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Am J Physiol Heart Circ Physiol 289: H330-H335, 2005; doi:10.1152/ajpheart.00674.2004
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Paradoxical attenuation of leukocyte rolling in response to ischemia- reperfusion and extracorporeal blood circulation in inflamed tissue

Stephan C. Schäfer,1 Desiree N. Sehrt,1 Markus Kamler,2 Heinz Jakob,2 and Hans-Anton Lehr1

1Institute of Pathology, University of Mainz Medical Center, Mainz; and 2Clinic for Thorax and Cardiovascular Surgery, University Clinic of Essen Medical Center, Essen, Germany

Submitted 7 July 2004 ; accepted in final form 24 January 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In contrast to acute preparations such as the exteriorized mesentery or the cremaster muscle, chronically instrumented chamber models allow one to study the microcirculation under "physiological" conditions, i.e., in the absence of trauma-induced leukocyte rolling along the venular endothelium. To underscore the importance of studying the naive microcirculation, we implanted titanium dorsal skinfold chambers in hamsters and used intravital fluorescence microscopy to study venular leukocyte rolling in response to ischemia-reperfusion injury or extracorporeal blood circulation. The experiments were performed in chambers that fulfilled all well-established criteria for a physiological microcirculation as well as in chambers that showed various extents of leukocyte rolling due to trauma, hemorrhage, or inflammation. In ideal chambers with a physiological microcirculation (<30 rolling leukocytes/mm vessel circumference in 30 s), ischemia-reperfusion injury and extracorporeal blood circulation significantly stimulated leukocyte rolling along the venular endothelium and, subsequently, firm leukocyte adhesion. In contrast, both stimuli failed to elicit leukocyte rolling in borderline chambers (30–100 leukocytes/mm), and in blatantly inflamed chambers with yet higher numbers of rolling leukocytes at baseline (>100 leukocytes/mm), we observed a paradoxical reduction of leukocyte rolling after ischemia-reperfusion injury or extracorporeal blood circulation. A similar effect was observed when we superfused leukotriene B4 (LTB4) onto the chamber tissue. The initial increase in leukocyte rolling in response to an LTB4 challenge was reversed by a second superfusion 90 min later. These observations underscore 1) the benefit of studying leukocyte-endothelial cell interaction in chronically instrumented chamber models and 2) the necessity to strictly adhere to well-established criteria of a physiological microcirculation.

leukotriene B4; animal model; intravital microscopy

THE MULTISTEP CONCEPT of leukocyte-endothelial cell interaction proposes early leukocyte tethering and rolling along the endothelial wall followed by firm leukocyte adhesion and transendothelial emigration; each consecutive step involves distinct adhesion molecules and distinct chemoattractant and/or adhesion-promoting mediators (5, 21, 23, 29). It is widely established that leukocyte rolling along the endothelium constitutes a prerequisite for subsequent leukocyte adhesion and emigration (22). However, Kanwar and co-workers (21) identified an enormous redundancy in this system: even an inhibition of leukocyte rolling by >90% with fucoidin or selectin antagonists exerted no significant effect on subsequent firm leukocyte adhesion.

In intact animal organisms, the study of leukocyte-endothelium interaction can be accomplished by intravital microscopy on acutely prepared tissues (e.g., hamster cheek pouch, cremaster muscle, and exteriorized mesentery) or on chronically instrumented tissues [e.g., the dorsal skinfold chamber model in hamsters (10, 28) and mice (27)]. Although in most acutely prepared tissues, there is always "spontaneous" leukocyte rolling under baseline conditions (7), baseline leukocyte rolling is virtually absent in carefully executed chronically instrumented tissues and is even considered a marker of tissue inflammation (for review see Ref. 28).

The present study was motivated by a laboratory accident: in a series of ischemia-reperfusion experiments, the strict criteria for a "good" chamber were not applied, and mildly or even overtly inflamed chambers were included in the experiments. Only when the data for leukocyte-endothelium interaction before and after reperfusion injury were reviewed did we discover that although in this series of experiments the stimulation of postischemic leukocyte adhesion was comparable to that in previous studies, there was virtually no postischemic increase in leukocyte rolling, which was in striking contrast to previous experience on the same model (25, 26). On analysis of the data obtained in individual postcapillary venules, we found that leukocyte rolling increased significantly in those vessels that had only few rollers at baseline (i.e., good chambers) but was virtually unchanged in vessels with moderately enhanced baseline rolling and even dropped paradoxically in vessels with robust baseline rolling (i.e., inflamed chambers). The present series of experiments was performed to validate this accidental observation and to extend it to another pathophysiological stimulus of leukocyte rolling under the condition of extracorporeal blood circulation (18, 19).

Furthermore, we saw parallels of this accidental observation with well-established differences in exogenously stimulated leukocyte rolling between acutely prepared and chronically instrumented microvascular beds. For instance, superfusion of an acutely prepared hamster cheek pouch microcirculation with leukotriene B4 (LTB4) significantly reduces baseline leukocyte rolling within only a few minutes, in parallel with an increase in leukocyte adhesion (7). Yet, in the same species, LTB4 superfusion onto a chronically implanted observation chamber conversely increases leukocyte rolling and leukocyte adhesion simultaneously (25). Despite this contrasting effect of LTB4 on leukocyte rolling, the adhesion-promoting effect of LTB4 on leukocytes is comparable in both situations (7, 25). Consistent with this line of reasoning, Ley and co-workers (30) observed a time-dependent alteration in leukocyte rolling flux after acute exteriorization of the cremaster muscle in wild-type mice as well as in other animal species. Leukocyte rolling flux reached its maximum 40–60 min after the beginning of the surgical procedure, in agreement with earlier findings in the hamster cheek pouch and the mouse and rat mesentery (30).

In this report, we provide experimental evidence that the attenuation of leukocyte rolling by LTB4 superfusion in acutely prepared tissues represents an unphysiological response that can be mimicked by a second superfusion of LTB4. This concept is further supported by the demonstration that leukocyte rolling in response to ischemia-reperfusion injury (25, 26) or extracorporeal blood circulation (18, 19) is significantly blunted and even paradoxically reversed in inflamed chambers, which show increased leukocyte rolling at baseline.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal model. The dorsal skinfold chamber preparation in awake Syrian Golden hamsters was used for intravital microscopy. The experimental preparation used in this study is very similar to that described previously in detail (10, 25, 28), with only minor modifications. Briefly, inbred 6- to 8-wk-old (55–70 g body wt) Syrian Golden hamsters were fed standard rodent diet and allowed water ad libitum; they were anesthetized by injection of pentobarbital sodium (60 mg/kg body wt ip; Narcoren, Merial, Hallbergmoos, Germany). The entire back of the animal was shaven, and two titanium frames were implanted so as to sandwich the extended double layer of the skin. One layer of the skin was completely removed in an 18-mm-diameter circular area, and the remaining layer, consisting of epidermis, subcutaneous tissue, and a thin striated skin muscle, was covered with a coverslip incorporated in one of the frames. The dorsal skinfold chambers were well tolerated by the animals, i.e., they showed no signs of discomfort and no adverse effects on feeding and sleeping habits. An indwelling catheter was implanted into the right jugular vein in the same session. The experiments were conducted in accordance with the national and institutional guides for the care and use of animals.

Intravital fluorescence microscopy. Between the implantation of the observation chamber and the microscopic investigation, a recovery period of 72–96 h was allowed to eliminate the effects of anesthesia and surgical trauma on the microvasculature. Epi-illumination (100-W xenon lamp attached to an Axiotech intravital microscope, Zeiss, Jena, Germany) and a x20 water immersion objective (total magnification x560; Zeiss) were used to select 10 regions of interest per chamber, each containing ≥1 characteristic draining venule with a diameter of 20–50 µm. Rhodamine 6G (Sigma-Aldrich Chemie, Taufkirchen, Germany) was administered intravenously immediately before the microscopic studies to visualize leukocyte rolling and firm adhesion to the vessel wall. To minimize the phototoxic effect, we used 1) rhodamine 6G, which is far less phototoxic than acridine orange (37), as a marker of leukocytes and 2) an intermediary amplifier as well as a fluorescent light dimmer. In this way, it is possible to dim light exposure to <20% of the output power of a 100-W fluorescent lamp (Fluo Arc HBO 100). In pilot studies, we tested the potential phototoxic effect of this setup with an observation period up to 4 h and a reapplication of rhodamine 6G after 2 and 4 h. Under these conditions, we observed no significant effect of light exposure on the number of rolling or sticking leukocytes. Leukocytes were classified according to their interaction with endothelial cells as rolling and adherent leukocytes as previously described in detail (25). The microscopic images were recorded with a high-resolution black-and-white camera (Kappa, Gleichen, Germany) on S-VHS video tape. A computer-controlled stepping motor (Märzhäuser, Wetzlar, Germany) was used to investigate the identical sites of interest at baseline and 2 and 4 h after 2 h of pressure-induced ischemia (25) or 20 min of extracorporeal circulation (18, 19), respectively, or at defined times after leukotriene superfusion (25).

Extracorporeal blood circulation. The experimental protocol was carried out as described elsewhere (18, 19). The experiments were performed in the laboratory of Markus Kamler (University of Essen Medical School). Briefly, extracorporeal blood circulation was introduced via a micro-roller pump (Alitea, Stockholm, Sweden) and Silastic tubing (1 mm ID, 60 cm long; Migge, Heidelberg, Germany) shunted between the carotid artery and the jugular vein in 20 animals. The sterilized extracorporeal circuit was primed with 1 ml of Ringer solution, and the flow rate was adjusted to 1 ml/min. Extracorporeal blood circulation was then continued for a total of 20 min (19). The animals tolerated these procedures very well and showed no signs of discomfort or changes in blood pressure or heart rate. The observation chamber temperature was 28°C, which declined only slightly during extracorporeal blood circulation. The percentage of the cardiac output represented by the extracorporeal blood circulation is ~3%/min. If the total blood volume of the hamster and the flow rate of the pump are considered, 21 ml of blood are transferred through the tube system within 30 min.

Ischemia-reperfusion. The experimental protocol was performed as described previously (25, 28). The experiments were performed in the laboratory of Hans-Anton Lehr (University of Mainz Medical School). In 20 animals, a 2-h period of ischemia was induced by application of gentle pressure on the muscle against a coverslip with a silicone pad and an adjustable screw that was just sufficient to empty the blood vessels, as described previously (25, 26). With the help of a computer-assisted stepping motor-driven microscope stage, the same vessel segments that had been recorded at baseline were investigated again at 2 h of reperfusion.

Topical leukotriene application. The experimental protocol was performed as described previously (25). LTB4 (Amersham Buchler, Braunschweig, Germany) or its vehicle (1% ethanol in 0.9% saline) was superfused directly onto the striated muscle within the observation window at 20 nmol/l for 3 min. The superfusate was then washed away with physiological saline solution, and stimulated leukocyte rolling was quantified by intravital microscopy at 15, 30, 60, and 90 min, as well as 15 min after renewed leukotriene superfusion at 20 nmol/l.

Image analysis. Unbranched venules (25–50 µm diameter, >250 µm long) were selected for observation. Fluorescently labeled leukocytes moving in the periphery of the axial stream across an imaginary line perpendicular to the axis of the vessel were considered rolling leukocytes. These cells were counted for 30 s and assessed as a fraction of the microvessel circumference, which was calculated from the microvessel diameter, which was assessed by Photoshop-based image analysis (4) and with the assumption of cylindrical vessel geometry. Leukocyte rolling velocity was assessed by quantifying the time (in seconds) needed by the leukocytes for a displacement of 200 µm. Leukocyte adhesion was quantified as cells that remained in the same spot on the endothelial lining during 30 s, expressed as the number of cells per endothelial surface (as calculated from the microvessel diameter and a defined length of 200 µm) (25, 26).

Statistical analysis. Values are means ± SD. Wilcoxon's test was performed for intergroup comparisons, and Spearman's rank correlation was performed to compare rolling and adherent leukocytes at baseline vs. the difference between stimulated minus baseline rolling leukocytes and adherent leukocytes in ischemia-reperfusion and extracorporeal blood circulation-treated animals. Leukocyte rolling velocity under baseline conditions was compared with that under stimulated conditions, and Wilcoxon's test was performed for intergroup comparisons. Differences of rolling leukocyte velocity under baseline and stimulated conditions were assessed and plotted against the change in the number of leukocytes rolling under baseline and stimulated conditions (see Fig. 4).



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  		Fig. 4. Correlation between alterations in leukocyte rolling velocity ({Delta}v, horizontal axis) and differences of stimulated minus baseline leukocyte rolling ({Delta}n, vertical axis) in 20 hamsters. Horizontal axis: difference between mean leukocyte velocity in single unbranched venules before and after 2 h of ischemia and 2 h of reperfusion. Positive values indicate decrease in leukocyte rolling velocity after ischemia-reperfusion. Vertical axis: difference between stimulated and baseline number of rolling leukocytes, normalized to vessel diameter. Each circle represents data from 1 animal.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The effect of extracorporeal circulation on leukocyte rolling is shown in Fig. 1. In analogy to previous observations (18, 19), extracorporeal blood circulation rapidly induced leukocyte rolling along the endothelium. However, this effect was seen only in optimal chamber preparations, in which <30 leukocytes were rolling under baseline conditions. When even mildly inflamed chambers were included in the experiments, the rolling-promoting effect of extracorporeal blood circulation was inconsistent and no longer statistically significantly different from baseline conditions (Fig. 1). When we included obviously inflamed chambers, in which ≥100 leukocytes rolled at baseline, in the experiments, extracorporeal blood circulation resulted in a paradoxical reversal of stimulated leukocyte rolling, with fewer leukocytes rolling after than before extracorporeal blood circulation. However, because of the enormous standard deviations of rolling responses in inflamed chambers, this effect reached no statistical significance. In agreement with previous observations (18, 19), extracorporeal blood circulation did not affect vessel diameters (not shown).



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  		Fig. 1. Leukocyte rolling, normalized to microvessel circumference at baseline (horizontal axis) and stimulated by 20 min of extracorporeal blood circulation. To better visualize dependency of extracorporeal circulation-induced leukocyte rolling vs. the extent of leukocyte rolling at baseline, postextracorporeal blood circulation leukocyte rolling is expressed as the difference between stimulated and baseline leukocyte rolling (vertical axis). A total of 200 vessels were examined in 20 hamsters, and data were stratified according to baseline leukocyte rolling in increments of 10 rolling cells (horizontal axis). Wilcoxon's test was used to test for statistically significant differences between baseline and stimulated leukocyte rolling: *P < 0.05. Spearman's correlation was calculated over the entire data set, and the calculated function is shown in the gray bar (r = –0.60, P < 0.01). Values are means ± SD.

 
The effect of ischemia-reperfusion injury on leukocyte rolling is shown in Fig. 2. In analogy with previous experiments (25, 26), ischemia-reperfusion resulted in a significant increase in leukocyte rolling along the endothelium. Consistent with the observations after extracorporeal blood circulation (Fig. 1), this effect was seen only in optimal chambers (<30 leukocytes rolling at baseline) and was subsequently lost in mildly inflamed chambers (30–80 rolling leukocytes) or even paradoxically reversed in obviously inflamed chambers (>90 rolling leukocytes at baseline). Baseline leukocyte rolling as a marker of chamber inflammation had virtually no significant impact on stimulated leukocyte adhesion to the endothelium, with a slight, albeit nonsignificant, trend toward reduced postischemic leukocyte adhesion in more inflamed chambers (Fig. 2, inset). In agreement with previous experiments (25, 26), ischemia-reperfusion injury had no effect on microvessel diameters (not shown).



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  		Fig. 2. Leukocyte rolling, normalized to microvessel circumference at baseline (horizontal axis) and stimulated by 2 h of pressure-induced ischemia followed by 2 h of reperfusion. To better visualize dependency of postischemic leukocyte rolling vs. the extent of leukocyte rolling at baseline, postischemic leukocyte rolling is expressed as the difference between stimulated and baseline leukocyte rolling (vertical axis). A total of 200 vessels were examined in 20 hamsters and stratified according to baseline leukocyte rolling in increments of 10 rolling cells (horizontal axis). Wilcoxon's test was used to test for statistically significant differences between baseline and stimulated leukocyte rolling: *P < 0.05. Spearman's correlation was calculated over the entire data set, and the calculated function is shown in the gray bar (r = –0.91, P < 0.01). Inset: effect of ischemia-reperfusion on firm leukocyte adhesion (20). Postischemic leukocyte adhesion is not significantly affected by the extent of baseline leukocyte rolling. For each subset of vessels, postischemic leukocyte adhesion is significantly different from baseline values (P < 0.01, not shown). Spearman's correlation yielded r = –0.26, which was statistically not significant. Values are means ± SD.

 
The effect of LTB4 superfusion on leukocyte rolling is shown in Fig. 3. In analogy with previous observations (25), leukotriene superfusion resulted in a rapid and statistically significant stimulation of leukocyte rolling along the endothelium, which remained at a rather high level during the subsequent 90 min. On repeated superfusion with LTB4 in the same mode and concentration, we observed a rapid and statistically significant drop in the number of rolling leukocytes (Fig. 3). This ~50% drop in the number of rolling leukocytes is virtually identical in terms of extent and time course to the decline in leukocyte rolling observed on initial LTB4 superfusion onto the cheek pouch preparation observed in the experiments of Dahlen and co-workers (7). In agreement with our previous observations (25) and observations by others (7), leukotriene superfusion did not affect microvessel diameters (data not shown).



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  		Fig. 3. Leukocyte rolling, normalized to microvessel circumference at baseline (–5 min) and at 15, 30, 60, and 90 min after superfusion of the microcirculation with leukotriene (LTB4; 20 nmol/l). Repeated superfusion after 90 min elicits a rapid reduction of leukocyte rolling (105 min). Microvessel diameters remain constant (not shown). Values are means ± SD of 35 postcapillary venular segments in 7 hamsters. *Significantly different from baseline (Wilcoxon's test). #Significantly different from 90 min (before repeated leukotriene superfusion) (Wilcoxon's test).

 
The effect of ischemia-reperfusion on leukocyte rolling velocity is shown in Fig. 4. Under the conditions of our experiment, ischemia-reperfusion had no significant effect on leukocyte rolling velocity in noninflamed or in borderline inflamed or blatantly inflamed chambers (Wilcoxon's test). When calculated for all chambers together, mean leukocyte rolling velocity was 45.3 ± 16.3 µm/s under baseline conditions and did not significantly change after ischemia-reperfusion (46.1 ± 17.9 µm/s). Subanalysis of data for noninflamed and borderline and blatantly inflamed chambers is shown in Table 1. It becomes apparent that the differences in postischemic leukocyte rolling in the three groups of chambers are neither associated with nor secondary to changes in leukocyte rolling velocity.


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  		Table 1. Effect of ischemia-reperfusion on leukocyte rolling and leukocyte rolling velocity in noninflamed, borderline, and blatantly inflamed chambers

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Chamber models have been used for intravital microscopy, because they allow prolonged observation periods (28, 32) and study of the microcirculation in the absence of anesthesia, which affects microcirculatory responses (9, 31), and in the absence of immediate surgical trauma (32). A classic application of intravital microscopy in chamber models has been the use of various fluorescent markers to study leukocyte-endothelial cell interaction (26, 28, 32).

The principal finding in the present study is that stimulated leukocyte rolling and, to a much lesser extent, stimulated leukocyte adhesion after ischemia-reperfusion injury and extracorporeal blood circulation are markedly influenced by the extent of leukocyte rolling under baseline conditions as a reflection of prior tissue injury (Figs. 1 and 2). In analogy to previous findings during experimental tissue preconditioning (8, 33, 40), baseline leukocyte rolling was, to a large extent, effectively protected from postischemic and extracorporeal blood circulation-induced leukocyte rolling in a "dose-dependent" fashion (Figs. 1 and 2). A quite similar effect has previously been observed in vitro in endothelial cells exposed to transient oxidative stress (41). Similarly, preconditioning with bradykinin superfusion significantly attenuated the postischemic leukocyte rolling in the rat mesentery by a mechanism that was thought to involve the preserved synthesis of nitric oxide during ischemia-reperfusion (38).

Davis and co-workers (8) proposed that P-selectin, an adhesion molecule that exists preformed in Weibel-Palade bodies and is rapidly translocated to the cell surface in response to appropriate inflammatory stimuli (15) and is centrally involved in postischemic leukocyte infiltration and tissue injury (24), is reduced as a consequence of ischemic preconditioning. Similarly, Nonaka and colleagues (33) observed in a retinal ischemia-reperfusion model that the maximum number of rolling leukocytes can be significantly reduced by ischemic preconditioning. Finally, Wang and co-workers (40) showed that preconditioning with morphine increases shedding from the neutrophil surface of gp100MEL14, the murine analog to human L-selectin and a key adhesion molecule involved in the initial steps of leukocyte rolling (39). On the basis of our present observation, we speculate that, in analogy to these earlier reports, "inflammatory preconditioning" may play an important role in our studies. The exact mechanisms that are operative in our experiments warrant further investigation.

In an effort to clarify the mechanisms by which elevated leukocyte rolling attenuates stimulated leukocyte rolling, we have performed superfusion studies with LTB4. LTB4 is a lipoxygenase product of arachidonic acid metabolism and is generated primarily by polymorphonuclear (3, 12) and mononuclear phagocytes (36). It has been identified, in chamber models (25) and other models of intravital microscopy (2, 20), as a key mediator of leukocyte-endothelial cell interaction in intact organisms after ischemia-reperfusion injury and as a potent chemoattractant responsible for the recruitment of neutrophils to the site of inflammation (34). LTB4 is of particular relevance for our present study because of an apparent discrepancy in the literature concerning the microcirculatory response under different experimental conditions. We report here that 20 nM LTB4 stimulates leukocyte rolling in vivo in the dorsal skinfold chamber model (Fig. 3). This finding is in agreement with data reported by Fox-Robichaud and co-workers (13), who described an almost twofold increase in leukocyte rolling on LTB4 stimulation of cat mesentery. In contrast to these findings, Dahlen and co-workers (7) observed the opposite effect: when they superfused 4 nM LTB4 onto the acutely exteriorized hamster cheek pouch microcirculation, leukocyte rolling was significantly reduced. Similarly, Martinsson and co-workers (31) observed a decrease of rolling leukocytes after superfusion of 10 nM LTB4 onto the cheek pouch of pentobarbital-anesthetized hamsters. Indeed, these previous observations of Dahlen and co-workers and also of Martinsson and co-workers are well reflected by our own experiment, in which LTB4 provoked a significant reduction of leukocyte rolling when superfused onto a microcirculation that had been exposed to LTB4 superfusion 90 min earlier and in which this prior superfusion had stimulated a marked increase in leukocyte rolling (Fig. 3). This suggests that prior leukocyte activation with LTB4 and potentially other mediators affords a fundamental manipulation of the renewed stimulation of inflammatory cells (e.g., L-selectin expression) (40) or endothelium (e.g., P-selectin expression) (8). Although multiple mediators work together in inflammation, LTB4 is considered one of the most potent mediators for the shedding of L-selectin (35), and it seems that it plays its role primarily in the early onset of inflammation (16). It is thus reasonable to assume that (at least part of) the paradoxical events observed in inflamed chambers in this study may be mediated through the action of LTB4. This notion is also supported by the recent demonstration that lipoxygenase products play a key role in ischemic preconditioning (6) and that lipoxygenase knockout mice fail to show protective effects on ischemic preconditioning (14). The rapid induction, within only 15 min, of leukocyte rolling by topical leukotriene superfusion in our present study makes it likely that P-selectin is involved, because this adhesion molecule is translocated to the endothelial surface within minutes, whereas most of the adhesion molecules involved in firm leukocyte adhesion require protein de novo synthesis and, hence, a much longer period of time (i.e., hours). Indeed, Zanardo and co-workers (42) showed by intravital microscopy on the internal spermatic fascia of rats that LTB4-induced leukocyte-endothelium interaction is associated with increased expression of P-selectin as well as several other alterations. Kanwar and co-workers (20) reported that blocking P-selectin with a functionally blocking antibody or administration of soluble sialyl Lewisx blocked leukotriene (albeit LTC4)-induced leukocyte rolling flux, but not velocity, in the mesentery microcirculation of rats, suggesting that P-selectin translocation and sialyl Lewisx are involved in LTC4-induced leukocyte rolling. In these studies, the time course of LTC4-induced leukocyte rolling was very similar to that in our present study: leukocyte rolling flux was rapidly stimulated, within only 15 min, and remained stimulated over the subsequent 60 min. Eppihimer and Schaub (11) reported reduced LTC4-induced leukocyte rolling after administration of a P-selectin antagonist in a mesentery venule model of intravital microscopy.

The observation that, despite drastic effects of inflammation on leukocyte rolling after ischemia and after extracorporeal blood circulation, we saw no comparable deleterious effects on postischemic leukocyte adhesion (Fig. 2, inset) underscores the fundamental differences in the mechanisms of leukocyte rolling and leukocyte adhesion (20) and is well in line with the concept that few rolling leukocytes are required for firm leukocyte adhesion (21). Further support for the validity of this observation can also be derived from a recent report in which leukocyte adhesion in response to platelet-activating factor was found unchanged in L-selectin-deficient mice (17).

Asako and colleagues (1) stressed that, in acute models of intravital microscopy, the extent of leukocyte rolling depends on the extent of the surgical trauma and that only a very careful surgical preparation results in very low levels of rolling. They concluded that only in carefully instrumented preparations can meaningful observations be made on leukocyte responses to inflammatory mediators [e.g., histamine (1)]. Our present report goes one step beyond this caveat: not only can such leukocyte responses be missed, but they can even be converted to paradoxical, contrary effects. In conclusion, our present study underscores our theory that when chronic chamber models are used for intravital microscopy, the established criteria for the absence of inflammation must be strictly applied to ensure a physiological response to ischemia-reperfusion injury, extracorporeal blood circulation, and potentially other microvascular stimuli of leukocyte-endothelium interaction (28).


   FOOTNOTES
 

Address for reprint requests and other correspondence: H.-A. Lehr, Institut Universitaire de Pathologie, Centre Hospitalier Universitaire Vaudois (CHUV), Rue du Bugnon 25, CH-1011 Lausanne, Switzerland (E-mail: Hans-Anton.Lehr{at}hospvd.ch)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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