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The aim of the current study was to evaluate the influence of platelet-derived growth factor (PDGF) on skin microcirculation during normal and impaired wound healing. Secondary healing wounds were created on the ears of hairless mice and treated once with 3 microg of PDGF-BB immediately after wound creation. Intravital fluorescence microscopy was used to quantify reepithelialization, revascularization, vessel diameters, vascular permeability, and leukocyte-endothelium interactions up to 24 days after wound creation. Microvascular perfusion was assessed by laser Doppler flowmetry. Wound healing was studied in normal (n = 15) and ischemic skin tissue (n = 15) as well as in mice (n = 17) rendered hyperglycemic by an intravenous injection of streptozotocin 7 days prior to wound creation. Treatment with PDGF accelerated reepithelialization and reduced the time for complete wound closure in ischemic skin from 14.9 +/- 2.5 (control) to 12.3 +/- 1.8 days (p < 0.03), and in hyperglycemic animals from 15.0 +/- 2.4 (control) to 12.0 +/- 3.0 days (p < 0.04). Revascularization of these wounds was also significantly enhanced after PDFG application. No other parameters were influenced by the treatment. Normal wound healing was not affected. This study confirms the positive influence of PDGF on wound healing under pathophysiological conditions. The effects in this model seem to be primarily due to the mitogenic potency of PDGF on keratinocytes and endothelial cells. A significant effect on leukocyte activation during the inflammatory process was not observed.

Aim: Recent studies have demonstrated that C-peptide is biologically active and might have a beneficial effect on late complications in diabetes mellitus. The aim of this study was to investigate the effects of systemically given C-peptide on dermal wound healing in diabetic mice., Methods: Experiments were carried out in male SKH-1 hr hairless mice. Dermal wounds were created (diameter 2.5mm) in streptozotozin-diabetic and normal control mice. Mice were randomized into three treatment groups (n = 10 each): Normal controls, diabetic mice with PBS or C-peptide injection twice daily. At various time points (prior wounding as well as days 4, 7, 10 and 15) microcirculation was quantitatively analyzed by intravital fluorescent microscopy to determine wound surface area, vessel diameter, red blood cell velocity, plasma leakage, functional capillary density. In addition, leukocyte/endothelium interaction was quantified by in vivo visualization of leukocytes., Results: Systemic administration of C-peptide showed no influence on wound healing or standard microcirculatory parameters. The leukocyte/ endothelium interaction revealed a significant (p<0.05) increase in the number of adherent leukocytes 15 days after wound creation in C-peptide treated diabetic mice., Conclusion: Except for the significantly increased number of leukocytes adherent to venular endothelium in the C-peptide group no alteration was observed in wound healing and microcirculation. Neutrophil recruitment after C-peptide injection is of interest because it may reduce the risk of infection in diabetes mellitus.

The aim of this study was to validate orthogonal polarization spectral (OPS) imaging against intravital fluorescence microscopy (IFM) for microvascular measurements in normal skin and during wound healing. Experiments were performed on the ears of hairless mice (N = 8). The diameter of arterioles and venules, red blood cell velocity in venules, and the functional capillary density were assessed under normal conditions using OPS imaging and IFM. After creation of a circular wound, these observations were repeated at the identical microvascular regions on days 4, 7, 10, and 15. Images were videotaped, and CapImage was used for off-line computer-assisted analysis. Using OPS imaging, the microcirculation of wounded skin in hairless mice could be observed successfully. The regression analyses against standard IFM revealed a significant (p < 0.001) correlation for measurements of all microcirculatory parameters investigated (venular diameter: r(2) = 0.98, N = 345; red blood cell velocity: r(2) = 0.51, N = 326; functional capillary density: r(2) = 0.44, N = 156). However, for diameter as well as for functional capillary density measurements, OPS imaging yielded lower absolute values compared with IFM. The authors were able to validate OPS imaging against IFM for the measurement of microvascular parameters in an animal model of skin wound healing. Such a device should now help to study the role of microcirculation in physiology and pathophysiology during wound healing in patients. First clinical investigations are promising.

Orthogonal polarization spectral (OPS) imaging is a new technique that can be used to visualize the microcirculation with reflected light. It uses hemoglobin absorption to visualize the red blood cells (RBCs). Thus the method could fail at low hematocrit (Hct). The aim of this study was to validate OPS imaging for quantitative measurements of diameter and functional capillary density (FCD) under conditions of hemodilution of varying degrees to achieve a wide range of Hcts. The validation was performed in the dorsal skinfold chamber of nine awake Syrian golden hamsters. Measurements of vessel diameter and FCD were performed off-line using Cap-Image on video sequences captured using OPS imaging and standard intravital fluorescence microscopy at baseline, 85, 70, 55, and 40% of the initial Hct. For hemodilution, isovolumic exchange of blood for 6% Dextran 60 was performed. Bland-Altman plots for the vessel diameter and FCD show good agreement between the two methods for both parameters at all studied Hcts. As expected, there was a systematic bias of approximately 4 microm in the diameter measurements since the RBC column was measured and not the intravascular diameter. In conclusion, OPS imaging can be used to measure diameter and FCD at a wide range of Hcts.

Little is known about the microvascular perfusion of the skin postnatally. Skin microvascular parameters can be assessed noninvasively with orthogonal polarization spectral imaging (OPS), a technique where, through the use of special optics, a virtual light source is created at a depth of 1 mm within the tissue. The light is absorbed by the Hb, yielding an image of the illuminated Hb-carrying structures in negative contrast. In nine term (weight 2100-4470 g) and 28 preterm infants (weight 550-2070 g; gestational age 24-33 wk) red blood cell velocity and vessel diameter and density were determined off-line with the Cap-Image program in vessels video-recorded by OPS near the axilla on d 1 and 5 of life. Blood pressure, heart rate, hematocrit, and body and incubator temperature were noted. Vessel diameter ranged from 6 to 24 microm, vessel density from 219 to 340 cm/cm(2) with no change between d 1 or 5 and no difference between term and preterm infants. Red blood cell velocity increased in preterm infants from d 1 [median 528 microm/s, 95% confidence interval (CI) 486-564 microm/s] to d 5 (median 570 microm/s; 95% CI 548-662 microm/s; p = 0.001) and correlated with the decrease in median hematocrit from 44% (CI 40%-60%) to 39% (CI 37%-43%) with r(2) = -0.37 with a 95% CI -0.59 to -0.11, p = 0.006. Hematocrit correlates with red blood cell velocity in the microvessels of the skin. The new technology can be used to assess quantitative changes in the microvessels and thus allows noninvasive assessment of tissue perfusion in term and preterm infants.

Adjunct diagnostic techniques might help surgeons to accurately analyse the depth of a burn. However, despite all technical innovations an ideal device for such an application has not been established for routine use as yet. OPS imaging implemented into the CYTOSCAN A/R is a new, recently introduced technique which allows to obtain high contrast images of the microcirculation without the necessity for fluorescent dyes. The aim of the study was to validate OPS imaging as a tool to study microcirculation in skin after burn injury. OPS imaging was applied by no-touch technique and capillary blood flow was videotaped. Subsequent measurements of the microcirculation were performed at the identical site of the burn. Quantitative analysis of the microcirculation was performed off-line using CapImage. OPS imaging produces high quality images of the microcirculation in a burn wound. Data is given as the number of perfused capillaries per observation area (functional capillary density; FCD) [n/cm2]. OPS imaging allows for direct in vivo visualization and quantification of the microcirculation in burned skin. Our preliminary results of the use of OPS imaging in assessing the microcirculation in burns appear promising, and we hope that this novel technique will allow to improve the knowledge of the dynamics of the microcirculation in the pathophysiology of thermal injury.

Unlabelled: Prolonged immobilization of severely ill, bed-ridden patients results in formation of pressure ulcers following inappropriate tissue blood perfusion, and local activation of leukocyte - endothelial cell interactions (L/EC). Various treatment modalities were implemented to improve local microcirculation with controversial results. The aim of our study was to investigate the influence of heparin and buflomedil, drugs improving the microcirculation, on local skin blood perfusion using intravital fluorescent microscopy (IVM) technique., Material and Methods: Experiments were carried out on 24 male hairless mice under inhalatory isofluran anaesthesia. Intravenous injection of FITC-dextran (150 kD, 5%) allowed to visualize capillaries during IVM, after immobilisation of hind limb, in an observation chamber. Observations were performed after i.v. injection of heparin (66 IU/kg b.w., n = 8) or buflomedil (6 mg/kg b.w., n = 8) 30, 120 and 210 minutes after chamber installation. Observations were recorded in 30 sec sequences on S-VHS tapes and evaluated using special software. Functional capillary density (FCD), defined as a total length of red cells perfused capillaries per observation field (expressed in cm/cm(2)), postcapillary venule diameters, and number of sticking leukocytes per 0.2 mm vessel length during 30 sec observation time, served as parameters of skin blood perfusion and activation of L/EC interactions., Results: A statistically significant decrease of FCD from 152.7 +/- 38.5 to 100.7 +/- 36.7 cm/cm (2) (p<0.05) was observed in control animals during 210 min lasting observation. Administration of heparin prevented decrease in FCD occurring in control animals during intravital microscopy, whereas buflomedil was found ineffective. Both drugs induced a nonsignificant reduction in the number of sticking leukocytes, whereas no changes in postcapillary venule diameters could be observed., Conclusion: The results suggest a protective effect of heparin in clinical therapeutic doses against impairment of skin perfusion during IVM. This observation may justify a trial on the effects of heparin in prevention of development of pressure ulcers.

Orthogonal polarization spectral (OPS) imaging was validated against intravital fluorescence microscopy (IFM) for microvascular measurements in skin flaps of hairless mice. Examinations were performed 1, 6, and 24 hours after elevation (n = 8) with both OPS imaging and IFM. A fluorescent dye was a prerequisite for IFM measurements but not for OPS imaging. Our findings show that OPS imaging can visualize the skin flap microcirculation independent from the application of fluorescent tracers. From these images, quantitative analysis of functional capillary density (FCD) was feasible. As expected, FCD was significantly lower in the distal part of the flap compared with its base (171.8 +/- 34.7 versus 62.0 +/- 25.6, mean +/- SD; 1 hour data). Comparison of OPS imaging and IFM revealed a significant correlation of FCD values (P < 0.001) at all time points. Given the success of this validation study on mouse skin flaps, clinical investigations will have to prove that OPS imaging can also successfully be used to monitor flaps in humans., (Copyright 2001 Wiley-Liss, Inc.)

Background: A newly developed observation chamber has been designed for comfortable limb immobilization during intravital microscopic analysis, which permits direct, repeated, long-lasting observations of the microcirculation in the various hindleg soft tissues., Methods: Experiments were performed under inhalation isoflurane/nitrous oxide anesthesia. Intravenously injected fluorescein isothiocyanate (FITC)-dextran (M, 150,000) and Rhodamine 6G (Sigma, St. Louis, MO) allowed for visualization of both microcirculatory phenomena in arterioles, capillaries, and venules and macrocirculatory structures as superficial saphenous artery and vein. Skin microcirculation analysis was performed from the epidermal side (group A, n = 7), whereas observation of deeper situated tissues was performed after oval skin excision on the medial surface of the tibial area (group B, n = 7). FITC-dextran (M, 150,000; group C, n = 8) injected into the foot pad permitted visualization of venous, arterial, and accompanying lymphatic vessels. With the aid of a computer-assisted microcirculation analysis system, functional capillary density, vessel diameter, edema formation, and leukocyte-endothelial cell interactions were evaluated. The ratio of rolling leukocytes, given as percentage of all leukocytes passing the vessel segment during a 30-second observation interval, and the number of sticking leukocytes per square millimeter of endothelial surface were determined., Results: This new model allows the analysis of the complex in vivo changes of macro- and microcirculatory parameters in the different (venous, arterial, lymphatic) vessels of the covering tissues (skin and muscle) of the mouse hindleg., Conclusion: The potential applications of this technique include the study of mechanical trauma, ischemia-reperfusion injury, and tissue compression mimicking both acute and prolonged venous stasis on both the microcirculatory and macrocirculatory levels in the different tissue compartments.

Intravital microscopy is widely used to study the microcirculation. However, the use of fluorescent dyes can induce phototoxic effects which may affect the measurements, particularly in tissue exposed to oxidative stress. The aim of the study was to determine the threshold light dose at which fluorescent microscopy is associated with phototoxic effects in the hamster dorsal skinfold chamber under normal and pathological conditions. The extent of phototoxicity in the microcirculation in the hamster skinfold chamber was investigated using intravital fluorescent microscopy during 60 min of illumination (1048 mW/cm2) applying two different concentrations of fluorescein isothiocyanate dextran under baseline conditions (groups A and B) and following 4 h of ischemia (groups C and D). In the second part of the study the microvasculature was analyzed regarding phototoxic effects during a standardized intravital microscopic examination after 4 h of pressure induced ischemia. Groups I and II (n=7) were studied using epiillumination after injection of fluorescein isothiocyanate dextran plus rhodamine 6G or rhodamine 6G only. In group III (n=7) only transillumination was used. Arteriolar vasospasm, microvascular perfusion failure, thrombus formation, and enhanced leukocyte endothelium interaction were observed as signs of a phototoxic effect in normal tissue. However, the light doses needed to induce these effects clearly exceeded those during standard examinations. The induction of a 4-h ischemia and reperfusion further enhanced these effects. Despite the predamage by ischemia/reperfusion the comparison of epiillumination and transillumination microscopy using a standard protocol showed no differences regarding the parameters analyzed at any time. This indicates that epiillumination and the fluorescent dyes per se did not affect the experimental results. These results show that ischemia/reperfusion studies in the dorsal skinfold chamber of the Syrian golden hamster can be carried out safely without the risk of inducing phototoxic effects by fluorescent microscopy. Nevertheless every laboratory using epiillumination and fluorescent dyes should take precautions to avoid these effects by the use of sensitive cameras to lower the light dose.

The objective of this study was to investigate the effects of the micronized purified flavonoid fraction Daflon 500 mg (90% diosmin and 10% hesperidin) on I/R-induced microvascular leukocyte-endothelium interaction and leakage of the high molecular weight plasma tracer FITC-dextran (relative molecular mass, 150 kd) as assessed in the striated skin muscle of the dorsal skin fold chamber model in the hamster. Intravital fluorescence microscopy was used for analysis of microvascular perfusion, leukocyte-endothelium interaction, and macromolecular leakage of FITC-dextran 150 kd in the striated skin muscle of the hamster. A tourniquet ischemia of 4 hours' duration was induced followed by reperfusion. Animals were treated with an oral administration of Daflon 500 mg (n = six) or its vehicle (5% Arabic gum solution, n = six) for 8 days at a daily dose of 30 mg/kg body weight. Measurements in the microcirculation were made before the 8-day feeding protocol before induction of ischemia and at 0.5, 2, and 24 hours of reperfusion. In the absence of I/R, no differences in microvascular perfusion, leukocyte-endothelium interaction, and macromolecular leakage were found in Daflon 500 mg and vehicle-treated control animals before and after administration of the drugs. Induction of ischemia and reperfusion, however, elicited a significant increase in venular leukocyte rolling and sticking in vehicle-treated animals, which was accompanied by enhancement of leakage of FITC-dextran 150 kd into the perivascular tissue. Treatment with Daflon 500 mg had no effect on postischemic leukocyte rolling and sticking, and macromolecular leakage of FITC-dextran 150 kd from arterioles and postcapillary venules was significantly reduced. These data indicate that Daflon 500 mg preserves the endothelial barrier function of striated skin muscle arterioles and venules after I/R, which appears to be independent of an action on postischemic intravascular leukocyte rolling and sticking.

The objective of this study was to investigate the effects of the purified, micronized, flavonoid fraction Daflon 500 mg (S 5682, 90% diosmin and 10% hesperidin) on tissue damage and leukocyte emigration in striated skin muscle after ischemia-reperfusion, as assessed by histomorphometric analysis. The experimental model used was the transparent dorsal skin fold chamber in the awake Syrian golden hamster. Sixty-four animals were randomly allotted to two treatment groups and time points of investigation. Animals were fed with 30 mg kg(-1) body weight Daflon 500 mg (n = 32) or its vehicle, 5% Arabic gum solution (n = 32), as control 8 hours before ischemia. Before induction of a tourniquet ischemia of 4 hours' duration and at 0.5, 2, and 24 hours of reperfusion, tissue sections were preserved for light and electron microscopic analysis (n = seven or eight animals per time point). The number of intravascular and extravascular leukocytes was determined by light microscopic analysis of esterase-positive leukocytes. For quantitative analysis of ischemia-induced endothelial cell damage, the endothelial thickness of capillaries was calculated by a computer-assisted imaging system, whereas the ischemic tissue damage was assessed by means of a score system (grade 0-3) by an independent investigator. The number of emigrated leukocytes was significantly reduced in Daflon 500 mg-treated animals compared with numbers found in control animals. The histomorphologic muscle fiber damage increased after reperfusion in both groups but was significantly reduced in the Daflon 500 mg-treated animals 2 and 24 hours after reperfusion. These results suggest that the emigration of leukocytes plays an important role in the development of postischemic reperfusion injury of striated skin muscle.

Hemoglobin-based oxygen carriers have been suggested to enhance the formation of oxygen free radicals, especially under conditions of ischemia-reperfusion (I/R), in which activation and adhesion of leukocytes play a pivotal role for propagation of reperfusion injury. This study investigates the effects of the hemoglobin-based oxygen carrier diaspirin-cross-linked hemoglobin (DCLHb) in an I/R model of hamster striated skin muscle. The dorsal skinfold chamber model in the awake Syrian golden hamster was used for analysis of the microcirculation and local tissue PO2 in striated skin muscle utilizing the technique of intravital fluorescence microscopy and a multiwire platinum surface (Clark type) electrode. Measurements were made before 4 h of pressure-induced ischemia and at 0.5, 2, and 24 h of reperfusion. Animals were treated with 5 ml/kg body wt of either 10% DCLHb (n = 8), 6% Dextran 60 (Dx-60; 60 kDa, n = 8), or 0.9% NaCl (n = 7), which was given intravenously 15 min before reperfusion. In animals treated with DCLHb or Dx-60, a significant decrease of leukocytes rolling along and sticking in postcapillary venules, associated with a recovery of functional capillary density and red blood cell velocity, was observed compared with saline-treated controls. In the early reperfusion period (0.5 h), DCLHb and Dx-60 efficiently restored local tissue PO2, whereas tissue PO2 decreased from 18.3 +/- 1.9 to 15.3 +/- 5.3 mmHg in 0.9% NaCl-treated animals. Electron microscopic analysis of the postischemic tissue at 24 h of reperfusion revealed markedly reduced tissue damage in animals treated with DCLHb compared with Dx-60 or isotonic saline. These results indicate that DCLHb attenuates postischemic reperfusion injury of striated skin muscle, presumably through alterations of leukocyte-endothelial cell interactions.

Oxaceprol, an established drug for treatment of degenerative joint disease, has recently been shown in vitro to reduce leukocyte adhesion to cultured endothelial cells and leukocyte extravasation in vivo in an arthritis animal model. The aim of this study was to examine the effects of Oxaceprol on the microcirculation of striated skin muscle. In the dorsal skinfold chamber of the awake Syrian golden hamster I/R injury was induced by applying a 4-hour complete pressure ischemia. Prior to the ischemia, after 30 minutes, 2 hours and 24 hours of reperfusion the following parameters were assessed in a blinded study: macromolecular leakage, leukocyte rolling fraction, adherent leukocytes, and functional capillary density (FCD). Rhodamine 6G to stain leukocytes in-vivo and FITC Dextran (MW 150,000) was used as a plasma marker. 15 minutes prior to reperfusion the animals received either an i.v. bolus infusion of Oxaceprol (50mg/kg) or an equivalent volume of saline, which was followed by a 45-minute continuous infusion at the same dose. At the conclusion of the experiment samples were collected from the chamber tissue for histological quantification of leukocyte extravasation using an esterase stain. Oxaceprol treatment resulted in a significant decrease of postischemic leukocyte adherence after 0.5 h and 2h of reperfusion. The histological sections revealed a significant reduction in the number of extravasated leukocytes. There was a reduction of macromolecular leakage and treatment also resulted in a preservation of tissue perfusion, as was indicated by a significant increase in FCD in the treatment group compared to the ischemia group. In summary, Oxaceprol was able to protect the tissue from ischemia/reperfusion injury.

Using the dorsal skin fold chamber model in the hamster, we analyzed local tissue partial oxygen pressure (PO2) in the striated skin muscle under nonischemic and postischemic conditions with a Clark-type multiwire oxygen surface electrode. Hypervolemic infusion (500 mg x kg(-1) I.V.) or isovolemic exchange transfusion (3.3 gm x kg(-1) I.V.; hematocrit 30%) with diaspirin-cross-linked hemoglobin (DCLHb) resulted in a slight decrease of the mean value of the local tissue PO2 (mm Hg) 1 hour after administration. Concomitantly, the frequency distribution curves of local tissue PO2 values were found to be more narrow (fewer values > 25 mm Hg and < 10 mm Hg). Resuscitation from severe hemorrhagic shock (bleeding of 33 ml x kg(-1) at 0.4 ml x min(-1)) with autologous blood (AuB), Dx-60, or DCLHb led to an increase of mean tissue PO2 values by 4.2-fold (p < 0.05 versus Dx-60), 1.9-fold, and 3.7-fold (p < 0.05 versus Dx-60), respectively, 2 hours after resuscitation. The reduction of tissue hypoxia (0-5 mm Hg) was significant only in the AuB- and DCLHb-treated animals. This study indicates that DCLHb effectively reverses tissue hypoxia after resuscitation from severe hemorrhagic shock by inducing a more homogeneous distribution of the local tissue PO2 levels.

Hemoglobin-based oxygen-carrying solutions are reported to exert vasoconstrictor effects and to enhance oxygen radical formation, particularly during ischemia-reperfusion. This study investigates whether diaspirin-cross-linked hemoglobin (DCLHb) affects the microvascular integrity of striated skin muscle. The microcirculation model in the hamster and intravital fluorescence microscopy were applied for investigation of the microvascular changes in striated skin muscle. Hypervolemic infusion (500 mg x kg(-1), I.V.) and isovolemic exchange transfusion (3.3 gm x kg(-1) I.V.; hematocrit 30%) with DCLHb (1) led to a short-lasting (0 to 2 minutes) arteriolar constriction (approximately 20% reduction in baseline diameter), (2) significantly influenced arteriolar vasomotion, (3) increased venular red blood cell velocity by 1.5-fold (p < 0.05 vs dextran, Mr 60,000), and (4) did not enhance microvascular leukocyte-endothelium interaction or endothelial permeability. Resuscitation from severe hemorrhagic shock with autologous blood (AuB) or DCLHb (33 ml x kg(-1), I.V.) immediately restored mean arterial pressure and heart rate, whereas 6% dextran (60 kd)(Dx-60) did not return these parameters to baseline. Venular red blood cell velocity was restored to 110% of baseline after DCLHb, to 90% of baseline after AuB, and to 45% of baseline after Dx-60. Leukocyte-endothelium interaction was significantly enhanced after resuscitation with AuB and Dx-60, whereas this phenomenon was absent after DCLHb. These data demonstrate that DCLHb increases venular red blood cell velocity under both nonischemic and postischemic conditions without inducing enhanced leukocyte-endothelium interaction in the microcirculation of striated skin muscle.

Objective: Venous congestion plethysmography enables noninvasive assessment of microvascular filtration capacity (Kf) in limbs. However, increases in fluid filtration might alter the balance of Starling forces: for example, progressive increases in interstitial fluid pressure (Pi) would reduce net fluid flux, thus underestimating Kf. Furthermore, elevation of cuff pressure to values close to diastolic blood pressure, as used in the protocol, may be itself impair tissue perfusion with unknown effects on the microvascular parameters investigated., Methods: Pi was measured in healthy volunteers (n = 14) with a modified "Wick in needle" technique during small (8 mm Hg) cumulative increases in venous pressure (0-95 mm Hg). Changes in the hemoglobin (Hb) concentration, oxygenated hemoglobin (HbO2) concentration and oxidized cytochrome aa3 concentration were assessed in the calf using noninvasive near-infrared spectroscopy. Skin red blood cell flux close to the strain gauge was evaluated by laser Doppler fluxmetry., Results: Pi at control was -0.89 +/- 0.8 mm Hg and during elevation of venous pressure remained constant until a cuff pressure of 30 mm Hg was reached. It rose thereafter to 1.57 +/- 1.3 mm Hg (mean +/- SD). Skin red cell flux was significantly reduced when cuff pressure exceeded 30 mm Hg and following cuff deflation, evidence of reactive hyperemia was obtained. Hb concentration increased significantly as a result of venous pressure elevation. No change in either HbO2 or cytochrome aa3 concentration was observed as long as cuff pressure remained under diastolic blood pressure., Conclusions: The small increase in Pi together with an absence of impaired tissue oxygenation during the venous congestion plethysmography protocol described by Gamble et al. supports the contention that this protocol enables accurate assessment of filtration capacity.

We have recently shown that the purified micronized flavonoid fraction (90% diosmin and 10% hesperidin) Daflon 500 mg attenuates reperfusion injury in the striated skin muscle of the hamster. Herein, we report on the action of Daflon 500 mg on postischemic macromolecular leakage of FITC-dextran 150 kD provoked by tourniquet ischemia. Intravital fluorescence microscopy was used for analysis of macromolecular leakage in the microcirculation model of the hamster. A tourniquet ischemia of 4 h duration was induced followed by reperfusion. Animals were treated by gavage of Daflon 500 mg (n = 6) for 8 days at a daily dose of 30 mg kg(-1) body weight. Control animals received equivalent volumes of the vehicle (5% Arabic gum solution, n = 6). Measurements of the microcirculatory parameters were made before induction of ischemia and at 0.5, 2 and 24 h of reperfusion. After induction of ischemia, macromolecular leakage from postcapillary venules was significantly enhanced in vehicle-treated animals. Treatment with Daflon 500 mg significantly attenuated macromolecular leakage of FITC-dextran 150 kD. Preliminary data from a histomorphometric analysis (n = 3/experimental group) indicated that the number of emigrated (extravascular) leukocytes after ischemia reperfusion was markedly reduced in Daflon 500 mg-treated animals as compared to controls. These data indicate that Daflon 500 mg prevents leakage of the macromolecular tracer FITC-dextran 150 kD from postcapillary venules after postischemic reperfusion, presumably through an inhibitory action on the emigration of activated leukocytes.

The relationship between systemic and microvascular Po2 in subcutaneous connective tissue and hemodynamics was investigated during 4-h hemorrhagic shock (40 mmHg) in conscious Syrian hamsters (n = 66) fitted with a dorsal skinfold window. Systemic blood gases, metabolic parameters, arteriolar, venular, and tissue Po2, microvascular red blood cell velocity, and blood flow were evaluated in survivors (S) and nonsurvivors (NS). Surviving animals were resuscitated with shed blood. Microvascular and tissue Po2 were measured by phosphorescence decay of Pd-meso-tetra (4-carboxyphenyl) porphyrin (30 mg/kg body wt iv). Shock caused a significant dissociation between systemic arterial and microvascular arteriolar Po2 levels. Arterial Po2 increased from 59.7 +/- 12.0 to 110.8 +/- 19.7 mmHg (S) and from 64.0 +/- 13.7 to 128.5 +/- 10.1 mmHg (NS), whereas Po2 in large arterioles decreased from 56.9 +/- 5.5 (control) to 29.5 +/- 20.1 (S) and 6.0 +/- 5.7 mmHg (NS). Correspondingly, tissue Po2 fell from 24.1 +/- 6.8 (control) to 0.9 +/- 0.6 (S) and 0.4 +/- 0.3 mmHg (NS). Venous Po2 decreased from 28.8 +/- 3.7 to 20.4 +/- 4.1 (S) and from 28.0 +/- 2.9 to 16.3 +/- 0.5 mmHg (NS). Shock outcome and tissue oxygenation were predicted by arterial blood gases and metabolic and microcirculatory conditions but not by central venous Po2.

Intravital microscopy is used for analysis of the microcirculation in various organs, e.g. mesentery, intestine, heart, liver and lung, requiring either exteriorization or in situ visualization techniques in anaesthetized animals. In contrast, the implantation of transparent chambers has been employed to allow chronic observation of the microcirculation in intact, non-anaesthetized animals. This paper reports results with two transparent chamber models: the skin-fold chamber model in the hamster and the mouse. An overview is provided of the technical development of the chamber technique and of the various experimental studies that have been performed using these models in ischaemia-reperfusion of striated muscle. Particular emphasis is given to a description of the methods used to induce ischaemia and reperfusion, namely pressure-induced or tourniquet-induced ischaemia. While the former allows simulation of the pathophysiological situation in compartment syndromes, the latter provides an appropriate simulation of the clinical situation in vascular, transplantation and reconstructive surgery. Future possibilities for these microcirculation models in research into the pathophysiology of ischemia-reperfusion are outlined, and potential therapeutic measures to preserve postischaemic tissue are discussed.

Leukocyte rolling in post-capillary venules is mediated by adhesion molecules of the selectin family expressed on both leukocytes (L-selectin) and endothelial cells (E- and P-selectin). With the use of intravital fluorescence microscopy, the effects of antibodies against these selectins were analyzed in the skinfold chamber model of BALB/c mice and the ear model of homozygous hairless mice (hr/hr) that permit chronic observation of striated muscle and skin microcirculation in awake animals, respectively. Mice were injected intravenously with monoclonal antibodies (MAb) to murine L-selectin and E-selectin and affinity-purified polyclonal antibodies to P-selectin. The antibodies, which are known to block cell adhesion, were tested by immunoprecipitation to selectively bind to L-, E-, or P-selectin. Leukocyte rolling was a constant finding in both microcirculation models in the absence of inflammatory stimuli. In both models, injection of anti-P-selectin antibodies completely prevented baseline leukocyte rolling over an observation period of 2 h (P < 0.01 vs. baseline), while no effects were seen after administration of either anti-L-selectin or anti-E-selectin MAb. Treatment with the isotype-matched control antibodies did not affect leukocyte rolling in either model. We conclude that leukocyte rolling in postcapillary venules of murine striated muscle and skin is a physiological process mediated via P-selectin, whereas L- and E-selectin appear not to play a significant role under these circumstances.

In random pattern skin flaps of mice, tissue fluorescence measured by means of videodensitometry at 24 h after flap elevation significantly correlates with intravital microscopically measured functional vessel density and viability of skin tissue as assessed by transmission light and electron microscopy. The correlation was found reproducible in non-ischaemic flaps (r = 0.86) and flaps being rendered ischaemic for 6 h after elevation (r = 0.98), indicating that increased microvascular permeability as a result of ischaemia/reperfusion does neither affect tissue fluorescence nor the accuracy of the fluorescein dye test. In addition, tissue fluorescence at 24 h after flap elevation accurately predicts ultimate flap survival on the 7th postoperative day with a sensitivity of 0.89 and a specificity of 0.85. These results suggest that in non-ischaemic as well as in ischaemic random pattern skin flaps tissue fluorescence can be used for assessment of nutritive blood flow, viability of skin tissue and ultimate tissue survival in these flaps.

The nutritional blood flow to the tissue is the principle factor determining the outcome of reconstructive procedures in plastic surgery. Beside vasoconstriction, thrombocytes aggregation, leukocytes activation, followed by production of oxygen free radicals and release of proteases, impaired fluidity of erythrocytes and swelling of the microvascular endothelium are responsible for impaired perfusion following ischemia/reperfusion. Therefore, vasoactive drugs known to improve nutritional blood flow are of particular interest in the prevention and therapy of skin flap necrosis. Several methods have been employed to quantify nutritional blood flow in skin flaps but none of them have elucidated the mechanisms underlying potential tissue protection by these drugs. We have developed two skin flap models in the hairless mouse, in which nutritional blood flow can be directly assessed at capillary level by means of intravital microscopy. In the arterial skin flap model, the efficacy of three vasoactive drugs (naftidrofuryl [4.5 mg/kg/day], pentoxifylline [17.0 mg/kg/day] and buflomedil [3.0 mg/kg/day]) to prevent and/or reduce tissue necrosis was investigated. Buflomedil provided the most efficient tissue protection, a phenomenon also verified in a random skin flap model. It was demonstrated that preoperative application of buflomedil preserves functional vascular density in the distal part of skin flaps more efficiently compared to postoperative application. However, there were no significant differences in amount of skin necrosis between the two groups. The tissue protection effect was also observed in random pattern flaps rendered ischemic for six hours after flap elevation.(ABSTRACT TRUNCATED AT 250 WORDS)

Neovascularization plays a major role in prosthetic vascular graft healing. New developments in manufacturing biomaterials have encouraged the use of biological and biosynthetic materials for both arterial replacement and bypass procedures. We have analyzed in vivo the process of neovascularization and incorporation of biolized bovine carotid artery (Solco P), a biological material, and the composite of ovine collagen and polyester mesh (Omniflow), a biosynthetic material. The synthetic fabric polytetrafluorethylene (e-PTFE), which is widely used in cardiovascular surgery, served as control. Using the dorsal skinfold chamber of the Syrian golden hamster as site for implantation (n = 15), angiogenesis and neovascularization were analyzed quantitatively by means of intravital fluorescence microscopy. In each chamber a piece (approximately 1 mm2) of each of the three vascular grafts was implanted. Five days after implantation neovascularization was ascertained in 90% of the Omniflow grafts, while only 40% of the PTFE and 20% of the Solco P implants revealed new ingrowing microvessels. On day 10 the density of newly formed microvessels was significantly higher (p less than 0.01) in Omniflow grafts (263.5 +/- 24.1 cm-1) as compared to PTFE (134.2 +/- 20.8 cm-1) and Solco P (153.2 +/- 32.0 cm-1). In addition, the biosynthetic composite revealed a larger extension of neovascularization into the perigraft tissue, and 12 days after implantation these grafts were most tightly incorporated.(ABSTRACT TRUNCATED AT 250 WORDS)

The reduction of postischemic reperfusion injury by exogenous adenosine has been ascribed to reduced oxygen radical generation and adhesion of leukocytes to the vascular endothelium. To provide in vivo evidence for this concept we investigated the effects of adenosine (110 micrograms.kg-1.min-1 iv) on postischemic leukocyte-endothelium interaction in the dorsal skinfold chamber model in awake hamsters by intravital fluorescence microscopy. Leukocytes were stained in vivo with acridine orange and classified according to their interaction with the endothelium as nonadherent, rolling, or sticking leukocytes. In control animals, reperfusion after a 4-h pressure-induced ischemia to the striated muscle in the dorsal skinfold chamber elicited a marked increase in leukocyte rolling and sticking. This phenomenon was significantly attenuated in adenosine-treated animals 30 min after reperfusion. Postischemic changes in vessel diameters and red cell velocities were not affected by adenosine. The data suggest that systemic adenosine administration reduces reperfusion injury by the inhibition of postischemic leukocyte adherence to the microvascular endothelium.

Intact blood supply by microcirculation to a wounded site is an indispensable prerequisite for normal tissue regeneration. However, microvascular changes taking place in the healing process of skin wounds are not understood due to the fact that only few models allow chronic in vivo studies on skin microcirculation. Therefore, we have modified the hairless mouse ear model with the purpose of a quantitative in vivo study of microhemodynamic changes throughout the healing process. Following the creation of a standardized skin wound on the ear of the homozygous hairless mouse (hr/hr), microvessel diameters, red blood cell velocities, wet weight, and leucocyte content of the ear tissue were determined. Surface area of the wound was assessed until complete closure was achieved. By repeated measurements at identical sites over the entire healing period, a distinct pattern of microvascular changes could be observed: microvessel diameters increased to a maximum a few days after wound creation, whereas red blood cell velocities reached their highest values at a later point in time and were still elevated after complete reepithelization of the wounds. Edema and leucocyte content of the ear tissue was most prominent in the early healing phase and gradually decreased to normal values thereafter. These results demonstrate changes of the microvasculature of the hairless mouse ear to injury, which are in accordance to other more indirect studies on this topic. Therefore, we conclude that the model presented is suitable for prolonged quantitative analysis of microcirculation during normal wound healing and may be used to assess microvascular changes taking place during wound healing in pathologically altered tissue.

The effect of buflomedil to protect skin tissue from ischemia and necrosis was studied in random cutaneous flaps. Measurements were performed by intravital microscopy on the microcirculatory level of capillary perfusion in a flap model in the hairless mouse. In 30 hairless mice, single-pedicle flaps measuring 6 x 16 mm were raised perpendicular to the spine of the animal. This flap develops a reliable amount of necrosis at its distal edge over a period of 7 days. A group of 10 mice received intravenous injections of buflomedil in doses of 3 mg/kg per day diluted in 0.1 ml normal saline beginning 4 hours before flap elevation and for 6 consecutive days postoperatively. In addition, 10 further animals received the same treatment except that it was started 5 minutes after flap elevation. In 10 mice serving as controls, normal saline in equal volumes as in the experimental groups was applied. By means of intravital microscopy, functional vessel density (FVD) was determined in 2.5-mm increments from the flap's base to its distal edge at 1, 6, and 24 hours after elevation. Skin-flap survival was quantified by measuring the necrotic area on day 7 by means of digital planimetry. Functional vessel density was preserved in the distal flap of animals pretreated with buflomedil, revealing a higher functional vessel density at 10.0 mm (p less than 0.01), 12.5 mm (p less than 0.05), and 15.0 mm (p less than 0.001) from the flap's base as compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The homozygous (hr/hr) hairless mouse ear was introduced in 1980 by Eriksson and coworkers as a model for in vivo studies of the skin microcirculation. Herein we expand on this work, presenting results of in vivo microvascular parameter measurements and morphologic studies in the intact ear. The in vivo measurements include microvascular diameter, RBC velocity, capillary density, and the frequency and amplitude of arteriolar vasomotion. In connection with the in vivo studies, a detailed anatomic description of the overall and vascular anatomy is given. Additionally, the preparation techniques for carrying out these in vivo and morphologic studies in the mouse ear are presented in detail.

The effects of pentobarbitone anaesthesia on the macrohaemodynamics and the microvasculature of subcutaneous tissue and skin muscle were studied in hamsters (n = 14) utilizing intravital microscopy, quantitative video techniques and a platinum multiwire electrode for local PO2 measurements. After achieving anaesthesia with 35 mg/kg b.w. pentobarbitone, changes in macrohaemodynamic parameters, blood cell velocity and vessel diameters were not observed. However, after 30 min of pentobarbitone anaesthesia, 25% of the arteriolar blood volume coursed through arteriovenous shunts. The increase in a-v shunting was accompanied by a significant decrease of functional capillary density. As a result, local PO2 values decreased slightly after 30 min of pentobarbitone anaesthesia. These findings provide evidence that 35 mg/kg b.w. pentobarbitone cause alterations in capillary density and perfusion. It is concluded that with a dose of up to 35 mg/kg b.w. pentobarbitone, these changes have little effect on local tissue oxygenation.

An animal model is described allowing for direct measurements of local tissue PO2, microhemodynamics and vascular density in the event of a prolonged non freezing cold injury. The model consists of implanting a transparent skin fold chamber in the dorsal skin fold in hamsters and of inserting two permanent indwelling catheters in jugular vein and carotid artery, respectively. The microcirculation was studied using a Wild Photomacroscope for photography and a platinum multiwire electrode for measurements of local PO2 in the conscious animal. After 72 h of recovery from anesthesia and surgery, the experimental was started with the animal immobilized. The decrease of local s.c. temperature was achieved by perfusing a heat exchanger with distilled H2O and Isopropanol 70% (1:1) at a rate of 81/min with the heat exchanger located directly beneath the aluminium frame of the chamber. With this technique, a decrease in local tissue temperature from 28 degrees C to 15 degrees C could be obtained within 15 min and was kept constant for 60 min. After photography of the microcirculation and local PO2-measurements, the local temperature was further reduced to 5 degrees C with 15 min. Sixty minutes later, the area exposed was slowly rewarmed from a level of 5 degrees C within 30 min. This procedure was repeated in intervals of 24 h over a period of five days. During the course of the experiments, local PO2 values shifted toward hypoxic or even anoxic values. Intravital microscopic observation revealed aggregate formation, stasis and obstruction of capillary flow associated with pronounced tissue anoxia after five cold exposures. This event resulted inevitably in tissue necrosis and scar formation after seven consecutive exposures to cold. It is concluded that this model can be used to study the effects of local non freezing cold injury in a precisely reproducible manner.

Spontaneous changes of diameter in small arteries and arterioles were studied in the hamster skin fold chamber preparation, which allows to observe the microcirculation without acute exposure and anesthesia. Continuous measurements of diameter were made when the preparation appeared to be physiologically normal according to preset criteria. 89% of the arterial vessels studied showed spontaneous changes in diameter. The activity was prevalently periodic with 29% of the vessels exhibiting random changes in diameter. The amplitude of the periodic activity expressed as a percentage of the mean diameter increases in proportion to the vessel size, arterial vessels in the 40-100 micron diameter range exhibit amplitudes of the order of 10-20%. The smallest arterioles show changes as large as 100% of mean diameter, which cause these vessels to become temporarily occluded. This phenomenon is analyzed in terms of its effect on peripheral vascular resistance by means of an analytical model that assumes vasomotion to be a periodic phenomenon. It is shown that the presence of time dependent changes in vessels diameter similar to those observed, has an important effect on peripheral vascular resistance.

An experimental model was designed for direct, quantitative studies of hemodynamic and morphologic parameters in the microcirculation. It consists of implanting a modified Algire chamber in the dorsal skin flap of hamsters and the implementation of two permanent catheters in jugular vein and carotid artery. The microcirculation was studied using intravital microscopy and television techniques for in situ measurements of blood cell velocity and vascular diameters. Due to the poor contrast between blood cells, blood capillaries and surrounding s.c. tissue, microvascular beds were visualized using fluorescent microscopy after i.v. injection of 0.2 ml of 5% FITC-Dextran 150. The combination of optical elements and low amounts of FITC-Dextran improved the contrast of the televised image without changing macro- and micro-hemodynamic parameters, and blood plasma was delineated as bright structure against the substantially darker background of red blood cells and surrounding tissue. This permitted the quantitative study of practically all blood vessels within a given field of s.c. tissue in unanesthetized animals. Blood cell velocity in arterioles was 0.7-1.1 mm/s, 0.2-0.7 mm/s in midcapillaries and reached 0.6 mm/s in collecting venules. Since i.v. injection of drugs and systemic pressure measurements are possible in this model, it provides a unique means for studying the reactivity of the microcirculation over a prolonged period.