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Laboratoire d'Etude de la Microcirculation, Université de Medecine Denis Diderot, Paris, France
Submitted 27 March 2007 ; accepted in final form 19 November 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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microcirculation
It should be stressed that most of the studies regarding the consequences of I/R in the microcirculation have examined venules (6, 18, 28). However, the study of possible changes in arterioles is very important for the understanding of changes in tissue perfusion and deleterious alterations of microvascular regulatory mechanisms.
Regarding the period during which ROS generation occurs, several controversial results have been reported. Some studies have suggested that a burst of ROS generation occurs during the first few minutes after ischemic tissues are reoxygenated, leading to the conclusion that the return of O2 to ischemic tissues is a critical event for the generation of ROS (5, 28). Some in vitro studies in human umbilical vein endothelial cells (HUVEC) have also reported the same conclusions (40, 41). However, growing evidence suggests that oxidant stress in vitro begins during ischemia, before reperfusion (29, 42). O2 may still be present during ischemia, so that ROS generation may also occur before reperfusion (4).
With regard to the source of ROS, several mechanisms have been identified. Xanthine oxidase (XO) reduces molecular oxygen, leading to the production of both superoxide and hydrogen peroxide (24). Endothelial nitric oxide synthase can produce considerable amounts of superoxide in the absence of sufficient tetrahydrobiopterin (43), whereas inducible nitric oxide synthase-dependent superoxide generation has been shown only for the L-arginine-depleted enzyme (31). Another important source of superoxide in vascular cells is NADPH oxidase (23).
More recently, the role of mitochondria as an important source of ROS in vascular cells has been reported (40).
Thus, in the present study, we aimed to answer the following questions. 1) Is there an increase in vivo of ROS production in the wall of arterioles submitted to ischemia and/or reperfusion? 2) If present, what are the sources of this ROS production, and more specifically are mitochondria a possible source of ROS?
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Six-week-old BALB/c mice (CERJ, Laval, France) were used in all experiments. The mice, weighing 22 ± 2 g (mean ± SE), were anesthetized by intraperitoneal injection of 90 mg/kg pentobarbital sodium (Sanofi). Mice were tracheotomized, and a cannula was inserted into the trachea to facilitate spontaneous breathing. The right carotid artery was catheterized with a PE-10 polyethylene cannula. This catheter was filled with 0.9% saline and connected to a pressure transducer (MP30, BIOPAC Systems) for continuous recording of systemic mean arterial blood pressure. A slow intra-arterial infusion (10 µl/min) of physiological salt solution was given throughout the experiment to prevent clotting in the catheter. A catheter was also inserted into the right jugular vein to inject antioxidant drugs [superoxide dismutase conjugated with polyethylene glycol (Peg-SOD) or superoxide dismutase (SOD)].
Tissue preparation.
The left cremaster muscle was prepared according to our previously described technique (44). Briefly, the muscle was detached from the scrotum. A transverse buttonhole slit 
5 mm long was made in the proximal part of the cremaster pouch. The testicle, epididymis, and the cremaster itself were then drawn out through the buttonhole. This procedure led to the invagination of the cremaster, which acquired a finger shape, with the cremaster pouch now turned inside out. The small pedicle that attaches the cremaster to the testicle was tied up with two stitches and cut between them, to separate the cremaster completely from the testicle. A flexible extensible ovoid ring was made with metal wire (diameter of 0.1 mm) and expanded gently, spreading out the cremaster, which acquired a racket shape. The ring was positioned so that the main cremaster artery was in the center of the racket's upper surface. Throughout these procedures, the muscle was continuously bathed with saline solution. This procedure involves minimal incision of the cremaster and so reduces considerably the risk of hemorrhage and lesions of the muscle and its microcirculation. Because the size of the ring adapts to the dimensions of the cremaster, extension of the muscle is sufficient to allow good optical resolution but does not affect the microcirculation. The muscle was continuously superfused at 1.2 ml/min with a modified Krebs-Henseleit solution at a temperature of 34.5°C in the muscle chamber.
Intravital microscopy. After preparation of the cremaster was completed, the stage was placed under a fluorescence microscope (Leitz) to allow for transilluminating and epifluorescence microscopy. The microscope was equipped with x25 water immersion lenses (Leitz) and a high-performance charge-coupled device camera (COHU, San Diego, CA). To elicit fluorescent images, the preparation was illuminated with a 200-W mercury lamp. The light was passed through a fluorescence microscope attachment with quartz collector, excitation filter (515–560 nm) for epi-illumination, and a band-pass filter (580 nm). During the intervening periods, the shutter for the excitation light was kept closed. The fluorescent images were recorded and stored in the memory of a computer (LG-3, Scion). The signal of a time generator (VTG-33; For-A-Company, Tokyo, Japan) was superimposed to facilitate evaluation of video images for later analysis. The fluorescence intensity was measured by image analysis software (Image-Pro Plus, Media Cybernetics).
Measurement of ROS generation in the cremaster microcirculation by using dihydroethidium. Dihydroethidium (DHE) was used as an oxidant-sensitive probe, to assess ROS generation in the cremaster microcirculation. DHE, a reduced and nonfluorescent precursor of ethidium bromide, can enter the cell. In the presence of oxidative challenge, hydroethidium is transformed intracellularly into the fluorescent compound ethidium bromide, which binds to DNA and can be detected by red fluorescence (47). Hydroethidium is especially sensitive to superoxide anion and to a lesser degree to H2O2 (42). ROS generation, in the cremaster arteriolar wall, was determined according to the modification of the method described by Suzuki et al. (39). The intensity of the fluorescent signal was measured as arbitrary units in two opposite walls along the vessel (100 µm length; see Fig. 1). The same section of the arteriole was studied under each experimental condition, and fluorescence intensity at time x (ITx) was expressed as difference between each time and baseline (change in intensity = ITx – IT0, where T0 is time zero). In previous experiments, we observed that different mercury lamps (even with the same technical characteristics) could induce significant changes in intensity values. To avoid any possibility of bias because of this phenomenon, when studying inhibitors, we used a total of four lamps and included systematically a control group and an I/R group in each series of experiments performed with each lamp.
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Experimental protocols. Two arterioles (52 ± 1.4 µm) were selected for measurement in each muscle. After an initial 45-min stabilization period, while the cremaster preparation was superfused with a standard buffer, a background autofluorescence image, in the selected tissue area, was recorded. After these measurements, mice were randomly allocated to different treatment groups. The preparation was then superfused with a buffer solution containing DHE (1 µM) for 105 min. Baseline fluorescent images were recorded 15 min after the onset of DHE superfusion (T0) and every 15 min until the end of the experimentation. At T0, ischemia was produced by occluding the arterial inflow to, and the venous outflow from, the cremaster muscle. Atraumatic microvascular clips were placed on the proximal part of the cremaster muscle to achieve complete ischemia for a period of 1 h. The clamp was then removed to restore circulation, and the microcirculation was observed during 30 min of reperfusion.
Changes in ROS generation in cremaster arterioles during ischemia and the recovery period. A total of 131 mice were studied in the present experiments. We studied the effect of I/R (n = 31 mice) on the intensity of fluorescence measured in the walls of cremaster muscle arterioles. This intensity was compared with that obtained under normal conditions of perfusion in the control group (n = 30 mice). To evaluate the source of superoxide generation, the cremaster muscle arterioles were treated on the one hand by inhibitors of cytoplasmic ROS production, allopurinol [100 µM, XO inhibitor (33)] or apocynin [100 µM, NADPH oxidase inhibitor (14)], and on the other hand by inhibitors of the mitochondrial respiratory chains, rotenone [10 µM, complex I inhibitor (32)], stigmatellin [1 µM, complex III inhibitor (2)], or antimycin A [10 µM, complex III inhibitor (10)]. Six mice were used for each inhibitor. A complementary series of experiments, decided a posteriori on the basis of the results obtained, was also performed with thenoyltrifluoroacetone [TTFA; 10 µM, complex II inhibitor, n = 5 mice (32)] or with simultaneous administration of allopurinol and stigmatellin (at the concentrations mentioned above; n = 6 mice). These concentrations were selected on the basis of previously reported inhibitory activity. All of the inhibitors were added to the Krebs-Henseleit suffusion of cremaster muscle at the same time as the fluorescent probe (DHE) and were present until the end of the experiment.
To test whether SOD, which degrades superoxide to H2O2, decreased the I/R-induced stress in the arteriolar wall, we injected a bolus of either SOD from bovine erythrocytes (n = 5) or Peg-SOD (n = 6). These injections were made at the beginning of the stabilization period, 1 h before ischemia was produced. The doses of antioxidants were given intravenously at 17 mg/kg (28) and 2,000 U/kg (15), respectively, in a bolus of 0.1–0.15 ml.
In complementary control experiments, we studied DHE fluorescence in control mice to which allopurinol or stigmatellin was administered during the usual treatment periods of the experiments (n = 4 mice for each inhibitor).
In addition, in four mice, we checked the possibility increased ROS production is responsible for vascular cell death using propidium iodide (PI), a fluorescent marker of cell death. PI was added to the superfusate at a final concentration of 1 µM, 30 min before I/R (21). Measurements were made at baseline, at the end of the ischemic and the reperfusion periods, and after 15-min exposure to 10% ethanol.
In our experimental model, the arteriolar wall shows an autofluorescence at the basal state. This autofluorescence was also studied in two complementary groups: after I/R and under conditions of normal perfusion (3 mice in each group).
Drugs and chemicals. Krebs solution, rotenone, antimycin A, TTFA, stigmatellin, allopurinol, PI, SOD, and Peg-SOD were purchased from Sigma. Apocynin was purchased from Calbiochem and DHE from Molecular Probes. Allopurinol was dissolved at 50 mg/ml in 1 M sodium hydroxide. DHE, rotenone, TTFA, apocynin, and stigmatellin were dissolved in DMSO and antimycin A in ethanol. These stock solutions of inhibitors and DHE were aliquoted and stored at –20°C. Dilutions from the stock were prepared freshly on the day of the experiment in Krebs solution.
Statistical analysis.
All results are reported as means ± SE, with n referring to the number of mice. Comparison of diameters before and after inhibitor and before and after ischemia were made by two-way ANOVA (i.e., factor group and factor time). When interaction between factors was significant, effect of time was analyzed in each group by paired t-test, with adjustment of significance for multiplicity. With regard to fluorescence, with the change of lamp, the intensities measured in control and I/R groups show significant differences between lamps. All statistical analyses were carried out lamp-by-lamp, and intensity variations (
I = ITx – IT0) were analyzed by repeated-measures two-way ANOVA. Post hoc analyses were performed when the two-way ANOVA was significant. A P value of <0.05 was considered statistically significant after adjustment for multiplicity made by the Tukey method. The same method was used for analysis of diameter. All calculations were made using either Statview 5.0 or SAS from SAS Institute.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The mean basal diameters of arterioles were not statistically different among groups (Table 1). Diameter results did not change during the experiment in the control groups, whereas they were reduced during the 1 h of ischemia in all other groups (–15 ± 2%) and returned to baseline values during reperfusion. Thus no significant differences among all groups were observed at minute 90. No significant changes in diameter were associated with pharmacological blockers except for apocynin and TTFA, for which a significant constriction was observed (P < 0.05).
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Figure 1 shows representative images of DHE fluorescence during I/R. As shown in Figs. 1 and 2, we found a significant increase in DHE fluorescence during ischemia in the I/R group compared with that shown in the control group. This increase occurred in the first minutes after ischemia (minute 15). No additional increase in fluorescence was observed during the 30 min of reperfusion in the I/R group.
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Identification of sources of ROS production in arterioles submitted to I/R. As shown in Fig. 3A, we found that allopurinol, an inhibitor of XO, significantly reduced the increase in ROS induced by ischemia. Apocynin, which inhibits NADPH oxidase, only exhibited a nonsignificant trend toward a decrease in ROS production.
With regard to the production of ROS by mitochondria (Fig. 3B), no significant reduction in fluorescence was observed in the arteriolar wall during the ischemia when we blocked complex I with rotenone or complex II by TTFA. In contrast, a significant inhibition of I/R-induced ROS production was found when we blocked the Qo site (oriented toward the intermembrane space) of complex III by stigmatellin. We observed the opposite effect under antimycin A, which blocks the Qi site (located in the inner membrane and facing the mitochondrial matrix); i.e., it markedly increased ROS production. The simultaneous administration of allopurinol and stigmatellin does not induce a reduction in fluorescence significantly different from that obtained with allopurinol alone.
It should be noted that we did not find any effect of these inhibitors on the basal value of arteriolar wall fluorescence measured 15 min after the topical administration (T0). In complementary experiments, we also checked that allopurinol or stigmatellin (the 2 inhibitors found to interfere with ischemia-induced increases in ROS production) did not affect DHE fluorescence in control vessels (data not shown).
In all experiments, we found that, during the reperfusion period, ROS fluorescence remained stable at the same level as that at the end of the ischemia period. During this period, no significant difference was observed between the I/R group and the I/R groups treated with inhibitors.
As illustrated in Fig. 4, we did not see any significant PI fluorescence during the ischemic period. Only rare, sparse fluorescence was observed during the reperfusion period in contrast to that observed after exposure to ethanol. In complementary experiments, we also checked that DHE did not induce cell death during I/R. Thus we compared the effect of I/R in the cremaster arteriolar wall treated by DHE alone vs. the effect with DHE+PI. We did not find any additional fluorescence when both probes were used simultaneously, thus confirming the absence of toxicity (data not shown).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In the pathological process of I/R injury, an increase in ROS production has been evoked as an important mechanism contributing to various types of microvascular dysfunction that trigger tissue injury, including impaired endothelium-dependent vasodilation, leukocyte plugging, and increased vascular permeability (6).
With regard to in vivo studies, most of those on I/R and ROS have been performed on the venular side of the microcirculation (18, 28). It has been shown that hydroxyl radical synthetized by XO is implicated in increased vascular permeability in I/R (27). In the same way, in 1989, Granger et al. (17) showed, in an intravital microscopy study, the role of ROS produced by XO in increasing leukocyte activation in cat mesenteric venules exposed to 1 h of I/R.
The present study represents, to our knowledge, the first direct demonstration of the influence of I/R injury on ROS production in the wall of arterioles. We could observe fluorescence in both smooth muscle cells and endothelial cells. However, the fluorescence was almost continuous in the endothelium and sparse in the smooth muscle layer. It was clear that the major contribution of fluorescence quantified in the present experiments was due to the endothelial layer. The increase in ROS is important because it can affect, in a dramatic way, all of the microvascular regulatory mechanisms. Although we did not test here NO-dependent dilation, our results are in line with the observations that SOD and other antioxidants restored the dilatory response in postischemic arterioles (23). They are also in line with findings by Bari et al. (3) showing the role of ROS in altered arteriolar responses to NMDA in a model of cerebral hypoxia or with findings by Rosenblum and Wormley (36) showing the same implication of ROS in the alteration of responses to endothelium-dependent dilators after hypoxia in pial arterioles of mice.
Considerable attention has focused on the role of ROS in the pathophysiology of I/R. Some studies have suggested that a "burst" of ROS generation occurs when molecular oxygen is returned to ischemic cells in vitro (33, 40) or in vivo (5, 28).
In contrast, the present study showed that, on the arteriolar side, an increase in ROS production occurs rapidly after the beginning of ischemia. In contrast, in the reperfusion period, ROS production did not increase any more and appeared relatively stable, at a high level. Because ROS strongly interact with all microvascular regulatory mechanisms that are crucial for adequation of oxygen supply to oxygen demand in the different organs, these results stressed the importance to prevent or to limit the oxidative stress generation as soon as a hypoxic situation is present. Our observations are in line with those of Steiner et al. (38), who demonstrated that hypoxia per se induced ROS production in rat mesenteric venules. Our observations are also in line with the results of Zuo and Clanton (48), obtained in a model of isolated rat diaphragm strips, which showed that ROS are generated in skeletal muscle during exposure to acute hypoxia, before reoxygenation. Finally, our observations are also in line with results obtained with cultured cardiomyocytes, in which ischemia before reperfusion generated significant amounts of ROS inside the cells (4, 11, 42).
One possible explanation for the discrepancies regarding the importance of an ischemic vs. reperfusion period in ROS production might be the differences in the type of ROS detected because of the different fluorescent probes used. Even if the specificity of each probe may be debatable (16), it is known that oxidation of dichlorodihydrofluorescein diacetate (to fluorescent dichlorofluorescein diacetate) is indicative of H2O2 or hydroxyl radical formation, whereas oxidation of DHE [to fluorescent ethidium bound to DNA (Eth-DNA)] provides a measure of superoxide formation. Indeed, all of the in vivo I/R studies cited that reported an increase in ROS only at the time of the reperfusion used dichlorodihydrofluorescein diacetate. Thus these studies did not exclude the possibility that an undetected increase in superoxide occurs during ischemia. This possibility is in line with the experiments by Vanden Hoek et al. (42) in cardiomyocytes exposed to I/R, which showed an increase in Eth-DNA fluorescence during ischemia and a significant increase in dichlorofluorescein diacetate fluorescence that occurred during reperfusion associated with a decrease in Eth-DNA fluorescence. Note, however, that recent results by Fernandes et al. (12), which examined the DHE-derived oxidation products by HPLC, showed that fluorescence was due to two products: 2-hydroxyethidium, known to be more specific for superoxide anion, and ethidium, a less specific product. Many interactions also exist between the different ROS possibly affecting DHE-derived fluorescent products (12). This makes it difficult to relate the present observed increase in DHE fluorescence exclusively to an increase in superoxide anion.
In the present experiments, we did not find significant vascular cell death either during ischemia or during the reperfusion period. It is likely that the duration of these periods was too short to allow the present increase in ROS production to be associated with cell death. This is in line with previous results by Harris et al. (22) who found significant cell death only after 3 h of I/R.
Early work by Zweier et al. (49) demonstrated that XO may be the major source of cytotoxic ROS in endothelial cells. The present study found a significant inhibition of I/R-induced ROS production by allopurinol. This observation supports the involvement of the XO pathway in this production and is in accordance with previous studies reporting involvement of XO in disturbances associated with I/R in venules. Indeed, several studies have shown that agents that scavenge or prevent the production of oxygen radicals via the XO pathway significantly reduce the leukocyte adherence and/or albumin leakage in venules after I/R (28, 35). Involvement of XO in endothelial dysfunction and blood flow deficits in the mesenteric microcirculation after resuscitated hemorrhagic shock was also found by Flyn et al. (13).
NADPH oxidase, originally characterized in neutrophils, is also present in endothelial cells (25) and is the principal source of superoxide production in several animal and human models of vascular diseases (19, 26, 45). However, in our experiments, we did not find a significant decrease in ROS production in the presence of apocynin, a NADPH oxidase inhibitor. A similar absence of effect of apocynin was also found in HUVEC submitted to I/R (40).
More recently, mitochondria have also been identified as an important source of ROS production. In isolated mitochondria, the ROS generation stimulated by the substrates NADH, ubiquinone, and succinate has been shown to involve respiratory chain complexes I and III (8).
Complex I is a membrane multiprotein complex that oxidizes NADH and uses coenzyme Q as an electron acceptor. It represents one of the two major entry points into the respiratory chain of mitochondria. In our experiments, when we blocked complex I with rotenone, no reduction in the fluorescence was observed in the arteriolar wall during ischemia. This result is similar to that found in HUVEC after I/R (40). However, it should be noted that rotenone blocks electron transfer in close proximity to the ubiquinone binding site. Some authors have reported the possibility that there may be more than one site of ROS production in complex I, including flavin or iron-sulfur centers upstream of the rotenone blocking site (1). Thus the absence of effect of rotenone cannot completely rule out the possibility that these complex I sites can be involved in ROS production. Another possibility showed by Paddenberg et al. (32) in the pulmonary vasculature is that, under hypoxic conditions, complex II may have an essential role in ROS production. Our results clearly showed the possible heterogeneity of the sources of ROS because, in muscle arterioles, we did not see any effect of TTFA used at the concentration demonstrated to inhibit ROS production in pulmonary vasculature. In contrast, we found a significant inhibition of superoxide anion production when complex III was inhibited by stigmatellin. This finding suggests that, in addition to XO, complex III is also involved in superoxide anion production by arterioles submitted to ischemia. Complex III of mitochondrial respiratory chain is an enzyme complex (cytochrome bc1) that oxidizes coenzyme Q and uses cytochrome c as electron acceptor. The oxidation of coenzyme Q proceeds as a set of reactions known as the "Q cycle" (see Ref. 1 for review). This cycle has two sites: the Qo site, oriented toward the intermembrane space, and the Qi site, located in the inner membrane and facing the mitochondrial matrix. In our study, we found that stigmatellin, a Qo-site inhibitor of cytochrome bc1, inhibited ROS production in the arteriolar wall during ischemia. By contrast, antimycin A, a Qi-site inhibitor, markedly increased this ROS production. Both of these observations are in line with those on the consequences of these two inhibitors on ROS production by isolated mitochondria (8). They are also qualitatively similar to findings in cellular preparations using hepatocytes (7), cardiomyocytes (49), and HUVEC (10, 40). The possibility of an absence of effect of some inhibitors in a specific preparation should always be kept in mind; however, it should be noted that we have only used inhibitors that have been extensively used by many others for inhibiting mitochondrial function, including in vascular cells. Thus these concordant results from cellular and subcellular studies, and here from in vivo study, lead to the general concept that complex III has a central role in mitochondria ROS production.
In summary, the present results demonstrate that, in the arteriolar wall, in vivo production of ROS was increased during ischemia. This production did not increase during reperfusion. The two main sources of ROS production were identified as XO and complex III of mitochondria.
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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is mediated by ceramide. Am J Respir Cell Mol Biol 24: 762–768, 2001.
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) and in the control group (without I/R; 
). Results are means ± SE. #P < 0.05, comparisons between groups. 
P < 0.01 and 
