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Multiframe particle tracking in intravital imaging: defining Lagrangian coordinates in the microcirculation.

The cellular composition of the microcirculation creates blood flow that can be unsteady and nonuniform. To obtain information about nonuniform cellular trajectories, we describe in vivo imaging techniques that provide both detailed tracking of individual particles as well as an approach to simultaneous multicolor particle tracking. Particularly relevant to biologic systems, Lagrangian methods provide information about the fate of individual particles and flow in the system.

Animals↗

Effect of metabolic acidosis on pulmonary microcirculation in the cat.

Using a new X-ray TV system, we studied the effects of metabolic acidosis, produced by intravenous injection of lactic acid, on internal diameter (ID; 100-500 micron), flow velocity, and volume flow in pulmonary microcirculation of anesthetized cats. We found that metabolic acidosis decreased the ID of arteries by 21%, whereas it increased flow velocity by 28% but not volume flow.

Acidosis, Lactic↗

Selective and tone-dependent vasodilation of arterial segment in rabbit and feline pulmonary microcirculation in response to ANP.

Using an X-ray television system, we directly measured the changes in internal diameter (ID; 100-600 microns) of small pulmonary arteries and veins in response to intravenous infusion of atrial natriuretic peptide (ANP) in anesthetized rabbits and cats. Under physiological conditions, ANP (0.01-1 micrograms/(kg.min) for 3 min) increased the ID of the small arteries in a dose-dependent manner by 4-9% and by 6-14% in the rabbit and cat, respectively. The maximum increase in ID occurred in the 400-600 microns arteries. In the small veins, however, the peptide had no significant effects on the ID. To simultaneously investigate the ANP-induced responses of pulmonary vessels with different vascular tone in a given lung region, we embolized a part of the rabbit pulmonary vascular tree with micro-beads (80-100 microns diameter) and measured ID responses to ANP (1 microgram/(kg.min) for 3 min) in the embolized vessels with elevated tone and the nonembolized vessels with baseline tone. In the embolized arteries, which had constricted by 31%, ANP induced 27% increase in ID, while in the nonembolized arteries it caused 8% increase in ID. This indicated that in the small arteries, the vasodilator activity of ANP is greatly enhanced under the conditions of elevated vascular tone. In the small veins, however, no significant vasodilation occurred even under the elevated-tone conditions. We concluded that ANP dilates the arterial segments in the rabbit and cat pulmonary microcirculation selectively and in a dose-dependent manner. Furthermore, the pulmonary vasodilator effect of ANP was dependent on the pulmonary vascular tone.

Animals↗

Involvement of superoxide in acute reaction of angiotensin II in mesenteric microcirculation.

Superfusion of angiotensin II (Ang II) ceased blood flow in rat mesenteric microcirculation, however, successive reflow occurred. When nitric oxide synthase inhibitor was present, the stoppage of flow occurred by the lower concentration of Ang II. Superoxide dismutase (SOD) significantly delayed the stoppage by Ang II and restored the successive reflow earlier. The acute reaction between Ang II and mesenteric artery induced immediate superoxide (O(2)(-)) production when observed by a chemiluminescence method using the Cypridina luciferin analog. The acute vascular O(2)(-) production on the addition of Ang II contributed to in vitro vascular contraction as it was significantly attenuated by SOD. The acute superoxide-producing effect is likely to be specific to Ang II because such significant modification by SOD was not observed for norepinephrine.

Angiotensin II↗

Platelet aggregation in the cerebral and mesenteric microcirculation of mice with genetically determined diabetes.

Platelet aggregates were induced in pial arterioles of the following strains of mice with a genetic predisposition for diabetes: ob/ob and db+/db+. Aggregation was compared with that in 6J+/+ mice, the nondiabetic controls for ob/ob animals, and in +m/+m as well as db+/+m, the nondiabetic controls for db+/db+ mice. Aggregation was also induced within mesenteric arterioles of db+/db+ animals and compared with that in db+/+m mice. Aggregation was monitored microscopically, by measuring the time required for a noxious stimulus to initiate aggregation in an injured arteriole. Platelet aggregation was initiated with equal ease in the pial arterioles of ob/ob mice and their 6J+/+ controls. However, the onset of aggregation in the pial arterioles of the db+/db+ group was significantly delayed when compared with the onset in either of the nondiabetic control groups, +m/+m or db+/+m. A similar prolongation in the time required to produce aggregation was also observed in the mesenteric arterioles of the db+/db+ mice when compared with db+/+m controls. The basis for reduced platelet aggregation in the microcirculation of db+/db+ mice is not explained. The results differ from those showing enhanced aggregation in many in vitro studies of platelets from human diabetics and from those of in vivo studies of two other animal models described in the literature. However, not all published studies have reported enhanced aggregation. The delayed aggregation in the present study may provide a basis for analysis of factors that regulate platelet aggregation in diabetes.

Animals↗

Disturbances of cutaneous microcirculation in patients with diabetic legs: additional parameters for a new therapeutic concept?

To study disturbed microcirculation involved in the pathogenesis of diabetic neuropathic plantar ulcers (DNPU), we recorded dynamic changes in laser-Doppler flux (LDF) and cutaneous oxygen tension (pcuO2) caused by short-time arterial occlusion and local heating at three different sites (forefoot, ulcer edge, lower leg) in patients (n = 14) with DNPU and healthy controls (n = 18). Significantly reduced dynamic pcuO2 parameters coincided with a significant increase of flux in the patient group. This post-stimulatory "hypoxic hyperemia" indicates a shifting of blood flux, reducing circulation of the nutritive capillaries comparable to an internal "steal effect". This may predispose to the development of DNPU when additional stresses influence the initial borderline balance, characterized by similar pcuO2 and increased flux values compared to controls. Abolishment of normal vasoconstriction in the shunt vessels by diabetic polyneuropathy is the assumed cause of increased arteriovenous perfusion and therefore raised flux values. Non-invasive testing of microcirculatory functions demonstrates characteristic disturbances in DNPU patients and could be used as additional parameters for new therapeutic concepts as the intravenous retrograde perfusion (RVP). After RVP therapy, applied to a subgroup (n = 7) of the patients, some dynamic microcirculatory parameters improved, allowing a preliminary quantitative evaluation of a therapeutic regimen.

Adult↗

Inflammatory responses to ischemia and reperfusion in the cerebral microcirculation.

Ischemia and reperfusion (I/R) has been shown to elicit an inflammatory response that is characterized by an increased production of reactive oxygen species, and the rolling, firm adhesion, and transendothelial migration of leukocytes in postcapillary venules. A rate-determining role for leukocyte-endothelial cell (L/E) adhesion in the initiation and propagation of reperfusion injury is supported by several reports that describe attenuated microvascular dysfunction and tissue injury following I/R in animals receiving neutralizing antibodies directed against certain leukocyte adhesion receptors and in mutant mice that are genetically deficient in these adhesion receptors The technique of intravital videomicroscopy has been applied to several tissues, including the brain, in order to directly observe the microcirculatory alterations and inflammatory responses that are elicited by I/R. The leukocyte- and platelet-endothelial cell adhesion responses to cerebral I/R are addressed and compared to responses observed in other postischemic vascular beds. The limited data available for the brain microcirculation support the potential of anti-leukocyte and anti-platelet strategies for stroke therapy.

Animals↗

The tracheal mucosa microvasculature and microcirculation. Intravital microscopic observations in rabbits and a histologic study in man.

A method for the intravital microscopic study of the tracheal mucosal microcirculation in the rabbit is described. A low tracheostomy is performed for ventilation, while a hole cut in the ventral portion of the trachea cranial to the tracheostomy is used for observations. The trachea is immobilized by a fixation device. Microscopic observations are made with a Leitz stereo microscope. The microvasculature of the rabbit tracheal mucosa is characterized by centripetally arranged arterioles which subdivide into a fairly sparse capillary network at the mucosal surface. The capillaries drain into venular networks at different levels of the mucosa and collect finally into circumferentially arranged veins which run mainly between the cartilages. For comparative purposes the microvasculature of the human tracheal mucosa has been analysed in clarified (Spalteholz) preparations. This investigation shows that the microvascular architecture is principally the same in the human and rabbit tracheal mucosa. There is, however, a much denser capillary network in the human tracheal mucosa.

Adult↗

Distribution of flow and red cell flux in the microcirculation.

The distribution of blood cells in the vascular system is not homogeneous, red cell concentration in the small vessel compartment being significantly lower than in the large vessel compartment. This is to some extent due to the difference in travelling speed between cells and plasma in the microcirculation (Fahraeus effect). However, even in the terminal vessel network haematocrit varies considerably as a result of plasma skimming phenomena. Thus, local (capillary) haematocrit is a function of flow rate distribution at bifurcations. Red cell concentration in the majority of capillaries is substantially lower than in any other vessel category, while a small number of capillaries carry an increased haematocrit, thereby satisfying mass balance. The haematocrit distribution as well as the average level of capillary haematocrit is a function of small vessel volume which in turn depends on pre-capillary resistance: vasodilation leads to increased capillary haematocrit and vice versa. Thus, one of the physiological functions of the resistance vessels is to determine the level of microvascular haematocrit. Alteration of red cell microrheology leads to a reduction of capillary O2-transport capacity mainly by reducing the haematocrit in nutritive capillaries.

Animals↗

Changes in gastric mucosal microcirculation and purine nucleotide metabolism during hemorrhagic shock in rats.

Intravital fluorescence microscopy and morphometry were used to study the microcirculation in the rat gastric mucosa during and after hemorrhagic shock. Under control conditions the circulation appeared homogeneous and unaffected by superfusion with 0.1 N HCl. During hemorrhagic shock, scattered areas of the mucosa lost circulation. Morphometry showed that the number of perfused mucosal vessels decreased significantly in the abluminal part of the mucosa both in perfused and ischemic areas, the reduction being most pronounced in the ischemic areas, where the number of perfused luminal vessels also decreased significantly. During reperfusion, bleedings occurred from the mucosa. A key finding was that the bleedings always had their origin at the border zone between ischemic and perfused areas. After hemorrhagic shock adenosine triphosphate and energy charge levels dropped significantly in both perfused and ischemic areas but with significantly lower levels in the ischemic areas. The hypoxanthine levels increased in both perfused and ischemic areas. The experiments show that local mucosal ischemia and accelerated nucleotide degradation are of pathogenetic importance in the development of stress ulcers after hemorrhagic shock. The border zone between circulated and ischemic areas seems to be an area of special interest.

Animals↗

Gastric mucosal microcirculation and purine nucleotide metabolism after retransfusion of rats in hemorrhagic shock.

Gastric mucosal microcirculation and purine nucleotide metabolism were studied after hemorrhagic shock and retransfusion in rats. The number of perfused microvessels and the concentration of adenosine triphosphate, adenosine monophosphate, inosine monophosphate, inosine, hypoxanthine, xanthine, and uric acid were investigated in mucosal biopsy specimens after 15 or 45 min of hemorrhagic shock and after 15 min of shock followed by 30 min of retransfusion. During shock a dephosphorylation of nucleotides and a decrease in the number of perfused microvessels occurred, the more the longer the duration of the shock period. Retransfusion led to an additional reduction in the number of perfused microvessels, but there was a partial restoration of high-energy phosphate metabolites in those areas of the mucosa which maintained a blood flow. The results indicate that there is a renewal of energy stores in gastric mucosa after hemorrhagic shock and reperfusion, although parts of the vascular bed are not reperfused.

Animals↗

Tumour microcirculation as a target for hyperthermia.

A great number of investigators have, independently, shown that tumour blood flow is affected by a hyperthermic treatment to a larger extent than normal tissue blood flow. While the majority of the studies on experimental tumours show a decrease and even a lapse in blood flow within the microcirculation during or after hyperthermia, the data on human tumours are less conclusive. Some of the investigators do not find a decrease in circulation, while others do. Obviously, this is an important field of investigation in the clinical application of hyperthermia because a shut down of the circulation would not only facilitate tumour heating (by reducing venous outflow, this reducing the 'heat clearance' from the tumour), but would also facilitate tumour cell destruction. The same holds for alterations that occur subsequently to the circulatory changes, like a heat-induced decrease of tissue pO2 and pH. If the frequently reported circulatory collapse of the tumour circulation could selectively be stimulated by, e.g. acidification or by vasoactive agents, hyperthermic treatment of patients would possibly be greatly facilitated and intensified. In hyperthermic tumour therapy a number of complex processes and interactions takes place, especially when the treatment is performed in combination with radiation therapy. One of them represents the group of processes related to the random probability of cell sterilization of individual tumour cells resulting in exponential survival curves which are typically evaluated with e.g. cell survival assays. This aspect has not been the issue of this paper. The other group of processes deals with the heat-induced changes in the micro-physiology of tumours and normal tissues which, as discussed before, may not only enhance the exponential cell kill, but which may also culminate in vascular collapse with the ensuing necrosis of the tumour tissue in the areas affected. If this takes place, a process of bulk killing of tumour cells results, rather than the random type of cell sterilization. At present it is not clear to what extent the various separate mechanisms contribute to the total effect of tumour control. With all these considerations in mind, one should be aware of the fact that effects, secondary to heat-induced vascular stasis alone will never be efficient enough to eliminate all tumour cells, even though a heat reservoir is created. This is so because some malignant cells will inevitably have already infiltrated normal, surrounding structures and will therefore not be affected by changes in the tumour vascular bed.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Garlic preserves patency and delays hyperthermia-induced thrombosis in pial microcirculation.

Three trials were carried out to study the effect of garlic on thrombus formation and patency in the mouse pial microcirculation in response to hyperthermia. Two different hyperthermic exposures, at 43 degrees C for 60 min and at 44 degrees C for 45 min, were applied to the brain surface of anaesthetized mice by heated artificial cerebrospinal fluid (ACSF). Garlic solutions were prepared 24 h prior to their injection, i.p., from a finely-ground powder in saline (pH 7.3) to deliver doses of 25, 50 and 75 mg/kg. Control groups of the three trials were injected with saline, pH 7.3. Garlic and vehicle solution injections were made 60 min prior to the intended hyperthermic exposure. Microvascular responses were monitored and were recorded by intravital videomicroscopy. With core body temperature kept at 37 degrees C and at the elevated ACSF temperatures, the first observed intravascular response was in the form of either passing emboli or as visible thrombosis in either arterioles or venules. Further thromboembolic events continued and throughout such exposures higher arteriolar patency was evident in the garlic-treated mice. Collectively, garlic significantly delayed the appearance of the first observable thrombo/embolic response. Data of this study evidenced that garlic delayed hyperthermia-induced platelet aggregation, in vivo. Such results could prove beneficial to those adversely affected by antithrombotic drugs, like aspirin.

Animals↗

Reactivity of the microcirculation in port wine stain hemangiomas evaluated by laser Doppler fluxmetry.

Twelve patients with port wine stains (both untreated and treated with argon laser) had their microcirculation monitored by laser Doppler flux at rest and after arterial occlusion lasting one minute. The blood cell flux was measured in normal, untreated and treated skin areas. The resting flux was significantly higher in untreated port wine stains than in the normal skin. The reactive hyperemia response was significantly impaired in the untreated skin, although a 60% increase in blood cell flux was achieved by the short arterial occlusion. In most of the recorded parameters the treated areas showed a less pathological reaction than the untreated ones. We conclude that peroperative vasodilation can be induced in port wine stains, and this may be used to improve the outcome of argon laser treatment.

Adult↗

Measurements of blood flow velocity in the microcirculation.

A method for the measurement of red blood cell velocity in the microcirculation is described. The technique is based on the on-line cross-correlation of the delay between optical signatures derived from photodetectors aligned in direction oflow. A specialised photodiode holder which can be inserted directly in place of an ocular is described. The interdetector delay data is computed automatically from the cross-correlation function by a peak detector designed to provide a voltage proportional to the delay to maximum cross-correlation, regardless of intermediate lower peaks. Application of this system to measurements in the rabbit ear chamber, mesentery and skeletal muscle is described.

Blood Flow Velocity↗

Functional study of the dermal microcirculation in systemic sclerosis.

To measure the effect of cooling on digital blood pressure we used a strain-gauge and photoplethysmograph, with an automatic cooling device. Eighteen patients were compared with 18 matched controls. Laser-doppler technique was used to measure the perfusion changes after heating a small area of the skin in 5 locations on the body, readings were given in perfusion units. Eighteen patients with systemic sclerosis were compared with 25 individuals with primary Raynaud's phenomenon and 30 healthy controls. Cooling to 10 degrees C caused a significant digital blood pressure drop of 58 mmHg in patients with systemic sclerosis and 61 mmHg in Raynaud's phenomenon, as compared with controls. The microcirculation in patients with systemic sclerosis responded in the same way to local heating as in the normal population, increasing the perfusion to the same extent. In conclusion, an unselected group of patients with systemic sclerosis have normal dermal microcirculatory response to heating in spite of severe cold intolerance.

Adaptation, Physiological↗

The interconnection between sympathetics, microcirculation, and insulin resistance in hypertension.

The pathophysiology of the frequent association of insulin resistance and hypertension has not been elucidated. The skeletal muscle is the major site of insulin resistance; when stimulated with insulin, the hypertensive skeletal muscles extract less glucose than the normotensive. We postulate that hypertension-related changes in the skeletal muscle microcirculation contribute to the impaired glucose uptake in hypertension. Vascular rarefaction in hypertension impairs the delivery of insulin and glucose to muscle cells. Insulin resistance has been described both in human and experimental hypertension and both conditions are associated with vascular rarefaction. Functional studies (response to whole body or forearm exercise) and anatomic investigations (conjunctival photography, mesenteric and muscle biopsies) show vascular rarefaction in human hypertension. In addition, patients with hypertension are known to have a larger proportion of insulin resistant, poorly vascularized fast twitch muscle fibers. A few interventions can increase or decrease insulin resistance and these effects can be explained on hemodynamic grounds. Beta adrenergic blocking agents aggravate insulin resistance, and their main hemodynamic effect is a decrease of cardiac output. Converting enzyme inhibitors, alpha adrenergic blocking agents and possibly calcium antagonists decrease the insulin resistance, and their major hemodynamic effect is vasodilation. Physical training decreases insulin resistance; a higher capillary density in skeletal muscles is the hallmark of physical training. A hypothesis ought to rest on sufficient supporting data and its validity ought to lend itself to experimental verification. We believe our hypothesis meets both criteria. After outlining the supporting evidence we propose a number of tests to prove or disprove the hypothesis. In addition to the testable hypothesis we also speculate on the possible cause of the frequent association between hypertension and insulin resistance. We propose that both insulin resistance and blood pressure elevation represent a facet of the "defense reaction" which might have offered an early survival advantage and may, over evolutionary times, have fostered natural selection of subjects with both conditions.

Animals↗

Diaspirin crosslinked hemoglobin: evaluation of effects on the microcirculation of striated muscle.

Hemoglobin-based oxygen carriers such as diaspirin-crosslinked hemoglobin (DCLHb) have been proposed for blood substitution due to their plasma expansion and oxygen transport capacity. This study investigates the effects of DCLHb on the microcirculation of striated muscle after moderate topload infusion and isovolemic exchange transfusion in awake hamsters. The skinfold chamber model in hamsters and intravital fluorescence microscopy were used for analysis of vessel diameter, red blood cell velocity (RBCV), leukocyte sticking to the microvascular endothelium, and macromolecular leakage in striated skin muscle. In each animal, arteriolar and postcapillary vessel segments were chosen and sequentially recorded on videotape (baseline). Animals were subjected to either topload infusion (10% of blood volume) or isovolemic exchange transfusion (hct 30%) of DCLHb followed by measurements at 10, 30, and 60 min thereafter. In vivo visualization of plasma and leukocytes was performed using FITC-dextran 150,000 and rhodamine 6G, respectively. No significant changes of vessel diameter and RBCV were observed after topload infusion or isovolemic exchange transfusion with DCLHb, either in postcapillary venules or in arterioles when compared with baseline values. Leukocyte sticking and macromolecular leakage were not found enhanced after administration of DCLHb. We conclude that the introduction of DCLHb-bound oxygen into the tissue does neither stimulate leukocyte adhesion nor impair endothelial integrity.

Animals↗