INTRODUCTION

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IT HAS BEEN WIDELY DEMONSTRATED that ischemia resulting from such clinical conditions as hemorrhagic shock, stroke, local trauma, or myocardial infarction, followed by reperfusion of the tissue with oxygenated whole blood, can compromise microvascular and cellular integrity by causing swelling of endothelial cells (20), increased microvascular resistance associated with reductions in arteriolar diameter (27), leukocyte-capillary plugging (7, 11), and leukocyte adhesion to postcapillary venules with attendant permeability changes (5, 14, 18), phenomena that collectively contribute to ischemia-reperfusion (I/R) injury. The response of a tissue to reperfusion after ischemia may also be characterized by a "slow-reflow" or "no-reflow" response in which blood flow is reduced despite the restoration of normal perfusion pressures. The mechanisms of the no-reflow phenomenon and I/R-mediated tissue injury are still poorly understood despite extensive investigation.

Several reports suggested that leukocytes at least partially mediate postischemic microvascular dysfunction (5, 14, 19). These studies documented both significant leukocyte infiltration after I/R and dramatic increases in resistance to tissue injury after I/R in leukopenic animals compared with animals with normal numbers of circulating leukocytes (19). The role of leukocytes in I/R is multifaceted; they have been shown to play a role in increasing permeability of postcapillary venules (14) and producing cytotoxic mediators such as O₂ and proteases (13). Furthermore, activation of leukocytes and consequent stiffening caused by increases in cytoplasmic viscosity can retard leukocyte passage through capillaries (35), increase microvascular resistance (9), and in certain cases effect permanent flow stoppage.

Reactive oxygen species (ROS), especially O₂, H₂O₂, and OH · , have been implicated in both recruitment and activation of leukocytes in I/R as well as I/R injury, on the basis of evidence that scavenging of these radicals inhibits leukocyte adhesion, infiltration, and the onset of tissue injury (2, 22). Support for the chain of events implicating ROS in I/R injury stems from in vitro and in vivo experiments in which application of specific ROS inhibitors attenuated I/R injury. However, many sources of ROS may exist, and the primary factors responsible for ROS production have not been clearly defined. For example, ROS are produced by leukocytes (13), mast cells (16), capillary endothelial cells, and muscle cells (1). Many of the intracellular enzymes involved, including xanthine oxidase (XO), have been identified in several different I/R models (6, 8, 30). However, the quantity of XO produced from its precursor xanthine dehydrogenase (XDH) in skeletal muscle, particularly after short-term focal ischemia, has not been extensively studied, and the contribution of XO to ROS production relative to the other sources mentioned above is also unknown.

Further complications in interpreting I/R data stem from the great number of indicators that have been used to assess tissue injury, including tissue-wide lipid peroxidation (17), increases in venular permeability (14), myeloperoxidase (MPO) activity (28), edema (15), functional capillary density (22), and uptake of nominally impermeant fluorescent dyes by nonviable cells (11, 12, 26, 31, 32). It is also worth noting that the utility of the latter indicators in assessing long-term, irreversible muscle injury has not been established. For example, propidium iodide (PI), a fluorescent DNA-binding marker, indicates that the nuclear membrane of stained cells is compromised (31), but it is unclear whether those muscle areas exhibiting significant nuclear fluorescence will experience long-term functional impairment or necrosis. A recent study indicates that an inverse correlation exists between muscle contractility and cellular uptake of PI after 1-h ischemia and up to 2-h reperfusion but does not prove a causal relation (32).

The study presented here was motivated in part by a recent investigation (11) in which a direct linear correlation was observed between the increases in microvascular resistance caused by leukocyte-capillary plugging and the extent of tissue injury as measured by the uptake of PI by myocytes. We used the short-term I/R model of Harris and Skalak (11) to examine several specific questions. First, we attempted to resolve whether plugging of capillaries by leukocytes is capable of injuring tissue or, conversely, whether tissue and capillary endothelium first damaged by other mechanisms restrict the passage of leukocytes and thereby effect increases in leukocyte-capillary plugging. Second, we attempted to resolve the contribution of all leukocytes (plugging, adherent, and extravasated) and tissue oxidants, specifically those derived from XO, to I/R-mediated tissue injury and microvascular resistance increases. Finally, we attempted to correlate tissue injury quantified by PI uptake with two standard biochemical injury markers, tissue MPO (28) and thiobarbituric acid-reactive substances (TBARS) (17).

	METHODS

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Surgical Protocol

Ischemia Protocol and Microvascular Measurements

In addition, collecting venules of at least 12.7-µm diameter and 100-µm length that drained the selected network were each videotaped for 2 min both before the induction of ischemia and after 1-h reperfusion, and the flux of rolling and firmly adherent leukocytes was counted. Firmly adherent leukocytes were defined as those that remained stationary for at least 30 s.

Measurements of Tissue Injury

Propidium iodide. PI, a fluorescent dye that has been used as a marker for cell death in skeletal muscle (11, 12, 31, 32), was added to the superfusate at a final concentration of 1 × 10⁶ M immediately before the network sketch. A minimum of 30 min was allowed for PI to diffuse through the tissue and intercalate into the DNA of damaged muscle cell nuclei. Quantification of network tissue injury was performed by determining the total number of PI spots (N_PI) in the entire volume (V) of tissue perfused by the vascular network selected for study (11). Fluorescence visualization was performed using a ×40 water-immersion objective, a microchannel plate intensifier, and a high-pass rhodamine filter with a cutoff frequency of 590 nm. Clues to the identity of a fluorescent spot were present in the characteristic oval shape of muscle cell nuclei as well as the appearance of several such spots linearly within the edge of a muscle fiber. The volume of tissue containing the network selected for study was examined sequentially one field of view at a time, and N_PI was counted.

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TBARS assay. To obtain a tissue-wide index of lipid peroxidation, the postischemic muscles of five animals from each experimental group were subjected to the protocol of Ohkawa et al. (25), except that an additional step to precipitate residual hemoglobin in the tissue homogenate was performed by application of Tsuchihashi's reagent (chloroform-ethanol, 2:3 vol/vol) followed by centrifugation at 15,000 g for 15 min. Spectrophotometric analysis was performed at 532 nm using 1,1,3,3-tetramethoxypropane as an external standard. As a positive control for this assay, nonischemic spinotrapezius muscles of four rats were homogenized (25) after a 1-h incubation of the tissue in a 1% solution of FeCl₃. The aqueous ferric ion produces sufficient lipid peroxidation in animal tissues to be quantified by the TBARS assay (33). Levels of TBARS are reported as nanomoles per milligram of wet tissue.

MPO activity. A previously published protocol was followed to quantify tissue MPO levels (28). Positive controls for the MPO assay were provided by the spinotrapezius muscles of four nonischemic rats 2-3 h after pretreatment with tumor necrosis factor- (5 µg ip) and 30 min after superfusion of the exposed tissue with 10⁶ M N-formylmethionyl-leucyl-phenylalanine (fMLP). Significant numbers of rolling, adherent, and extravasated leukocytes were observed in these preparations, as expected.

XO activity. Tissue XO and XDH activities were determined for each treatment group using a previously published protocol (30). To ensure the specificity of the assay, oxypurinol (100 µM) was added to each sample before spectrophotometry. Values of XO and XDH are reported as units per gram of wet tissue; 1 unit was defined as the quantity necessary to convert 1 µmol of xanthine to uric acid in 1 min.

Collection of plugging data and calculation of increase in network resistance. Plug duration and network dimensional data were obtained from the recordings as previously reported (9). The total time a bifurcation was plugged was divided by the 2-min observation period to determine a plugging fraction for each bifurcation. These plugging fractions were weighted in a computer model of the actual network (34) to determine the overall increase in network resistance (%R) caused by leukocyte-capillary plugging. A separate model network was created for each experiment, because each yielded a different network topology. Two major assumptions were made for these simplified networks. First, the pressure in the two collecting venules was presumed to be equal in the model, which includes only one output node. Second, variation in a vessel's diameter over its length was neglected; measurements taken at its entrance, midpoint, and exit were averaged to produce the diameter used by the model. Under control conditions, these diameters were approximately equal; however, after I/R, capillary narrowing often occurred, which significantly altered the diameter of the vessel at one or more locations. Cross-connections in the vasculature were included in the analysis. %R caused by leukocyte-capillary plugging was not determined under control conditions, because this value has previously been established as ~1% in rat spinotrapezius muscle single networks (9).

I/R protocols. Four separate groups were studied. In nonischemic animals (n = 7) used to control for damage caused by surgical trauma and prolonged muscle exteriorization, surgical exteriorization of the spinotrapezius muscle was performed but no further manipulations were made. Tissue injury along the lateral edge of the muscle was quantified 45 min and 2.5 h after stabilization to provide baseline measurements for the preischemia and postreperfusion (1 h) measurements in the following protocols. Leukocyte rolling and adhesion were assessed at both time points.

Statistical Analyses

	RESULTS

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Nonischemic Controls

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View larger version (21K): [in this window] [in a new window]   Fig. 1.   Correlations between resistance increase (%R) caused by leukocyte-capillary plugging and tissue injury [no. of propidium iodide spots (NPI) per total volume of perfused tissue] from 4 ischemia-reperfusion (I/R) groups. Error bars indicate SD. A: nonischemic control. B: I/R + saline, including linear regression (r2 = 0.834). C: I/R + superoxide dismutase (SOD), including linear regression (r2 = 0.094). D: I/R + oxypurinol, including linear regression (r2 = 0.044).

I/R + Saline Group

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I/R + SOD Group

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I/R + Oxypurinol Group

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Correlations of Tissue Injury With Capillary Network Resistance

ANOVA and Student's t-tests both indicate that no significant difference exists in (N_PI/V)_mean among the nonischemic control, I/R + SOD, and I/R + oxypurinol groups (all P > 0.05). Thus treatment of the muscles with SOD or oxypurinol appears to have reduced tissue injury to the level of the nonischemic control after 1.5-h I/R, and the level of injury appears to be constant and independent of %R.

Leukocyte Rolling, Adhesion, and Emigration

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Mean leukocyte rolling flux fractions after 30-min ischemia and 1-h reperfusion. Error bars indicate SD. No significant difference exists among the 4 groups (all P > 0.05). Oxy, oxypurinol.

In Fig. 3, the mean number of leukocytes firmly adherent to 100-µm sections of the venular endothelium is reported. The mean number of adherent leukocytes was 1.4 ± 0.37, 4.5 ± 1.26, 2.36 ± 0.51, and 4.0 ± 0.95 for the nonischemic control, I/R + saline, I/R + SOD, and I/R + oxypurinol groups, respectively. The range of collecting venule diameters studied fell between 25 and 30 µm for every group, and thus differences in vessel surface area were small. No significant difference existed between the nonischemic control and I/R + SOD groups (P > 0.05), but the I/R + saline group exhibited a significant increase in leukocyte adherence relative to the nonischemic control and I/R + SOD groups (both P < 0.05). In addition, the I/R + oxypurinol group exhibited a significant increase in leukocyte adherence relative to the nonischemic control group (P < 0.01). Thus oxypurinol prevented tissue injury, whereas SOD effectively prevented both tissue injury and leukocyte adhesion.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Mean number of adherent leukocytes/venule after 30-min ischemia and 1-h reperfusion. Error bars indicate SD. * P < 0.05 relative to nonischemic control group, P = 0.059 relative to I/R + SOD group. ** P < 0.05 relative to nonischemic control and I/R + SOD groups.

Giemsa staining of fixed, whole-mount tissues and intravital tracking of individual adherent leukocytes labeled with the fluorescent marker acridine red revealed no extravasated leukocytes in any treatment group by the end point of the study (data not shown).

Lipid Peroxidation

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Quantity of thiobarbituric acid-reactive substances in skeletal muscle tissues subjected to 30-min ischemia and 1-h reperfusion and control group. Error bars indicate SD. No significant differences exist among the 4 treatment groups, but all groups show significant decreases relative to positive control muscles. * P < 0.0001 relative to control.

XO Activity

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Mean activity of xanthine oxidase (open bars) and total xanthine dehydrogenase + xanthine oxidase (filled bars) after 30-min ischemia and 1-h reperfusion. Error bars are SD. Significantly higher levels of XO were detected in I/R + saline and I/R + SOD groups relative to both nonischemic control and I/R + oxypurinol groups. * P < 0.05 relative to nonischemic control group.

Tissue MPO

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Tissue myeloperoxidase (MPO) activity after 30-min ischemia and 1-h reperfusion. Data are reported as means ± SD. No differences exist among treatment groups (all P > 0.05), suggesting that leukocyte infiltration was not altered because of I/R or pharmacological interventions. However, all groups exhibit significantly decreased MPO relative to control muscles treated with a combination of N-formylmethionyl-leucyl-phenylalanine (fMLP) and tumor necrosis factor- (TNF) to induce leukocyte infiltration (all * P < 0.001).

	DISCUSSION

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Role of XO

In nonischemic tissues, XO exists primarily in the NAD⁺-reducing XDH form (36) that is incapable of oxidant production. However, conversion of XDH to XO (D-O form conversion) has been shown to occur in liver (36), mesentery (8), and skeletal muscle after 2-h ischemia and 30-min reperfusion (30). We show here that after short-term (30-min) ischemic insult and 1-h reperfusion, significant conversion of D-O form occurs and approaches 50-60% of the total enzyme present (Fig. 5). These values are smaller than the 70-80% conversion reported by Smith et al. (30) after 2-h ischemia and 30-min reperfusion, suggesting that a more severe ischemic insult may be required to cause greater D-O conversion. However, short-term I/R was sufficient to effect a significant increase in XO beyond that of nonischemic controls in both the I/R + saline and I/R + SOD groups (Fig. 5), suggesting that a quantity of XO sufficient to generate superoxide radicals may be present in these two groups.

XO inhibition with oxypurinol was as effective as SOD infusion in reducing tissue injury as measured by N_PI/V to levels of the nonischemic control group (Fig. 1, C and D). Concentrations of oxypurinol similar to that used here have also been used to effectively reduce tissue injury in a canine gracilis muscle model (30), and the present data support this report of its effectiveness in this regard. It is worthwhile to note, however, that although N_PI/V was reduced in the I/R + oxypurinol group to the level of the nonischemic control group, the dose of oxypurinol administered did not abolish all XO activity. The residual activity, ~20% of the total XDH + XO, is again comparable to that of the nonischemic control group. There are at least two explanations for these observations. First, free XO present in the blood may bind some quantity of infused oxypurinol, reducing the free stream pool available for diffusion into endothelial cells and parenchymal cells. Second, the sensitivity of the assay is 1 mU/g (21); thus the oxypurinol levels may be indistinguishable from controls with this signal resolution. In any case, both tissue damage and XO activity in I/R + oxypurinol were reduced to the level of the nonischemic control group, suggesting that despite the possibility of incomplete XO inhibition, the quantity of oxypurinol administered had a functionally prophylactic effect, a possibility supported by the finding that administration of higher doses of oxypurinol did not result in further decreases in N_PI/V or further reduction of XO activity (data not shown).

Microvascular Resistance Changes

Leukocyte Adhesion and Emigration

As noted (see RESULTS), none of the experimental treatments resulted in quantifiable levels of leukocyte emigration. This observation is confirmed by the MPO measurement, which suggests that total leukocyte infiltration did not change as a result of I/R. However, N_PI/V was significantly lower in the I/R + SOD and I/R + oxypurinol groups relative to the I/R + saline (cf. Figs. 1, A-C, 3, and 6). Together, these observations indicate that leukocyte infiltration, including that which might not be reflected in the MPO assay (because of sensitivity), did not contribute to elevated tissue injury. Furthermore, these data collectively suggest that tissue XO, not adherent or emigrated leukocytes, is responsible for the production of ROS that ultimately led to the tissue injury reflected by PI uptake.

Propidium Iodide and Spatially Distributed Tissue Injury

Because some confirmation of the efficacy of PI as an injury measure relative to standard biochemical methods is warranted, the present study used both N_PI/V and TBARS as indicators of tissue injury; although N_PI/V changed significantly in the treatment groups relative to controls, the level of TBARS (Fig. 4) was not significantly different between and among treatment groups. Although the spectrophotometric assay used is not as sensitive as methods such as HPLC, other studies have shown its ability to measure increases in products of lipid peroxidation relative to nonischemic controls for ischemia of 3 h or longer (17). This suggests that the lack of discrepancy in the level of TBARS is not a sensitivity issue but, rather, the 30-min ischemic insult was not sufficient to produce a measurable level of lipid peroxidation. It is therefore conceivable that PI uptake may be indicative of short-term, reversible cell damage; however, PI-positive cells may also be more susceptible to a longer-term, more permanent damage that would in fact result in lipid peroxidation.

Previous reports of the mostly distributed nature of PI spots (11, 31) were confirmed by the present study; a representative image is shown in Fig. 7a. In addition, qualitative tissue studies revealed that a fiber possessing a significant number of fluorescent nuclei tended to exhibit PI fluorescence along the majority of its length, whereas a neighboring fiber showed little, if any, PI uptake (Fig. 7b). Although this observation was not investigated extensively in this study, it lends support to the hypothesis of Suematsu et al. (31), who presented immunohistochemical evidence of a correlation between muscle fiber type and uptake of PI after 1-h low-flow ischemia and 1-h reperfusion of rat spinotrapezius muscles. Those authors observed elevated PI uptake by type IIB (fast glycolytic) fibers relative to type I (slow oxidative) fibers and argued that fiber susceptibility to low oxygen states may play an important role in postischemic complications. A more recent study describes preferential PI uptake after 2.5-h I/R by myocytes with low mitochondrial volumes, as assessed by uptake of the fluorescent marker rhodamine 123, and further supports this hypothesis (32).

View larger version (52K): [in this window] [in a new window]   Fig. 7.   Epifluorescent images of rat spinotrapezius muscles after 30-min ischemia and 30-min (a) or 1-h (b) reperfusion. In a, injury is distributed spatially, whereas in b, injury is localized to 2 nonadjacent muscle fibers. Scale bar = 50 µm.

The leukocyte adherence, plugging, and MPO data, as well the XO assay, suggest further that the distributed injury is produced primarily by local XO-mediated ROS release, because XO is localized to most parenchymal and vascular endothelial cells (1). The alternative leukocyte-mediated mechanisms are not supported by the current data. For example, it would be expected that PI fluorescence would cluster around postcapillary venules if adherent, activated leukocytes caused cell damage, but PI clustering was not observed. Second, ROS release by leukocytes plugging capillaries may produce a distributed injury by local diffusion of ROS and proteases; in fact, a weak correlation between plugging sites and PI-positive cells has been demonstrated (11). However, a previous study failed to yield such a correlation (31). The lack of data concerning the activation state of plugging leukocytes after I/R and the further possibility that the incidence of leukocyte-capillary plugging is related not to their activation state but to tissue injury (as discussed earlier), make it difficult to lend support to the second hypothesis.

Third, the role of extravasated leukocytes is still undetermined, and focal injury caused by a leukocyte migrating through the tissue may be possible. A report on leukocyte extravasation in rat mesentery in response to topical application of two chemotactic stimuli, fMLP and histamine, showed that the velocity of a crawling leukocyte was ~19.8 µm/s and that emigrated leukocytes did not possess migration paths directed toward any specific location in the tissue (24). If the crawling velocity in skeletal muscle is similar, one leukocyte could conceivably account for many PI spots. Two factors make this mechanism of tissue injury unlikely in our model. First, because of the increased number and density of parenchymal cells in skeletal muscle relative to mesentery, the crawling velocity of a leukocyte may be expected to be lower, making it less likely that one leukocyte could account for many injury locations. Second, as previously noted, we were unable to detect extravasation of leukocytes after 1.5-h I/R. Hence, it is probable that the short-term I/R insult reported here does not result in extravasation of adherent leukocytes over the time frame studied and thus that the speculated focal injury does not occur. Our MPO data also argue against such a mechanism operating in this I/R model, but a study tracking leukocyte emigration and a possible correlation with newly fluorescent cellular nuclei is needed in a longer-term I/R setting (4 h) to more clearly define the ability of emigrated leukocytes to produce focal injury in skeletal muscle.

The present study supports the hypothesis that endothelial cell XO, not plugging or adherent leukocytes, is primarily responsible for tissue damage observed after short-term I/R. This hypothesis is in accordance with previous reports of leukocyte-independent postischemic damage after longer-term skeletal muscle ischemia (4, 29). Furthermore, pharmacological intervention with SOD and oxypurinol was effective in attenuating microvascular resistance increases caused by leukocyte-capillary plugging. Finally, damage quantified by PI uptake was spatially distributed throughout the tissue. Further work is warranted to fully explore the role of leukocytes in producing this distributed injury after both short- and long-term ischemia.