No abstract

Leukotriene (LT)B4 promotes leukocyte chemotaxis and adhesion to the endothelium of postcapillary venules. The cysteinyl leukotrienes, LTC4, LTD4, and LTE4, elicit macromolecular leakage from this vessel segment. Both leukocyte adhesion to the endothelium and macromolecular leakage from postcapillary venules hallmark the microcirculatory failure after ischemia-reperfusion, suggesting a role of leukotrienes as mediators of ischemia-reperfusion ijury.Using the dorsal skinfold chamber model for intravital fluorescence microscopy of the microcirculation in striated muscle in awake hamsters and sequential RP-HPLC and RIA for leukotrienes, we demonstrate in this study that (a) the leukotrienes (LT)B4 and LTD4 elicit leukocyte/endothelium interaction and macromolecular leakage from postcapillary venules, respectively, that (b) leukotrienes accumulate in the tissue after ischemia and reperfusion, and that (c) selective inhibition of leukotriene biosynthesis (by MK-886) prevents both postischemic leukotriene accumulation and the microcirculatory changes after ischemia-reperfusion, while blocking of LTD4/E4 receptors (by MK-571) inhibits postischemic macromolecular leakage.These results demonstrate a key role of leukotrienes in ischemia-reperfusion injury in striated muscle in vivo. (J. Clin. Invest. 1991Invest. . 87:2036Invest. -2041.) Key words: intravital fluorescence microscopy* microcirculation * striated muscle * leukocyte/endothelium interaction * macromolecular leakage

The aim of this study was to establish a simple and safe method of anaesthesia for intravital microcirculatory observations in small laboratory animals. The usefulness of isoflurane inhalation anaesthesia has been investigated in different strains of mice commonly used in experimental medicine. These were the hairless (hr/hr, n = 12), the BALB/c (n = 12) and the nude mouse (nu/nu, n = 3). Anaesthesia was maintained by mask inhalation of isoflurane vaporized at concentrations of up to 4% in the induction phase, at 1.5% during acute surgical procedures and at 0.8-1.3% during prolonged experimental observations. Isoflurane was vapoured in a N(2)O/O(2) mixture and saturated with 32-36% F(i)O(2). During observations the body temperature was kept constant at 37 degrees C. The tail artery was cannulated for monitoring of mean arterial blood pressure (MAP) and heart rate (HR). To maintain the body fluid balance, isotonic saline was administered at a constant rate of 0.2 ml/h. Arterial blood samples were drawn for blood-gas analysis at the end of the experiments. All animals survived the anaesthesia protocol lasting between 3 and 6.5 h. During isoflurane inhalation, no breathing complications or changes in systemic circulatory parameters were observed. Mean values of MAP and HR were 79+/- 3 mmHg and 486+/- 13 min(-1), respectively, over the entire observation period. A moderate acidosis was recorded in animals under isoflurane anaesthesia, with alterations of arterial blood pH, p(a)O(2) and pCO(2) values (7.29+/- 0.06, 130+/- 19 mmHg and 35.6+/- 4.7 mmHg, respectively). In conclusion, inhalation anaesthesia with isoflurane is useful for experimental studies in the mouse due to (1) the simplicity of administration of the anaesthetic, (2) the rapid induction of anaesthesia, (3) easy control of the depth of anaesthesia, (4) the low percentage of complications, and (5) stable MAP and HR during observations lasting several hours. The proposed technique is especially suitable for observations of the microcirculation under intravital fluorescence microscopy.

Functional capillary density (FCD) is one of the parameters obtained by intravital microscopy using epi-illumination of the tissue surface or trans-illumination of thin tissue layers. FCD, defined as the length of red cell-perfused capillaries per observation area (cm^-1), has been used as an indicator of the quality of tissue perfusion in various animal models. Quantitative analysis of FCD in randomly selected regions of the tissue is performed by means of a computer-assisted video analysis system which allows calculation of the length of RBC-perfused capillaries. Basically, two different mathematical approaches can be employed: the first approach is based on the addition of the distances between two neighboring points (pixels) on the video screen (Pythagorean principle). The second approach uses the superimposition of a grid system that allows estimation of the capillary length by counting the number of intersections between the capillaries and the grid lines (stereological approach). The immanent error has been calculated in our laboratory to be ± 1 % with the Pythagorean and ± 5% with the stereological method. Beside these systematic errors of computerized measurement, the individual (user-dependent) errors occurring during recognition and redrawing of the capillaries on the video image with use of a digitizing tablet are in the range of ± 10% (intraindividual) and ± 70% (interindividual) for the recognition and ± 3% (interindividual) for the redrawing procedure. Our studies indicate that the errors resulting from the use of a computer-assisted calculation (Pythagorean or stereological approach) or the user-assisted redrawing of the capillaries are negligible when compared to the errors made during recognition of the capillaries on the video screen. The methods are applied for assessment of FCD in two different microcirculation models of skin muscle and pancreas yielding highly reproducible, user-independent results under physiologic conditions and the pathophysiologic conditions of ischemia-reperfusion.

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.

The underlying mechanisms of the beneficial therapeutic effects of small-volume resuscitation with hyperosmolar solutions for treatment of hypovolemic shock are still poorly understood. Using the dorsal skinfold chamber model and intravital fluorescence microscopy, we investigated the effects of hyperosmolar saline dextran on ischemia-reperfusion injury in striated skin muscle of awake normovolemic golden hamsters. Test solutions (4 ml/kg body wt i.v.) were administered 2 min before reperfusion after 4 h of pressure-induced ischemia. In animals receiving 0.9% saline (control), we observed a drastic enhancement of leukocyte rolling along and sticking to the endothelium of postcapillary venules 0.5 h after reperfusion. Postischemic leukocyte rolling and sticking were significantly reduced when animals were treated with 7.2% saline alone (HSS), 10% Dextran 60 in 0.9% saline (HDS), or 10% Dextran 60 in 7.2% saline (HHS). In control animals, capillary perfusion was reduced to approximately 60% of preischemic values 0.5 h after reperfusion. Concomitantly, leakage of the macromolecule fluorescein isothiocyanate-dextran (5 mg in 0.1 ml saline i.v., M(r) 150,000) into the perivascular space increased from 0% before ischemia to approximately 12% at 0.5 h reperfusion. In contrast, when animals were treated with HSS, HDS, or HHS before reperfusion, capillary perfusion decreased to a significantly minor extent of approximately 15%, and macromolecular leakage was slightly increased to approximately 5%. Our results suggest that hyperosmolar saline dextran effectively attenuates postischemic microvascular disturbances elicited by ischemia-reperfusion, presumably through reduction of postischemic leukocyte-endothelium interaction and capillary swelling.

A novel model for the investigation of the microcirculation in synovial tissue of the mouse knee joint is presented. The mouse knee joint was exposed on a specially designed plexiglass stage with a slight flexion. After partial resection of the skin, the patella tendon was cut transversally, which allowed for visualization of the "Hoffa's fatty body", an intraarticular fatty tissue containing synovial cells on the interior surface of the joint. An intravital fluorescence microscope was adjusted to observe the microcirculation of this intraarticular synovial tissue without opening of the joint capsula. For staining of the plasma, fluorescein isothiocyanate (FITC)-dextran was used, and for the staining of leukocytes rhodamine 6G was used. The tissue investigated presents with a high-density honeycomb-like capillary network, containing some postcapillary venules and a few arterioles. The following parameters were assessed off-line using a computer-assisted microcirculation analysis system: flow and diameter of arterioles and postcapillary venules, as well as functional capillary density. Moreover, leukocyte-endothelial cell interaction was quantified by counting the number of rolling cells and cells adhering to the endothelium in postcapillary venules. As an indication of endothelial leakage, macromolecular extravasation was also assessed. To validate the model, we investigated these parameters at three time points during an observation period of 60 min. There was no change in functional capillary density, nor in vessel diameter after 60 min of observation. Moreover, there was neither a change in the number of rolling cells, nor in the number of cells adhering to the endothelium nor in extravasation of FITC-dextran, thus indicating the stability of the preparation. The new model allows the quantitative analysis of the intraarticular microcirculation of the synovial fatty tissue in vivo. It provides insight into the dynamics of synovial microcirculation and leukocyte-endothelial cell interaction in acute or chronic joint inflammation.

The leukocyte beta 2-integrin Mac-1 (CD11b/CD18) and its endothelial ligand intercellular adhesion molecule 1 (ICAM-1) are involved in leukocyte adhesion to and macromolecular leakage from postcapillary venules during inflammatory reactions. Both events are also encountered after ischemia-reperfusion of striated muscle, suggesting a central role of both adhesion proteins in reperfusion injury. Using intravital fluorescence microscopy and a microcirculation model in awake BALB/C mice, we investigated the effects of monoclonal antibodies (MAb) and Fab fragments to Mac-1 and MAb to ICAM-1 on leukocyte-endothelium interaction and macromolecular leakage of fluorescein isothiocyanate-dextran (1.5 x 10(5) mol wt) in striated skin muscle after 3 h of ischemia followed by reperfusion. We demonstrated that administration of MAb and Fab to Mac-1 before reperfusion was as effective as administration of MAb to ICAM-1, which was found to be significantly upregulated in the postischemic tissue by immunohistochemical analysis, in preventing postischemic leukocyte adhesion to and macromolecular leakage from postcapillary venules, whereas postischemic leukocyte rolling was not affected after MAb administration. Postischemic capillary perfusion was efficiently preserved in animals treated with anti-Mac-1 and anti-ICAM-1 MAb compared with animals receiving the isotype-matched control antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)