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Consiglio Nazionale delle Ricerca Institute of Clinical Physiology, Faculty of Medicine, University of Pisa, Pisa, Italy
Submitted 6 May 2004 ; accepted in final form 22 September 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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oxygen free radicals; vasodilation; NG-monomethyl-L-arginine; nitric oxide donor
Furthermore, it has been firmly established that diminished oxygen delivery to tissue in response to hypoxia is countered by an increased functional capillary density in the microcirculation (24). Previous data (4) have demonstrated that the administration of SOD maintains vasodilation but also has an effect on capillary perfusion during postischemic reperfusion, characterized by low oxygen tension, whereas NO blockade decreases capillary perfusion and increases lipid peroxides.
Therefore, we hypothesize that delivery of oxygen to tissue in response to short-term hypoxia is regulated by NO availability and ROS formation at the capillary level.
It is widely accepted that a large amount of oxygen is released by the arteries rather than capillaries during normoxia (9, 18, 27, 28). This gradual PO2 decrease has been observed in many animal models in normoxia and has been related to the extensive oxygen permeability of blood vessel walls, a factor that enhances normal tissue oxygenation. Analysis of the distribution of oxygen tension during control conditions in the microcirculation has demonstrated that there is significant drop in PO2 in the arterioles, whereas capillaries contribute little oxygen to the tissue and are at a vascular oxygen bottom in terms of oxygen content (4, 6).
Until now, however, changes in oxygen delivery and capillary flow regulation in response to short-term hypoxia have been poorly understood. Therefore, the objective of our investigation was to study the effects of systemic hypoxia on capillary perfusion, specifically on changes in oxygen delivery at the tissue level. In addition, to determine whether oxygen delivery to the tissue can be modulated by NO during systemic hypoxia, we first measured oxygen distribution and capillary perfusion in addition to the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA). We then used the NO donor 2,2'-(hydroxynitrosohydrazono)bis-etanamine (DETA/NO) to reestablish the NO level after L-NMMA treatment during hypoxia. The in vivo oxygen tension distribution after 20 min of exposure to 10% O2-90% N2 in the hamster cheek pouch microcirculation was measured by using noninvasive phosphorescence quenching microscopy that allows instantaneous on-line PO2 analysis in the microvessels and in the tissue (4). The effects of hypoxia, L-NMMA, and DETA/NO on ROS formation and capillary perfusion were also investigated (4, 5).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental protocols. Experiments were divided into three separate protocols. In the first group (HY, n = 10), cardiovascular variables were measured continuously while the animals breathed 21% O2 for a 20-min period followed by a period of 20 min of hypoxia.
The second group (L-NMMA, n = 14) of animals, after the initial stabilization period, were treated with L-NMMA (10 mg/kg in 500 µl of 0.9% saline; Sigma) and infused intravenously for 20 min during air breathing. Seven animals of this group were treated with L-NMMA infused intravenously during air breathing, and then the inspirate was changed for 20 min to 10% O2.
The third group (DETA/NO, n = 7) was continuously infused with the NO donor, 2,2'-(hydroxynitrosohydrazino)bis-etanamine DETA/NO (Inalco, Milan, Italy) 20 min after the administration of L-NMMA after hypoxia. DETA/NO was dissolved in 0.5 ml saline and administered intravenously over 20 min into the femoral vein. Animals were treated with saline solution or L-NMMA or L-NMMA plus DETA/NO under normoxic conditions (room air, 21% PO2) and then exposed to 20 min of hypoxia and immediately observed. The groups were subjected to PO2 measurements during baseline and after hypoxia, breathing 10% O2-90% N2. Hypoxia was produced by having the animal breathe from a bag containing a mixture of 10% PO2-90% N2. After 20 min of exposure, systemic parameters and blood gas analysis were repeated. Four to six arterioles and venules were chosen for investigation and were followed throughout the protocol.
The hamster cheek pouch was prepared for intravital microscopy as a single layer (4). The cheek pouch was spread out over a Plexiglas microscope stage. A region of 
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.
Measurement of microvascular parameters.
Perfused capillary length (PCL) was assessed in a region of 0.5 mm2 of capillary segments that had red blood cell (RBC) transit in at least a 30-s period. PCL (cm–1) was also evaluated by measuring and adding the length of capillaries that had RBC transit within the total length of RBC-perfused capillaries divided by the area of the microscopic field of view (5). The relative change in PCL from baseline levels after the intervention is a relative indicator of capillary perfusion. PCL was measured by using our laboratory imaging software system.
Microvascular diameters (D) and RBC velocity were analyzed online using the photodidode/cross-correlator system (model 102B velocity tracker; Vista Electronics, San Diego, CA). The measured centerline velocity was corrected according to vessel size to obtain the mean RBC velocity [V, centerline RBC velocity (mm/s)/1.6] (21). Blood flow (Q) was calculated from measured parameters as Q = VD2·
/4.
Measurement of microvascular PO2.
Oxygen tension measurements were made by using the Pd-phosphorescence quenching method (4). The decay rate of the light-excited phosphorescence is inversely proportional to the partial PO2 according to the Stern-Volmer equation (18, 28): 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 (Porphyrin Products; Logan, UT) 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 (0 = 600 µs, kq = 325 Torr–1·s–1 at pH 7.4 and 37°C) and was 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 model 9855B; Knott Elektronik, Munich, Germany). The decay curves were averaged, visualized, and saved by a digital oscilloscope (100 MHz, Hitachi oscilloscope V-1065). Decay time constants were determined by a computer fitting the averaged decay curves to a single exponential using the Stern-Volmer equation. PO2 was initiated 2 min after injection, whereas interstitial tissue PO2 was measured after a period of 15-min, allowing enough porphyrin to extravasate for sufficient extravascular phosphorescence signal strength.
Measurement of lipid peroxides. To measure plasma hydroperoxides, the analytic method d-ROMs (Diacron; Parma) was used (4). It is based on Fenton's reaction or on radical formation during lipid peroxidation. The oxyradical species we produced, whose quantity is directly proportional to the quantity of plasma peroxides, are trapped by alchylamine, a phenolic compound that forms a colored, stable radical detectable spectrophotometrically at 505 nm. The concentration of the colored complex is directly correlated to the concentration of hydroperoxides. We mixed 10 µl of a chromogenic substance and 1 ml of the kit buffer with 10 µl of blood for 1 min at 37°C. The results are expressed in arbitrary units (au; 1 au = 0.08 mg/100 ml H2O2). Blood samples were taken at baseline and after hypoxia from the cannulated carotid artery.
All reported values are means ± SD. GraphPad Software (San Diego, CA) was used to analyze statistical differences. Data were analyzed by the Mann-Whitney U-test to examine differences between groups. The Friedman two-way ANOVA by ranks test was used to determine differences between groups at different times. Where significant differences were indicated by this test, further comparative analysis was undertaken by using Dunnett's test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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l/s; n = 20), A3 (diameter: 30.0 ± 9.0 µm, RBC velocity: 1.80 ± 0.10 mm/s, blood flow: 1.40 ± 1.00 l/s; n = 15), and A2 arterioles (diameter: 48.0 ± 10.0 µm, RBC velocity: 2.14 ± 0.27 mm/s, blood flow: 4.90 ± 3.00 l/s; n = 15). The venules were V2 (diameter: 18.0 ± 7.5 µm, RBC velocity: 0.8 ± 0.5 mm/s, blood flow: 0.39 ± 0.08 l/s; n = 20) and V3 (diameter: 50.0 ± 10.0 µm, RBC velocity: 1.2 ± 0.8 mm/s, blood flow: 4.1 ± 1.2 l/s; n = 20). Microvascular distribution of PO2 in microvessels and tissue is reported in Figs. 1 and 2. The intravascular PO2 was lower in A3 and A4 arterioles by 25 and 40% versus A2 arterioles, respectively.
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Inspiration of 10% O2-90 N2 caused a significant vasodilation of the A2, A3, and A4 arterioles (Fig. 3). A4 arterioles dilated more than A2 and A3 arterioles (P < 0.05). Large venules increased also by 20%, whereas in V2, the diameter changed by 4%. Hypoxia caused a significant decrease in RBC velocity in all the arterioles (Fig. 4). The RBC velocity in V3 decreased and remained unchanged in V2 venules. Hypoxia reduced blood flow significantly in the A3 and A4 arterioles (Fig. 4). In V2, blood flow did not change significantly, whereas in V3 blood flow decreased significantly during hypoxia.
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L-NMMA reduced the diameter of all the arterioles, whereas venules dilated (Fig. 3). The effect of L-NMMA was greater in A4 arterioles versus larger arterioles. RBC velocity decreased in venules, but flow did not change significantly versus baseline (Fig. 4). PCL decreased significantly by 39% versus control animals (Fig. 5). L-NMMA significantly increased ROS formation by 34% versus control animals (Fig. 6).
Effects of L-NMMA and hypoxia. With L-NMMA administration and hypoxia, the intravascular PO2 was similar and lower than during hypoxia in all the arterioles (Fig. 1). The tissue PO2 was reduced by 33% when compared with the HY group (Fig. 2). L-NMMA reduced the diameter in arterioles and terminal arterioles (Fig. 3). RBC velocity and blood flow decreased significantly in all the arterioles (Fig. 4). PCL also significantly decreased after hypoxia by 65% versus control animals (Fig. 5). L-NMMA significantly increased ROS formation by 104% during hypoxia (Fig. 6).
Effects of L-NMMA-DETA/NO and hypoxia. Intravascular PO2 increased significantly after DETA/NO in all vessels compared with the L-NMMA-HY group (Fig. 1). Tissue PO2 was significantly increased by 50% during DETA/NO infusion when compared with the HY group (Fig. 2). Venular PO2 was significantly increased during DETA/NO when compared with the L-NMMA-HY group. Infusion of DETA/NO after L-NMMA significantly increased arteriolar and venular diameter (Fig. 3). RBC velocity and blood flow also significantly increased (Fig. 4). DETA/NO after L-NMMA increased PCL significantly by 20% versus the HY group (Fig. 5). DETA/NO significantly decreased ROS formation versus HY, L-NMMA, and L-NMMA-HY groups (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The reduction in tissue PO2 is due to the reduction in arterial inflow and therefore, in oxygen delivery, but it is aggravated by the reduction in capillary perfusion when L-NMMA is injected in addition to hypoxia. The NO donor administration after L-NMMA, by increasing the concentration of NO, restored the capillary perfusion and decreased the formation of ROS during hypoxia. These phenomena were accompanied by a significant increase in tissue PO2. NO blockade during 21% O2 caused vasoconstriction and reduction in capillary perfusion and increased ROS formation more than that observed during control conditions.
Our findings are in agreement with Suematsu et al. (31), indicating that endogenous NO modulates oxidative stress in mast cells and arteriolar and venular endothelium. Moreover, our results are consistent with the previous observations that the administration of NO donors is beneficial in preventing the increases in ROS formation during early reperfusion characterized by low oxygen tension (4). Therefore, the increase in NO plays a significant ROS scavenger effect in hypoxia. The results obtained in this study led us to suggest that the concentration of NO may execute the function to regulate capillary perfusion by limiting the oxidative stress of endothelial cells caused by hypoxia.
Our finding showed a gradual PO2 decrease from the larger arterioles to the terminal arterioles during normoxia in the hamster cheek pouch, thus confirming our previous observation (4). The physical reason behind this significant exit from the arterial circulation before arriving to the capillaries is that blood vessels are not a barrier to the process of diffusion. Consequently, a significant amount of tissue oxygen is contributed by the arterioles during normoxia. During hypoxia, however, arteriolar intravascular PO2 tended to be uniform throughout the arterial network showing that little oxygen was released from the arterioles in this condition. Therefore, the oxygen delivery was shifted from arterioles to capillaries during hypoxia as also suggested by the significant increase in the capillary perfusion and the reduction in blood flow in arterioles.
Many studies have documented that NO has a shear stress-dependent dilator response on arteriolar resistance vessels and veins, but this effect diminishes in small resistance arteries in normoxia (13). It is generally accepted that PO2 modulates NO formation; however, the identity of the signal for hypoxic vasodilation and its interaction with NO is not known (26). Our findings show that short-term hypoxia causes a decrease in flow associated with vasodilation. Therefore, the decrease in flow/shear stress does not necessarily cause vasoconstriction in hypoxia (12). However, there is an interaction between NO and oxygen tension, because further lowering of NO levels by administration of L-NMMA during conditions of low PO2 was associated with vasoconstriction, decrease in capillary perfusion, and increase in lipid peroxide formation. DETA/NO eliminated the vasoconstrictor stimulus, thus showing that the level of NO is crucial to maintaining vasodilation. Therefore, it would appear that the origin of hypoxic vasodilation may not necessarily be dependent on a shear stress-mediated mechanism, because it is modified by the concentration of NO.
Recent findings (15) suggest that NO donors produce a NO-like bioactivity and induce vasodilation in vivo. In fact, the nitrite pool is converted to NO by reacting with deoxyhemoglobin and its leaving group is the met(ferric)heme protein that limits scavenging and inactivation of NO. Another mechanism to explain the hypoxic vasodilation controlled by NO level is related to a regulated release of NO by the RBCs at hypoxic regions. The reaction of NO with heme is competitive with O2; however, during low PO2 hemoglobin, instead of irreversibly consuming NO through conversion to nitrate, heme could actually preserve it through formation of S-nitrosothiols (7, 17). The origin of the hypoxic vasodilation may not necessarily be dependent on shear stress-related NO-release but on the enhanced NO concentration related to the direct interaction of NO/O2 where oxygen acts as an NO scavenger (16). Therefore, it appears that the presence of nonenzymatic origins of NO in the microcirculation may contribute to vasodilation on the reduction of local tissue PO2.
Hypoxia induces vascular relaxation through changes in superoxide production and cGMP formation in microvascular coronary endothelial cells during baseline and on postischemic reperfusion (1). Recent experiments indicate that SOD, which metabolizes superoxide anions, causes vasodilation during ischemia reperfusion (4). Steiner et al. (29) showed that NO generation is impaired in hypoxia and that tissue NO levels are depleted by the increased ROS during hypoxia. Therefore, superoxides interfere with NO-dependent maintenance of blood flow in hypoxia. Thus the hypoxia induced after L-NMMA blood flow decrease may lead to increased superoxide formation and subsequent, increased lipid peroxidation. Conversely, the administration of the NO donor increased tissue perfusion, potentially decreasing superoxide production and lipid peroxidation. The lowered lipid peroxide formation may not be a direct effect of NO but a consequence of the increased blood flow caused by NO.
Changes in blood flow/shear stress have been demonstrated in arterioles, venules, and capillaries that are the largest part of the endothelium tissue. Capillaries sense rate, magnitude, and pattern of shear stress as assessed by changes of capillary filtration to changes in blood flow that occur within the microvascular network (8). Whereas NO inhibition limited the ability to maintain tissue PO2 during hypoxia, the NO donor restored the capillary perfusion and tissue PO2. NO blockade also decreased capillary perfusion and augmented the large drop in PO2 in A4 terminal arterioles in normoxic conditions. Notwithstanding the large drop in PO2 in these arterioles, the tissue PO2 decreased significantly. Interestingly, Kashiwagi et al. (20) have shown that expression of endothelial NOS (eNOS) occurs in endothelium of capillaries and venules in normal conditions, whereas the smaller arterioles express neuronal NOS but little of eNOS, if any. Considering that capillaries are the main source of NO and the effect of NO on capillary flow induced by hypoxia, it is not unreasonable to speculate that NO has the function to regulate the oxygen supply to the tissue.
We suggest that NO may be important to preserve capillary perfusion through its ability to decrease endothelial cells' lipid peroxidative products leading to the deterioration of capillary function. Our results show that L-NNMA causes a significant reduction in capillary perfusion accompanied by an increase in ROS formation. Conversely, NO donors reduce the ROS-to-baseline values. Evidence that the lipid peroxide formation may directly induce loss of membrane integrity and that the alteration in fatty acid composition impairs capillary flow by the reduction of membrane fluidity was found in brain capillaries (25). Fluid shear stress reflects mechanical forces exerted on endothelial cells, is a stimulus of eNOS activity, and increases membrane fluidity in endothelial cells, thus facilitating capillary flow (14, 33). Furthermore, NO donors have been shown to increase RBC deformability, whereas decreased deformability resulted in impairment of capillary microcirculation (2, 24). In a previous study (3), we found that blockade of NO by L-NMMA caused a significant reduction in the number of perfused capillaries due to trapping of RBC. Furthermore, after postischemic reperfusion, the no-reflow phenomenon was negligible after both L-arginine and NO donors and capillary blood cell velocity was significantly higher than that observed in control animals. We hypothesize the possibility that hypoxia may alter the lipid composition of the endothelial cells through the increase in ROS after NO inactivation, resulting in the reduction of the number of perfused capillaries.
In conclusion, our findings show that oxygen is partly supplied by arterioles during normoxia, whereas during hypoxia capillaries appear to be the major suppliers of the oxygen delivery to the tissue. The blockade of NO causes a significant reduction in capillary perfusion and increases lipid peroxides while decreasing tissue PO2. These alterations are corrected by NO administration, which increases capillary perfusion and tissue PO2. These alterations are corrected by NO concentration, which increases capillary perfusion and tissue PO2. We hypothesize that NO concentration is a determinant of lipid peroxide formation and the regulation of the number of patent capillaries during hypoxia. Conditions leading to low NO concentration may promote the production of ROS damaging the capillary endothelial cells and decreasing oxygen delivery to the tissue through the decrease of capillary perfusion.
Study limitation. Oxidative stress was evaluated by measuring the concentration of hydroperoxides in plasma; however, because these materials have a comparatively long half life in the circulation, they may be produced in other parts of the organism and not be fully representative of events in the tissue under observation.
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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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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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