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Consiglio Nazionale delle Ricerca Institute of Clinical Physiology, Medical School, University of Pisa, 56100 Pisa, Italy
Submitted 10 February 2003 ; accepted in final form 14 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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oxygen tension; oxygen free radicals; capillaries; nitric oxide
Alterations in oxygen metabolism and ROS cause changes in arteriolar resistance and inflammatory reactions in I/R (43). A relevant radical in biological regulation is superoxide formed by NAD(P)H oxidases, xanthine oxidase, mitochondria, cytochrome P-450, cyclooxygenase, and isoforms of nitric oxide (NO) synthase (NOS) (25, 42, 43). It has been suggested that SOD protects many tissues from I/R-induced injury (20, 37, 44). SOD, which scavenges a superoxide anion, normally prevents significant decomposition of released NO. However, NO has an ambiguous role because many experimental studies showed that NO has beneficial effects on platelet aggregation and leukocyte adhesion but can also have toxic and harmful consequences in I/R (2, 9, 22). NO inhibition partially decreased the damage in I/R in the hamster cheek pouch microcirculation (3).
Our first aim was to study the relationship between oxygen availability, ROS formation, and blood flow during postischemic reoxygenation in the microcirculation. Reoxygenation could promote increased ROS production that would lead to enhanced NO degradation. We reasoned that the administration of a scavenger and NO inhibition would provide insight into the mechanism of oxidative stress-NO interactions in microvascular responses to reperfusion. Therefore, our second aim focused on the prevention of ROS production by controlled ROS scavengers. Because ROS, superoxide anion, NO, and oxygen maintain vascular tone and blood flow, their measurements must be assessed locally and in real time to understand the pathophysiological process of reperfusion damage.
We measured the in vivo oxygen tension during baseline and reperfusion by using noninvasive phosphorescence quenching microscopy that allows instantaneous on-line PO2 analysis and rapid sequences of measurements in the microvessels and in the tissue (8, 15, 18, 19, 38). Our model was the hamster cheek pouch microcirculation during I/R (3, 4). We measured ROS formation during I/R in the microcirculation. Lipid peroxides were assayed as an index of oxidative stress during baseline and reperfusion by using a new method allowing measurements in the systemic blood (7, 10). Furthermore, we evaluated the ability of SOD and an NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), to inhibit ROS formation during I/R. The microvascular functional damage that occurs during reperfusion was quantified by changes in arteriolar diameter, red blood cell (RBC) velocity, increased leukocyte adhesion, and perfused capillary length (PCL), namely, the capillaries perfused by blood (3, 6).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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We used four groups of animals subjected to I/R. The first group (O2I/R, n = 10) was subjected to oxygen measurements during baseline and after reperfusion. The second group was used as a control group (I/R, n = 7), whereas the third group (SOD, n = 7) was injected with a bolus of 9.0 mg/kg SOD from bovine erythrocytes dissolved in 0.9% saline (Sigma; St. Louis, MO) and administered 30 min before I/R, and 0.15 mg · kg–1 · min–1 were infused intravenously during reperfusion. The fourth group (L-NMMA, n = 7) was treated with L-NMMA (1 mg/100 g body wt in 500 µl of 0.9% saline) (Sigma) infused intravenously 15–30 min before I/R and during the period of reperfusion. In two subgroups of animals (SOD-L, n = 3; L-NMMA-L, n = 3), the hamster cheek pouch was superfused with a superfusate solution containing SOD (167–500 U/ml) or with a solution containing L-NMMA at a final concentration of 10–5 M. The cheek pouch was superfused 15–30 min before ischemia and during the period of reperfusion. Topical administration was used to avoid systemic effects. Control animals received equivalent amounts of physiological saline. Ischemia was produced by occluding the arterial inflow to, and the venous outflow from, the cheek pouch. Atraumatic microvascular clips were placed on the proximal part of the cheek pouch to achieve complete ischemia for a period of 30 min. The clamp was then removed and the microcirculation was observed after 30 min. We measured ROS formation in all the groups, whereas arteriolar responses, RBC velocity, PCL, and leukocyte adhesion on venules in the I/R, SOD, and L-NMMA groups were measured during baseline and I/R.
The hamster cheek pouch was prepared for intravital microscopy as a single layer (7, 12). The cheek pouch was spread out over a Plexiglas microscope stage. A region of about 1 cm2 in area was prepared as a single layer for intravital microscopic observations. The cheek pouch was covered with transparent plastic film to prevent both desiccation of the tissue and gas exchange with the atmosphere. Observations were made with an intravital microscope (Orthoplan, Leica Microsystem; Wetzlar, Germany), and the transillumination technique was used. All selected microvessels and interstitial tissue segments were also recorded by a video camera (COHU; San Diego, CA) displayed on a monitor and transferred to a video recorder. The hamster's body temperature and cheek pouch temperature were maintained at 37°C with circulating warm water. An intravenous injection of pentobarbital sodium (300 mg/kg) was used as the method of euthanasia.
For the I/R, SOD, and L-NMMA groups the cheek pouch was prepared as previously reported (3, 6). Epi-illumination was provided by a xenon 150-W lamp using the appropriate filters for fluorescein isothiocyanate, bound to dextran, molecular weight of 150,000 [Sigma; 50 mg/100 g body wt intravenously injected as 5% (wt/vol) solution in 5 min], and for acridine red. A heat filter was also used. The area of interest was televised with a COHU 5253 SIT low-light level camera and observed on a Sony PVM 122 CE monitor. Video images were videotaped and microvascular measurements were made off-line by a computer-assisted imaging software system (MIP Image, CNR, Institute Clinical Physiology; Pisa, Italy).
PO2 measurement system. The oxygen-dependent
quenching of the phosphorence decay technique method is based on
phosphorescence emitted by albumin-bound palladium
meso-tetra(4-carboxyphenyl)porphyrin (Pd-porphyrin)
(7,
18). Pdporhirin (Porphyrin
Products; Logan, UT) is excited to its triplet state by exposure to pulsed
light after which phosphorescence intensity is reduced by emission and energy
transfer to O2. The relationship between phosphorescence lifetime
and oxygen tension is given by the Stern-Volmer equation supplied from
different arterioles branching off the selected transverse arteriole: 1/
= 1/o + kqPO2, where
o and are the phosphorescence lifetimes in the absence
of molecular oxygen and at a given PO2, respectively,
and kq is the quenching constant, with both factors being
pH and temperature dependent. Pd-porphyrin bound to serum albumin and diluted
in saline (0.9% sodium chloride, Elkins-Sinn) to a final concentration of 15
mg/ml was used as a phosphorescent dye (o = 600 µs,
kq = 325 Torr–1 ·
s–1 at pH 7.4 and 37°C) and intravenously
injected (15 mg/kg body wt). Phosphorescence was excited by light pulses (30
Hz) generated by a 45-W xenon strobe arc (EG&G Electro Optics; Salem, MA).
The pulsed light illuminated a round area of 
140-µm diameter, whereas
PO2 measuring sites were microscopically selected by an
adjustable slit with a fixed size at 15 x 20 µm. For microvascular
PO2 measurements, the slit was longitudinally fitted
within the vessel, whereas for the analysis of interstitial
PO2, it selected intercapillary spaces avoiding
interference with blood vessels. Filters of 420 and 630 nm were used for
porphyrin excitation and phosphorescence emission, respectively.
Phosphorescence signals were captured by a photomultiplier (EMI, 9855B, Knott
Elektronik; Munich, Germany). The decay curves were averaged, visualized, and
saved by a digital oscilloscope (Hitachi Oscilloscope V-1065, 100 MHz,
Hitachi, Denshi). Decay time constants were determined by a computer fitting
the averaged decay curves to a single exponential, using the Stern-Volmer
equation (18).
PO2 was initiated 2 min after the injection, whereas
interstitial tissue PO2 was measured after a period of
15 min, allowing enough porphyrin to extravasate for sufficient extravascular
phosphorescence signal strength. Measurements were made before the occlusion,
after 30 min of ischemia, and after 5, 15, and 30 min of reperfusion.
Measurement of lipid peroxides. To measure plasma hydroperoxides, the analytic method d-ROMs (Diacron; Parma Italy) was used (7, 10). The d-ROMs assay is based on Fenton's reaction or on radical formation during lipid peroxidation. The produced oxyradical species, the quantity of which is directly proportional to the quantity of plasma peroxides, were trapped by alchylamine, a phenolic compound that forms a colored stable radical that can be detected spectrophotometrically at 505 nm. The concentration of the colored complex is directly correlated to the concentration of the hydroperoxides. Ten microliters of a chromogenic substance and 1 ml of the kit buffer were mixed with 10 µl of blood for 1 min at 37°C. The results were expressed in arbitrary units (au; 1 au = 0.08 mg/100 ml H2O2). In a pilot study, we measured the variability of ROS production in a group of hamsters during baseline conditions for 180 min. The level of lipid peroxides did not change significantly during the time (220 ± 55 au). Blood samples were taken at baseline and at 5, 15, and 30 min of ischemia and reperfusion for each hamster from the cannulated carotid artery.
Measurements of microvascular parameters. PCL, defined as the
total length of capillary segments that have at least one RBC passing through
them in a 30-s period, was analyzed from four to six different microscopic
fields. Microvessel diameters were measured by an image-shearing system
(Digital Image Shearing Monitor model 907, IPM). The RBC velocity was
determined by using dual-slit cross correlation (velocity tracker Mod 102 B,
IPM; San Diego, CA). The blood flow (Q) was measured with the measured
parameters Q = V · 
(D/2)2,
where V is the velocity and D is the diameter of the
microvessels. The measured centerline velocity was corrected according to
vessel size so that the mean RBC velocity could be obtained.
Animals in all the groups received an intravenous injection of acridine red (1 mg/100 g) to visualize the leukocytes at baseline and after reperfusion (3). The number of adherent leukocytes was expressed as the number per 100-µm length of venule (diameters: 16 ± 8 µm, length > 250 µm).
Mean arterial blood pressure (MAP) (Viggo-Spectramed P10E2 transducer; Oxnard, CA) and heart rate (HR) were measured by a Gould Windograf recorder (model 13-6615-10S, Gould, OH).
Statistical analysis. Data were expressed as means ± SD. When two groups were compared the unpaired Student t-test was used. Comparisons between multiple groups were conducted using one-way analysis of variance followed by a Tukey-Kramer test to compare all treatment groups. The statistical significant was determined at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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PO2 measurements. At baseline, the mean PO2 for arterioles of diameter 14.5 ± 5.5 and 43.5 ± 9.5 µm was 38.9 ± 9.1 and 42.5 ± 7.9 mmHg, respectively. The number of observations was 25 for each vessel. Mean PO2 was measured in venules with a diameter of 18.8 ± 7.0 and 50.0 ± 4.5 µm, and PO2 was 22.0 ± 3.5 and 30.0 ± 10.5 mmHg, respectively. Data showing intraarteriolar, capillary, and intersitium PO2 at different time points during baseline were shown in Fig. 1. The PO2 measured above the centerline of arterioles at the beginning of reperfusion was significantly lower than the baseline values (diameter of arterioles: 43.5 ± 9.5 µm). Thirty minutes after reperfusion began, PO2 had returned to the baseline values. The interstitial PO2 values were significantly lower than intravascular PO2 values and did not change significantly with respect to the vessel order. The capillaries were characterized by a great variability of PO2 during reperfusion thus showing very low values close to zero (range: 7–0 mmHg) in some capillaries (Fig. 1).
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ROS measurements. Lipid peroxide concentration did not change during ischemia, whereas ROS formation increased by 90 and 72% within 5 and 15 min of reperfusion (P < 0.05). After 15 min of reperfusion ROS levels began to decrease significantly. All the values measured were reported in the Fig. 2.
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In contrast to the untreated hamster cheek pouch, those treated with SOD before I/R showed no significant increase in ROS formation (Fig. 2). SOD treatment inhibited the ROS formation at all the times studied. We obtained the same protective effects with SOD either intravenously injected or when SOD was locally administered. We locally superfused the hamster cheek pouch with SOD during I/R to show that the increased ROS production was formed by the cheek pouch, and it was not related to a rise in systemic hydroperoxides.
L-NMMA reduced the ROS formation within 5 min of reperfusion compared with the I/R group, whereas ROS increased by 72% after 15 min of reperfusion compared with the baseline (Fig. 2). When L-NMMA was superfused on the hamster cheek pouch, ROS production was maintained at normal levels after 5 min, whereas ROS increased by 52% after 15 min of reperfusion compared with baseline.
Microvascular parameters during reperfusion. In Figs. 3, 4, 5, we report the percent changes of diameter, RBC velocity, and flow in arterioles (baseline diameter: 43.50 ± 9.5 µm; baseline RBC velocity: 2.0 ± 0.35 mm/s; baseline blood flow: 22.5 ± 9.0 nl/s) for each of the treatment groups during the time. In the I/R group the diameter, RBC velocity, and blood flow of arterioles decreased significantly after 30 min of reperfusion compared with the baseline (P < 0.05). Capillary RBC velocity was 0.211 ± 0.028 mm/s before ischemia and 0.028 ± 0.034 mm/s (n = 21 capillaries) within 30 min of reperfusion (P < 0.01). In the I/R group, PCL decreased significantly when compared with the baseline (9,500 ± 450 µm, P < 0.01) (Fig. 6). The number of adhering leukocytes increased significantly after 30 min of reperfusion (Fig. 7).
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In the SOD group, arterioles of the hamster cheek pouch (baseline diameter: 49.0 ± 7.0 µm) did not show vasoconstriction during reperfusion when compared with the baseline. The RBC velocity and blood flow in arterioles (baseline RBC velocity: 2.2 ± 0.5 mm/s; baseline blood flow: 24.8 ± 10.5 nl/s) did not change significantly after reperfusion compared with the baseline. In the L-NMMA group during the baseline the diameter of arterioles (baseline 47.0 ± 9.0 µm), RBC velocity and blood flow (baseline RBC velocity: 2.1 ± 0.5 mm/s; baseline blood flow: 23.1 ± 10.8 nl/s) decreased by 20% (P < 0.05), 5%, and 10% (P < 0.05), respectively, after L-NMMA injection. In the L-NMMA group, after 15 min of reperfusion, the decrease in diameter was more marked than in I/R group, whereas the decrease in RBC velocity was more evident in the I/R group (Figs. 3 and 4). This behavior could be explained with a vasomotion activity observed during early reperfusion in the L-NMMA group.
In the SOD group, PCL did not change compared with the baseline, whereas it was significantly reduced in the L-NMMA group compared with the baseline (SOD, 9,050 ± 680 µm; L-NMMA, 7,980 ± 500 µm) (Fig. 6).
The number of leukocytes adhering to the venular wall decreased significantly at the end of reperfusion with SOD (P < 0.05). With L-NMMA there was a significant increase in the leukocyte adhesion to venules compared with the number observed after 30 min of reperfusion in the I/R group (P < 0.05, Fig. 7).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Our measurements of PO2 during baseline showed a large drop from arterioles to capillaries, whereas the capillary PO2 distribution was characterized by pronounced heterogeneity. These results are consistent with those obtained during normoxia in different preparations. These results imply that the oxygen transfer from erythrocytes to tissue occurs in arterioles. Capillaries are not the sole source of oxygen supply to the surrounding tissue because a consistent reduction in PO2 levels was found from the arterioles to capillaries (18, 35).
In our study we demonstrated that during early reperfusion there was low PO2 in arterioles, whereas capillaries were characterized by low values close to zero throughout reperfusion. There was a significant improvement in arterial blood flow after reperfusion, which may explain the increase in PO2. Within 20–30 min of reperfusion, the tissue oxygenation recovered notwithstanding the fact that capillary perfusion decreased significantly.
The main source of microvascular functional damage during reperfusion appears to originate in the capillaries. In agreement with our findings, it has been shown that anoxic zones develop in the rat heart by using surface NADH fluorescence photography during hypoxia or ischemia. These zones always start at the capillaries thus indicating the onset of irreversible myocardial dysfunction from the capillaries (17). Other studies of the rat myocardium showed the origin of heterogeneity in tissue oxygenation due to low flow ischemia. Moreover, most NADH-fluorescent areas were found to have developed in capillaries despite sustained flow during early reperfusion (41).
Our experimental data indicated that the administration of a scavenger before I/R maintained all measured microvascular parameters at the baseline values. ROS scavenging by SOD markedly decreased ROS production and improved the arteriolar blood flow, thus increasing capillary perfusion during reperfusion. A reduced level of NO attenuated ROS production within 5 min of reperfusion, whereas arteriolar flow decreased significantly but to a lesser extent than in the I/R group. After 30 min of reperfusion, the observed changes in flow and capillary perfusion were relatively minor in the I/R group compared with those observed in the L-NMMA group.
The peroxidation of membranous phospholipids induced by I/R was inhibited by SOD in different models (23, 27). However, there are a significant number of studies that show how antioxidants either fail to prevent injury or have merely an early protective effect that waned with increased duration of reperfusion (31, 40). We suggest that a failure to adequately scavenge ROS is related to the difficulties in protecting capillaries. Capillary endothelial cell could be more susceptible to injury caused by the low oxygen state at early reperfusion. We suggest that the increased lipid peroxides caused by hypoxia during reperfusion may alter the capillary membrane function, including endothelial volume control. There are many reports showing that endothelial swelling of capillary vessel wall contributes to the "no-reflow" phenomenon during reperfusion (16).
The PO2 time course followed an inverse pattern compared with real-time ROS production; there was a low level of PO2 in the blood and tissue at the beginning of reperfusion that recovered toward the baseline values 15–30 min after reperfusion began. ROS returned to baseline values when PO2 recovered completely. Moreover, after 30 min of reperfusion the arterial blood flow did not recover the baseline values thus showing dissociation between blood flow and PO2. The low oxygenation at the beginning of reperfusion does not appear to be a protective mechanism to reduce ROS formation. Conversely, the conditions of low-flow hypoxia appear to cause endothelial injury to the arteries. At the beginning of reperfusion the reduced oxygen availability may produce endothelial cell injury and promote the activation of O2 to reactive species. An interpretation of our findings is that the increased production of ROS, combined with decreased perfusion, may contribute to the endothelium damage with the impairment of oxygen consumption and subsequent loss of vasomotor tone control. In agreement with our hypothesis, Suematsu et al. (33) showed xanthine oxidase-dependent oxidative stress after 20 min of low flow hypoxia in the perfused rat liver. It had long been assumed that overproduction of ROS during reperfusion could be related to overoxygenation, whereas it appears that hypoxia in the early reperfusion may be a crucial factor in determining an increased production of ROS that impairs oxygen consumption by the endothelial cells.
An alternative hypothesis could be that NO, ROS, and PO2 constitutes a regulation system for oxygen consumption during reperfusion. It was shown that the formation of NO, and its activity in resistance arteries, differs significantly depending on the local concentration of oxygen and it is potentiated by hypoxia and anoxia (36). Coste et al. (11) developed a phosphorescent probe PdTCPPNa (4), of which its luminescence properties are affected by local variations of intracellular PO2 on living human umbilical venous endothelial cells. These authors (11) showed that activation by acetylcholine or endothelial NOS induces a significant decrease in PO2 of which its amplitude is dependent on the acetylcholine dose, i.e., the eNOS activity level. Consistently with this finding, a decline in the PO2 profile near the arteriolar wall was measured under physiological levels of PO2 and of shear stress in different preparations. A significantly higher tissue permeability to O2 or oxygen consumption by endothelial cells than would be expected from in vitro studies was hypothesized (18, 30). Although these effects should be further investigated, it is conceivable to assume that oxygen consumption and transport may be associated with the local ROS formation, changes of shear stress on the vessel wall, and levels of PO2.
Our findings showed that NO inhibition caused an increased leukocyte adhesion on postcapillary venules in a relationship with increased oxidative stress during reperfusion. Moreover, capillary perfusion decreased significantly and the trend was toward a more marked decrease than in the I/R group. Kashiwagi et al. (24) showed that NO availability in arteriolar endothelium and mast cells appeared to be maintained mainly by nonendothelial origins involving NOS1 (neuronal synthase) present in nerve terminals and mast cells, whereas venules depend on the endothelial NOS3 (endothelial synthase) as a major source. Our previous observations (5) showed an increased P-selectin expression at venular bifurcations in the hamster cheek pouch microcirculation. Furthermore, we observed that increased oxidative stress before I/R blunted the protective effects of anti-P-selectin treatment with marked leukocyte adhesion on postcapillary venules, thus suggesting that the mechanism of leukocyte adhesion is due to dysfunctional endothelium after oxidative stress. This hypothesis is consistent with the results obtained in the rat mesenteric circulation by Suematsu et al. (34) showing that NG-nitro-L-arginine methyl ester (L-NAME) caused cellular oxidant formation mainly in venules with adhering leukocyte. All these data are consistent with our findings showing that NO levels may contribute more greatly to inhibiting leukocyte adhesion on venules than other factors during reperfusion.
Our findings showed that antioxidant treatment blocks both ROS production and leukocyte adhesion and increased flow. Conversely, inhibition of NO increased ROS production and leukocyte adhesion and decreased microvascular flow. These results imply that increased leukocyte adhesion to venules during reperfusion is linked primarily to the generation of ROS during hypoxic conditions. The hypoxic conditions maintained at reperfusion by promoting ROS formation may lead to decomposition of NO and to increase leukocyte adhesion (34, 45). Superoxide could also be reduced by L-NMMA that initiates the expression of adhesion molecules (14, 26–28). SOD may attenuate leukocyte adhesion by inhibiting the breakdown of NO by increased ROS production during the period of low oxygenation. Moreover, the increased flow observed with SOD during reperfusion is an important contributor to the decreased adherence of leukocytes to postcapillary venules, which could decrease leukocyte-endothelial interactions by increasing venular shear rate, i.e., the force generated at the vessel wall by the movement of blood (32).
The moderate inhibition of oxidant stress observed with L-NMMA during early reperfusion is not protective against microvascular perfusion. The decrease in ROS production may be due to decreased formation of peroxynitrite that causes lipid peroxidation. However, at early reperfusion we observed a significant influence on arteriolar RBC velocity in the L-NMMA group that could be due to an increase in vasomotion activity. These results support previous observations in the hamster cheek pouch showing an increased vasomotor activity during reperfusion after L-NMMA treatment (4). However, inhibition of NO production was associated with vasoconstriction throughout reperfusion leading to greater reduction of capillary perfusion.
The local control mechanism of vasomotor tone by NO is crucial but very complex because NO is oxidized by both oxygen and the superoxide anion. Recent findings indicate that oxygen selectively modulates NO production and endothelium-dependent relaxation in aorta smooth muscle (36). Hypoxia has a significant modulating effect on NO synthesis, and it induces vascular relaxation through changes in superoxide production and cGMP formation in microvascular coronary endothelial cells during baseline and on reoxygenation (1). Therefore, it is reasonable to further propose that the mechanism responsible for vasoconstriction or vasodilation during reperfusion is dependent on the presence of NO and/or its decomposition due to oxidative stress.
In conclusion, our findings demonstrate that the early reperfusion is characterized by low concentration of oxygen linked to increased production of ROS. After this initial transient in arterioles, the oxygen tension and production of ROS return to normal after reperfusion, whereas the blood flow and capillary perfusion decrease. The early increased ROS production, in turn, may impair oxygen consumption by endothelial cells, thus further promoting activation of oxygen to ROS. This event is substantiated by the finding that treatment with SOD maintains ROS at normal levels, which, in turn, should be effective to increase the production of endothelial NO. Conversely, a decrease in NO levels leads to decreased ROS production during early reperfusion, which, however, increased later during reperfusion, ultimately causing vasoconstriction and greatly increasing venular leukocyte adhesion on postcapillary venules during hypoxic conditions. Therefore, low flow hypoxia is primarily responsible for vascular endothelial damage during reperfusion through changes in the ROS and NO production.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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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