanthine oxidase in the skin of rats (N = 16), pigs (N = 7), and humans (N = 8) was measured after varying intervals of ischemia and in the rat also following reperfusion. Control pig and human skin were found to contain minimal enzyme activity, almost 40 times less than that of the rat. In the rat, xanthine oxidase activity was stable throughout a prolonged period of ischemia, and a significant decrease in activity was found after 12 hours of reperfusion (p less than 0.05). In humans, xanthine oxidase activity was unaffected by ischemia time, and in the pig, it did not increase until 24 hours of ischemia (p less than 0.05). The potential sources of free radicals and the mechanism of action of xanthine oxidase and its inhibitor allopurinol in improving flap survival in different species are reviewed.     

Effects of opioid peptide dalargin on reparative processes in wound healing

At intraperitoneal injection and local application of opioid peptide dalargin induces fibroblast proliferation (3-fold increase in the mitotic index) and growth of capillaries, accelerates the maturation of granulation tissue and of scar, epitheliazation of the defect, and considerably reduces the period of healing of skin wound in rats. The stimulating action of dalargin is associated with its effect on the microcirculation system and activation of the macrophage-fibroblast interaction. Possessing the triggering mechanism, the drug induces a cascade of inflammatory-reparative reactions, which reduce the duration of all healing stages.