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Am J Physiol Heart Circ Physiol 294: H1914-H1922, 2008. First published February 22, 2008; doi:10.1152/ajpheart.01371.2007
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Intermittent hypoxia modulates nitric oxide-dependent vasodilation and capillary perfusion during ischemia-reperfusion-induced damage

Silvia Bertuglia

Consiglio Nazionale delle Ricerche Institute of Clinical Physiology, University of Pisa, Pisa, Italy

Submitted 27 November 2007 ; accepted in final form 13 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The microvascular function of nitric oxide (NO) during ischemia-reperfusion (I/R) in intermittent hypoxia (IH)-pretreated hamsters was analyzed using 20 mg/kg of the nonselective NO inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) and 5 mg/kg of the preferential inducible NO inhibitor S-methylisothiourea sulphate (SMT) injected before I/R. Studies were made in the hamster cheek pouch microcirculation (intravital fluorescence microscopy). IH consisted of 6 min of 8% O2 breathing followed by 6 min of 21% O2 for every 8 h for 21 days. Normoxia controls (NCs) were exposed to room air for the same period. The effects were characterized in terms of systemic hemodynamics, diameter, flow, wall shear stress in arterioles, capillary perfusion, and the concentrations of thiobarbituric acid-reactive substances (TBARS) and plasma NO, assessed as nitrite/nitrate (NOx) levels. IH did not change arterial blood pressure and increased hematocrit and shear stress. IH increased NOx and TBARS levels and reduced arterial diameter, blood flow, and capillary perfusion versus the NC. Conversely, TBARS and NOx were lower during I/R in IH-pretreated hamsters, resulting in vasodilation and the increase of capillary perfusion and shear stress. After IH, capillary perfusion was reduced by 24% (2.3%) and enhanced by 115% (1.7%) after I/R (P < 0.05). Both modalities of NO blockade decreased NOx generation and increased TBARS versus IH. L-NAME and SMT induced a significant decrease in arteriolar diameter, blood flow, and capillary perfusion (P < 0.05). L-NAME enhanced TBARS more than SMT and aggravated I/R damage. In conclusion, we demonstrated that preconditioning with IH greatly reduces oxidative stress and stimulates NO-induced vasodilation during I/R injury, thus maintaining capillary perfusion.

nitrite; nitrate; S-methylisothiourea; N{omega}-nitro-L-arginine methyl ester; shear stress; hematocrit

ISCHEMIC PRECONDITIONING IS a potent inducible endogenous mechanism against ischemia-reperfusion (I/R) injury (13). Intermittent hypoxia (IH), with repeated episodes of hypoxia and normoxia, causes preconditioning-like cardioprotective effects and increases exercise tolerance in elderly men with and without coronary artery disease (14, 17, 34). Beguin et al. (2) found that rats treated with 40 s of 10% O2 plus 20 s of 21% O2 for 4 h had reduced myocardial infarction induced by subsequent global I/R after 24 h of IH treatment. Cai et al. (15) reported that 24 h after the treatment of mice with five cycles of 6 min of 6% O2 plus 6 min of 21% O2, the heart was protected against myocardial injury induced by 30 min of global ischemia followed by reperfusion. Nitric oxide (NO) and reactive oxygen species (ROS) levels have been proposed to be involved in the protective mechanisms provided by IH against I/R injury (18, 23, 27); however, the mechanisms in which IH preconditioning increases resistance to I/R injury are not well understood.

Previous studies have shown that the beneficial effects of treatments that reduce I/R injury are inversely correlated with enhanced oxidative stress that subsequently modulates shear stress (SS), thus reducing NO bioavailability and endothelium-dependent vasodilation in postischemic reperfusion (9, 11, 12, 31). Blood vessels are constantly exposed to SS, a frictional force exerted on the vessel that can influence vascular function by stimulating the production of NO, endothelial NO synthase (eNOS) expression, and oxidant signaling mechanisms involved in the control of fundamental physiological processes (42). The interaction between ROS and NO during hypoxia may reduce NO bioavailability and endothelium-dependent vasodilation. As a result of the interaction between ROS and NO, the highly toxic reactive oxidant peroxynitrite is formed, which increases oxidative stress and results in the impaired function of multiple proteins including vascular K+ channels that are critical for vasodilation (27, 38, 39). Recent studies suggested that inducible NOS (iNOS) induction, leading to NO overproduction, is in part responsible for I/R injury and nitrite-derived NO being a potential mediator of vasodilation (24, 32). We hypothesized that in I/R after IH preconditioning, there is an increased activity of eNOS due to increased SS and the regulation of oxidative stress, thereby increasing endothelium-dependent vasodilation.

Our first aim was to test whether IH increases resistance in I/R injury. The second aim was to investigate whether NO levels are related to the effects on vascular function and microvascular perfusion during I/R events after IH pretreatment. Therefore, we studied the influence of various NOS inhibitors in hamster cheek pouch microcirculation during I/R damage after IH preconditioning. A nonselective NOS inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) or the iNOS inhibitor S-methylisothiourea sulphate (SMT) was used before I/R treatment. In addition, the third aim was to determine whether exposure to IH improved endothelium-dependent dilation, via the evaluation of the vasodilator response to acetylcholine (ACh) (4, 17).

The in vivo microcirculation of the hamster cheek pouch, extensively used in studies of I/R-induced injury, has been observed using intravital microscopy to characterize changes in microvascular diameter, blood flow, and wall SS in arterioles (4–12). In addition, we measured the changes in perfused capillary length [PCL; length of capillaries perfused by red blood cells (RBCs)] after postischemic reperfusion. We also correlated IH with the changes in the levels of thiobarbituric acid-reactive substances (TBARS), a marker of oxidative stress, and plasma NO assessed as nitrite/nitrate levels (NOx) during I/R damage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental protocol. Male Syrian golden hamsters (80–100 g; Charles River, Calco, CO, Italy) were anesthetized with intraperitoneal injections of pentobarbital sodium (50 mg/kg). Heart rate (HR) and mean arterial blood pressure (MAP; Viggo-Spectramed P10E2 pression transducer system, model 13-6615-10S; Gould Instrument Systems) were measured before the IH protocol, and microcatheters were inserted into the right carotid artery and left femoral vein. The catheters were guided subcutaneously to the dorsum, exteriorized through the skin, and tied to the skin. Arterial and venous blood was sampled into heparinized capillary tubes and analyzed for PO2, PCO2, and pH at 37°C using a pH/blood gas analyzer (Ciba Corning pH/blood gas analyzer, model 248; Essex, UK). Hematocrit was measured from centrifuged arterial blood taken in heparinized capillary tubes (Hettick Hct 20 Centrifuge; Tuttlingen, Germany). All animal studies were approved by the Animal Care and Use Committee of the National Council of Research, Italy.

IH protocol. Animals were subjected to IH 1 day after catheter implantation. The chamber containing the animals was vented with 100% nitrogen delivered via a timed solenoid valve. The O2 concentration was rapidly reduced to 8% O2 and kept at this level for 6 min. The chamber was then vented with compressed air elevating O2 to 21% for 6 min. This procedure was repeated five times in 1 h, and the whole cycle was repeated every 8 h for 21 days. Normoxia control (NC) hamsters were placed in a Plexiglas box flushed with compressed air at 6-min intervals, instead of nitrogen. Baseline recordings (systemic hemodynamics, hematocrit, and blood sampling) were obtained before and after IH preconditioning. After the IH pretreatment was completed, the animals were anesthetized, intubated endotracheally, and injected with L-NAME or SMT or saline. After 60 min, the animals were prepared for cheek pouch observation. After a 30-min stabilization period and baseline observation (systemic hemodynamics, blood sampling for NOx and TBARS measurements, arteriolar diameter, and RBC velocity), hamster cheek pouch underwent 30 min ischemia followed by 30 min reperfusion. Ischemia was induced by applying atraumatic microvascular clips on the proximal part of the cheek pouch for a period of 30 min. The clamp was then removed, and the microcirculation was reperfused for 30 min. Microcirculatory measurements and the arteriolar response to ACh and sodium nitroprusside (SNP) were made before and after I/R.

Experimental groups. Fifty animals were assigned to six groups. After IH preconditioning, the animals were randomized to receive 1) the NOS inhibitor L-NAME (20 mg/kg, n = 10; Sigma, St. Louis, MO), a nonselective eNOS inhibitor; 2) the iNOS inhibitor SMT (5 mg/kg, n = 10); or 3) an equivalent volume of saline (1 ml/kg), control for IH (IH group, n = 10), injected intravenously 60 min before the cheek pouch preparation (4). NC (n = 10) and NC1 (20 mg/kgL-NAME, n = 5) and NC2 (5 mg/kg SMT, n = 5) groups were kept under normoxic conditions for 12 days and injected with an equal volume of saline or with L-NAME (20 mg/kg) or SMT (5 mg/kg), respectively, before I/R. In two separate groups of five animals each, the arteriolar response was assessed in NC and IH by adding ACh and SNP to the bath solution.

Intravital microscopy. The cheek pouch was spread out and fixed to a Plexiglas microscope stage (3–12). A thin black blade was then inserted through a small incision between the top and bottom layers of the pouch. The cheek pouch was superfused with a 37°C Ringer solution (4 ml/min), with 5% CO2-95% N2 adjusted to pH 7.35. Microvascular images were transmitted through a fluorescence microscope (Orthoplan; Leica Microsystem, Wetzlar, Germany) and a filter block (Ploemopak; Leica) fitted with a long working distance objective [x4, numerical aperature (NA) 0.14; and x20, NA 0.25] and a x10 eyepiece. Epi-illumination was provided by a xenon 150-W lamp using filters for fluorescein isothiocyanate, bound to dextran (50 mg/100 g, intravenously injected as 5% wt/vol solution in 5 min; molecular weight 150,000; Sigma Chemical), and for acridine red. A heat filter was also used. A closed-circuit system consisting of a Cohu high-performance charged-coupled device camera (model 4922-5010; Cohu, San Diego, CA), a Sony video recording system (model VO 5800), and a Sony trinitron color monitor were used to view and record the microscopic images.

Measurement of microvascular parameters. RBC velocity was measured online by using the photodiode/cross-correlator system (PhotoDiode/Velocity Tracker model 102B; Vista Electronics, San Diego, CA) (10). A video image-shearing method (digital image shearing monitor, model 907; IPM, San Diego, CA) was used to measure vessel diameter. The center line velocity was corrected according to the vessel size to obtain the mean RBC velocity (V) (7). Blood flow (Q) was calculated according to the formula Q = V x {pi} (D/2)2; SS was calculated according to the following equation: SS = 8 {eta}·Vm/D, where {eta} is the blood viscosity and D the diameter of the vessel. The PCL, defined as the length of capillaries that are perfused by at least one RBC in a 30-s period, was assessed in a region of ~0.5 mm2 (10). The PCL was measured by using imaging analysis software (Project Engineering, Florence, Italy).

Lipid peroxide and nitrite/nitrate determinations. Lipid peroxidation was measured in plasma using an assay to estimate levels of TBARS (ZeptoMetrix, Buffalo, NY). TBARS, generated during the peroxidative process and including mainly lipid peroxides and malondialdehyde, were determined using 100 µl of plasma and 60 min incubation under acidic conditions at 96°C; measurements were made spectrophotometrically at 532 nm. TBARS values are expressed in terms of malondialdehyde equivalents as nanomoles per milliliters.

NOx levels in plasma were measured as previously reported (12). Blood samples were collected before and after I/R with a 5-ml syringe containing 0.14 ml of sodium citrate (26.35 mg/ml) per milliliter of withdrawn blood. The blood samples were centrifuged and separated from RBCs, and plasma was stored at –70°C. After ultrafiltration of the plasma through a 10-kDa membrane, the samples were incubated with nitrate reductase and enzyme cofactors for 3 h for the conversion of nitrate to nitrite. Absorbance was read at 540 nm using a plate reader after the addition of the Griess reagents that convert nitrite into a deep purple azo compound.

Response to ACh and SNP. ACh (10–7 M; Sigma) and SNP (10–4 M; Sigma) were topically applied on the cheek pouch microcirculation to assess arteriolar responses (4). ACh was topically applied after IH and 30 min of postischemic reperfusion to assess the endothelial function of arterioles [baseline diameter in NC, 48.0 (3.20) µm; and IH, 37.3 (1.90) µm; n = 10, number of vessels observed for each group]. Topical administration was used to avoid systemic effects. Vessel diameters were recorded for at least 5 min in each period after ACh was topically applied. After the administration of ACh, the cheek pouch was washed out with a 36°C Ringer solution (4 ml/min) with 5% CO2-95% N2 adjusted to pH 7.35. At the end of the experimental protocol, SNP (100 µm) was topically applied to the cheek pouch to measure the maximal arteriolar vasodilation. All diameter changes in response to ACh were expressed as percent change = [(D – Db)/Db] x 100, where Db is the baseline diameter (17).

Statistics. All reported values are means (SD). GraphPad software (San Diego, CA) was used to analyze statistical differences. Bivariate correlation analyses were performed with the calculation of Pearson R correlation. Differences between groups at the same time points were determined by the Kruskall-Wallis test, followed by the Dunnett's test. The Friedman two-way ANOVA by rank test was used to evaluate changes between groups at different times, followed by the Dunnett's test. Two-way ANOVA for repeated measures, followed by Tukey's test, was used to detect for differences among the groups. P < 0.05 was used to establish statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hematological and hemodynamic variables in animals treated with IH are reported in Tables 1 and 2. IH did not increase MAP and HR in preconditioned hamsters. IH resulted in a significantly decreased PaO2-PaCO2 (P < 0.05) and an increased pH (P < 0.01); hematocrit was elevated in IH, L-NAME, and SMT groups when compared with the NC group (P < 0.05). L-NAME and SMT increased MAP, and HR did not change significantly (P < 0.05; Table 1).


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  		Table 1. Hemodynamic variables

 

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  		Table 2. Hematological variables

 
Effects of IH. After the IH preconditioning protocol, NOx and TBARS levels were significantly enhanced in the IH group versus the NC group (P < 0.05; Figs. 1 and 2). Conversely, they were lower after I/R versus the NC group (P < 0.05). In all the animals, the observed arterioles were classified according to the branching order and were selected A2 arterioles [values before I/R in NC, diameter, 48.0 µm (SD 3.20); RBC velocity, 2.14 mm/s (SD 0.42); blood flow, 2.41 nl/s (SD 0.783); and SS, 0.99 Pa (SD 0.10); and IH, diameter, 37.3 µm (SD 1.90); RBC velocity, 2.0 mm/s (SD 0.38); blood flow, 1.34 nl/s (SD 0.9); and SS, 1.30 Pa (SD 0.18); n = 25, number of arterioles]. IH reduced the arteriolar diameter by 22%, the RBC velocity by 13%, and blood flow by 26%, and SS increased by 11% versus the NC group (P < 0.05; Figs. 3, 4, 5, and 6). Conversely, they were significantly enhanced after I/R compared with the NC group (P < 0.05). In the IH group, NOx and ROS did not show a statistically significant relationship to diameter (r coefficient = 0.39–0.34 and P = 0.2–0.7, Pearson coefficient for NOx and ROS, respectively, in I/R), RBC velocity (r coefficient = 0.11–0.10 and P = 0.7–0.66, Pearson coefficient), and blood flow (r coefficient = 0.2–0.2 and P = 0.5–0.4, Pearson coefficient), a result indicating that the effects due to NOx and ROS are not likely to be a cause for the observed changes. During the IH protocol, the PCL was significantly reduced [values before I/R, NC, 1.78 cm–1 (SD 0.34); and IH, 1.35 cm–1 (SD 0.23)] by 24% of control (P < 0.05; Fig. 7). After postischemic reperfusion, there was a significant increase in PCL by 115% versus the NC group (P < 0.05). ACh arteriolar responses after I/R from the NC [baseline diameter, in NC, 49.0 µm (SD 2.70); and IH, 38.5 µm (SD 1.50); n = 10, number of vessels observed for each group] showed a progressive constriction (Fig. 8). ACh-induced dilation was not enhanced by pretreatment with IH, whereas this significantly improved ACh-induced dilation after postischemic reperfusion such that the response was similar to that in the NC before I/R (P < 0.05). The responses to SNP, the endothelium-independent vasodilation, were significantly similar in both groups (Fig. 9). SNP caused a significant dilation of arterioles in all groups (P < 0.01). This indicates the presence of similar microvascular vasodilatory reserve in all groups and that vascular smooth muscle function was not impaired.


Figure 1
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  		Fig. 1. Changes in nitrite/nitrate (NOx) in plasma of hamsters treated with ischemia-reperfusion (I/R) plus saline [normoxic control (NC)] or N{omega}-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg iv; NC1 group) or S-methylisothiourea sulphate (SMT, 3 mg/kg iv; NC2 group) and intermittent hypoxia (IH; IH group) or IH plus treatment with L-NAME (L-NAME group) or IH plus treatment with SMT (SMT group) before and after 30 min of reperfusion. Values are means (SD); n = 7 animals in each group. *P < 0.05 vs. the NC group; °P < 0.05 vs. IH; #P < 0.05 vs. the NC group after I/R; +P < 0.05 vs. the IH, SMT, and L-NAME groups; ^P < 0.05 vs. the L-NAME group.

 

Figure 2
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  		Fig. 2. Changes in thiobarbituric acid-reactive substances (TBARS) in plasma of hamsters treated with I/R plus saline (I/R) or L-NAME (20 mg/kg iv; NC1 group) or SMT (3 mg/kg iv; NC2 group) and IH (IH group) or IH plus treatment with L-NAME (L-NAME group) or IH plus treatment with SMT (SMT group) at baseline (Bsl), before I/R, and after 5, 15, and 30 min of I/R, respectively. Values are means (SD); n = 7 animals in each group. *P < 0.05 vs. the NC group; °P < 0.05 vs. IH; #P < 0.05 vs. the NC group after I/R; +P < 0.05 vs. the IH, SMT, and L-NAME groups; ^P < 0.05 vs. the L-NAME group.

 

Figure 3
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  		Fig. 3. Changes in diameter of A2 arterioles in the hamster cheek pouch microcirculation in response to I/R in NCs (NC, NC1, and NC2 groups) plus treatment with saline or L-NAME or SMT and in hamsters pretreated with IH (IH group) or IH plus treatment with L-NAME (L-NAME group, 20 mg/kg) or IH plus treatment with SMT (SMT group, 3 mg/kg iv) before and after 30 min of reperfusion. Data are means (SD) of percent changes of corresponding resting values given in the text. Each point represents an average of at least 30 measurements from 10 animals. *P < 0.05 vs. the NC group; °P < 0.05 vs. IH; #P < 0.05 vs. the NC group after I/R; +P < 0.05 vs. the IH, SMT, and L-NAME groups; ^P < 0.05 vs. the L-NAME group.

 

Figure 4
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  		Fig. 4. Changes in red blood cell (RBC) velocity of A2 arterioles in the hamster cheek pouch microcirculation in response to I/R in NC hamsters (NC, NC1, and NC2 groups) plus treatment with saline or L-NAME or SMT and in hamsters pretreated with IH (IH group) or IH plus treatment with L-NAME (L-NAME group, 20 mg/kg) or IH plus treatment with SMT (SMT group, 3 mg/kg iv) before and after 30 min of reperfusion, respectively. Data are means (SD) of percent changes of corresponding resting values given in the text. Each point represents an average of at least 30 measurements from 10 animals. *P < 0.05 vs. the NC group; °P < 0.05 vs. the IH; #P < 0.05 vs. the NC group after reperfusion; +P < 0.05 vs. the IH, SMT, and L-NAME groups; ^P < 0.05 vs. the L-NAME group.

 

Figure 5
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  		Fig. 5. Changes in blood flow of A2 arterioles in the hamster cheek pouch microcirculation in response to I/R in NC hamsters (NC, NC1, and NC2 groups) plus treatment with saline or L-NAME or SMT and in hamsters pretreated with IH (IH group) or IH plus treatment with L-NAME (L-NAME group, 20 mg/kg) or IH plus treatment with SMT (SMT group, 3 mg/kg iv) before and after 30 min of reperfusion, respectively. Data are means (SD) of percent changes of corresponding resting values given in the text. Each point represents an average of at least 30 measurements from 10 animals. *P < 0.05 vs. the NC group; °P < 0.05 vs. the IH; #P < 0.05 vs. the NC group after reperfusion; +P < 0.05 vs. the IH, SMT, and L-NAME groups; ^P < 0.05 vs. the L-NAME group.

 

Figure 6
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  		Fig. 6. Changes in shear stress of A2 arterioles in the hamster cheek pouch microcirculation in response to I/R in NC hamsters (NC, NC1, and NC2 groups) plus treatment with saline or L-NAME or SMT and in hamsters pretreated with IH (IH group) or IH plus treatment with L-NAME (L-NAME group, 20 mg/kg) or IH plus treatment with SMT (SMT group, 3 mg/kg iv) before and after 30 min of reperfusion, respectively. Data are means (SD) of percent changes of corresponding resting values given in the text. Each point represents an average of at least 30 measurements from 10 animals. *P < 0.05 vs. the NC group; °P < 0.05 vs. the IH; #P < 0.05 vs. the NC group after reperfusion; +P < 0.05 vs. the IH, SMT, and L-NAME groups; ^P < 0.05 vs. the L-NAME group.

 

Figure 7
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  		Fig. 7. The perfused capillary length (PCL) changes in the hamster cheek pouch microcirculation in response to I/R in NC hamsters (NC, NC1, and NC2 groups) plus treatment with saline or L-NAME or SMT and in hamsters pretreated with IH (IH group) or IH plus treatment with L-NAME (L-NAME group, 20 mg/kg) or IH plus treatment with SMT (SMT group, 3 mg/kg iv) before and after 30 min of reperfusion, respectively. Data are means (SD) of percent changes of corresponding resting values given in the text; n = 10 for each group. *P < 0.05 vs. the NC group; °P < 0.05 vs. the IH; #P < 0.05 vs. the NC group after reperfusion; +P < 0.05 vs. the IH, SMT, and L-NAME groups; ^P < 0.05 vs. the L-NAME group.

 

Figure 8
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  		Fig. 8. Effect of I/R in NCs and in IH-pretreated (IH group) hamsters on dilation [baseline diameter, in NC, 49.0 (2.70) µm; and IH, 38.5 (1.50) µm; n = 10, number of vessels observed for each group] to acetylcholine (ACh). Arterioles of hamster cheek pouch of IH-pretreated hamsters after I/R dilated in a similar manner to NC before I/R. All diameter changes in response to ACh were expressed as percent change = [(D – Db)/Db] x 100, where D is diameter and Db is the baseline diameter. Data are means (SD). °P < 0.01 vs. baseline; *P < 0.05 vs. NC group before I/R; #P < 0.05 vs. NC after I/R.

 

Figure 9
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  		Fig. 9. Effect of I/R in NC and in IH-pretreated (IH group) hamsters on dilation [baseline diameter, in NC, 49.0 µm (SD 2.70); and IH, 38.5 µm (SD 1.50); n = 10, number of vessels observed for each group] to sodium nitroprusside (SNP). Data are percent changes in diameter from baseline. Arterioles of hamster cheek pouch of NC and IH-pretreated hamsters dilated in a similar manner to endothelium-independent agonist SNP. Data are means (SD). *P < 0.01 vs. baseline.

 
Effects of L-NAME and SMT. In NC1 and NC2 groups, TBARS and NOx did not change significantly at baseline after an L-NAME and SMT injection, whereas TBARS increased and NOx decreased significantly in I/R (P < 0.05; Figs. 1 and 2). In the L-NAME and SMT groups, both L-NAME and SMT decreased NOx levels in I/R injury at comparatively higher levels than in the IH group (P < 0.05; Fig. 1), but the reduction in NOx plasma concentration was more marked with L-NAME versus SMT (P < 0.05). TBARS increased significantly in both groups when compared with the IH group (P < 0.05; Fig. 2). Furthermore, L-NAME was more able than SMT to increase TBARS accumulation in I/R. In the NC1 and NC2 groups, arteriolar diameters, RBC velocity, and blood flow [NC1, baseline, 47.0 µm (SD 7.0) and 2.0 mm/s (SD 0.5) and 2.31 nl/s (SD 10); and SS, 0.81 Pa (SD 0.13); and NC2, baseline, 47.9 µm (SD 5.8) and 2.1 mm/s (SD 0.3) and 2.20 nl/s (SD 8.5); and SS, 0.84 Pa (SD 0.10)] decreased after an L-NAME and SMT injection (P < 0.05 vs. baseline conditions; Figs. 3–6). The inhibitory effects of L-NAME and SMT on these parameters were also evident during postischemic reperfusion in I/R (P < 0.05 vs. the NC group).

In the L-NAME and SMT groups, L-NAME and SMT reduced the arteriolar diameter, RBC velocity, blood flow, and SS versus those in the NC group. The decrease in arteriolar diameter, SS, blood flow, and capillary perfusion was significantly greater with L-NAME than with SMT (P < 0.05). PCL also decreased significantly after L-NAME [values before I/R, L-NAME, 1.05 cm–1 (SD 0.32); and SMT, 1.10 cm–1 (SD 0.19)] and SMT, respectively, versus the IH group (P < 0.05; Fig. 7).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our main finding is that IH prevented I/R damage by reducing NOx levels during postischemic reperfusion, showing that NO plays a crucial role in preserving capillary perfusion against I/R injury since the interference with NO production was largely negative. Our data showed that IH preconditioning increased the levels of plasma NOx and TBARS compared with those in NC hamsters; however, the levels of NOx and TBARS during postischemic reperfusion were reduced and resulted in a substantial increase in blood flow, SS, vasodilation, and capillary perfusion. Exposure to IH increased the hematocrit, which may contribute to a sustained SS stimulus on the endothelial cells. Moreover, vasodilatory function was impaired during I/R in NCs, whereas IH preserved vasodilation in I/R. IH prevented the impairment of cheek pouch vascular endothelium-dependent responses, as shown by the ACh-dependent vasodilation. Remarkably, the use of the NO synthesis inhibitor L-NAME in IH-preconditioned hamsters completely abolished the IH protective effects. Conversely, vasodilation was only partially restored with the iNOS preferential blocker. Therefore, both eNOS and iNOS may participate in IH protective effects against I/R. The major source of NO during postischemic reperfusion may be eNOS, which can be upregulated in response to elevated wall SS as found in the groups treated with IH. Moreover, the reduction in oxidative stress may be responsible for the improvement in vascular function during I/R damage.

Our data showed that hematocrit increased by 21%, which implies an increase of at least 21% in blood viscosity. However, the arterial blood pressure did not change during IH preconditioning. Martini et al. (30) showed that a 20% change in hematocrit and blood viscosity is the point at which there are no changes in blood pressure, cardiac output, and peripheral vascular resistance. These authors suggested that this is the hematocrit increase at which the increase of NO due to increased SS is balanced by increased NO scavenging and increased blood viscosity. Nevertheless, persistent hypertension is a common response maladaptation to severe, sustained IH, and central cardiorespiratory network responses to IH are mediated by the level of oxidative stress (20, 23). However, our results are in agreement with Tamisier et al. (40) who showed persistent sympathoexcitation following continuous hypoxia but not following IH, possibly because the hypoxic protocols were hypocapnic, and hypocapnic hypoxia does not lead to a significant poststimulus sympathoexcitation. Moreover, Manukhina et al. (29) suggested that an efficient NO storage during hypoxia may prevent hypotension, a detrimental effect of NO overproduction. IH can also have an important role in terms of increasing viscosity by stimulating the formation of erythropoietin. Recent data have suggested that a 1-wk administration of erythropoietin changes vasomotor tone regulation without causing hypertension (33). Moreover, there is a transgenic mouse line with severe polyglobulia that, due to an increased expression of eNOS associated with enhanced NO bioavailability, does not develop hypertension and myocardial infarction (37).

Our data showed that both NOx and lipid peroxide levels were higher during IH adaptation compared with those of NCs. This response was associated with increased SS, vasoconstriction, and a small reduction in capillary perfusion. The increase in SS during IH could be explained by the increase in oxidative stress (8, 31). Indeed, IH changes the susceptibility of the heart to oxidative stress, and elevated superoxide production during exposure to IH attenuates vasoconstrictor responsiveness to norepinephrine and myogenic activation in skeletal muscle resistance arteries (35, 36). Therefore, the vasomotor response to IH strikingly depends on the level of oxidative stress. Previous findings have also shown that the suppression of endothelial NO formation during conditions of hypoxia induces an acute imbalance in favor of vasoconstriction, reduction of capillary perfusion, and leukocyte adhesion (10). Moreover, a source of NO from iNOS is not a potential mechanism to induce vasodilatation in conditions of alkalosis such as during IH. NOS-independent NO production seems to complement the endogenous NO production especially during ischemia and acidosis when oxygen availability is low and the NOSs operate poorly (32). Therefore, our results suggest that exposure to IH elicits vasomotor changes that can be at least partly looked up as a physiological response to pH changes and enhanced ROS production, thereby reducing NO bioavailability and NO-induced vasodilation.

There was an increase in arterial diameter, blood flow SS, and capillary perfusion after I/R, within minutes of reperfusion in IH-preconditioned hamsters, which can be explained by an increased activity of eNOS in vascular endothelial cells. We found that both L-NAME and SMT per se influenced I/R injury and that the protective effect of IH was abrogated by L-NAME and by SMT (a selective blocker of iNOS). L-NAME increased the damage more than SMT. The increase in the hematocrit during IH would allow the increase in SS and eNOS expression, thus ensuring an additional increase in NO levels during postischemic reperfusion. Indeed, elevated SS is known to modulate eNOS expression in arteries and to control oxidative stress (30, 31, 41). Koti et al. (28) have reported protective effects of ischemic preconditioning of the liver as a result of the enhancement of eNOS expression as well as Cai et al. (15), who have shown that IH results in the upregulation of eNOS expression in the heart. Phillips et al. (35) have shown that impaired vasodilation in rat cerebral and skeletal muscle resistance arteries is due to altered mechanisms of endothelium-dependent vascular relaxation after exposure to IH rather than altered sensitivity to NO. Our data indicate that NO-mediated, endothelium-dependent vasodilation during IH is not compromised and is similar to NCs. Nevertheless, our data showed that I/R can impair agonist-induced NO production and subsequent NO-mediated dilation in the hamster cheek pouch. This could also explain the deficiency of capillary perfusion observed in I/R injury. Conversely, IH prevented the impairment of cheek pouch vascular endothelium-dependent responses, as shown by the increased ACh-dependent vasodilation during I/R. The responses to SNP, the endothelium-independent vasodilation, were significantly similar in NC and IH groups, showing that endothelial dysfunctions are basically responsible for the arteriolar responses. Therefore, our results indicate that increased intraluminal SS induces eNOS expression and improves endothelium-dependent dilation in I/R by increasing NO production during IH preconditioning.

Accumulating evidence suggest that NOx level is a determinant in I/R and that the application of nitrite as a direct NO donor for treatment of ischemic disorders confers a substantial dose-dependent cytoprotective effect, limiting necrosis and preserving organ function (19, 24). Tissue acidosis and relative hypoxia is present during I/R, and in this metabolic state, the bioactivation of nitrite is likely enhanced (32). Therefore, the increase in NO can partially explain the vasodilatory effect observed during I/R in preconditioned hamsters.

Our data show that a moderate but significant vasodilation was still present in SMT group, although in these animals, oxidative stress was higher than that in IH-preconditioned animals, thus indicating that NO levels are more important than oxidative stress in the mechanism of the protective effects of IH. However, I/R damage was decreased in the IH group through a reduction of NOx versus NCs during postischemic reperfusion; this may mean that NO produced in massive outbursts may form a stronger oxidant peroxynitrite with resultant I/R damage. A small increase in NO production can act as a protective mechanism, whereas a large increase in NO production may be proinflammatory, resulting in systemic and local damage. Recently, Ding et al. (18) showed that IH upregulated the baseline level of iNOS mRNA and protein expression, leading to an increase in NO production in the isolated heart. Cuong et al. (16) studied changes in NO and mitochondrial superoxide during anoxic preconditioning in isolated hearts and single cardiomyocytes. They concluded that the increase in NO production during reoxygenation in the preconditioned animals was attenuated by the iNOS inhibitor. Nitrite can be involved in IH-preconditioning effects, because SMT showed a vasodilator effect and enhanced capillary perfusion. The fact that SMT promoted a different inhibition in NOx levels can be attributed to the inhibition of iNOS rather than eNOS activity, since we had a less-marked decrease in SS, blood flow, and capillary perfusion when compared with L-NAME. The ability of SMT to partly prevent I/R damage may indicate that NO activity was not completely blocked. In summary, the IH protective effects against I/R injury may also be determined by the balance between NO generation from iNOS and the reduction of oxidative stress during I/R injury. Although we have demonstrated a role for iNOS/eNOSs in the response to IH preconditioning against I/R, there are issues that need to be addressed by future studies such as the direct quantification of iNOS/eNOS, which was beyond the scope of the present study.

The significant loss in capillary perfusion found with I/R is the most critical microvascular damage that ultimately leads to irreversible and lethal conditions in I/R injury (3–5, 12, 26). Oxygen free radicals and endothelial dysfunction are the most likely causes of the noted vasoconstriction, which decreases RBC velocity and blood flow in I/R, leading to a limited extraction of toxic catabolites. Conversely, IH increased microvascular perfusion immediately after reperfusion with the beneficial outcome reflected by the reduction in NOx and oxidative stress. The increased eNOS, and the improvement of the endothelium-dependent vasodilation in I/R in IH-preatreated hamsters, could explain the increase in capillary perfusion. Moreover, our data showed that the eNOS inhibition caused a marked capillary perfusion loss during I/R compared with IH. This is consistent with previous findings, indicating that increases in endogenous NO levels and NO donors are beneficial in preventing the no-reflow phenomenon in I/R injury (8, 11). Hence, apart from the well-established NO regulation of vascular tone, NO controls capillary perfusion during I/R injury.

The capillary lumen and its glycoprotein and protein constituents are continuously subjected to changing SS that impacts the composition of the glycocalyx (1). Glycocalyx perturbation may contribute to enhanced vulnerability of the vessel wall under hypoxic or I/R conditions (22). Kashiwagi et al. (25) have shown that the expression of eNOS occurs in the endothelium of capillaries and venules in normal conditions, whereas the smaller arterioles express neuronal NOS but little of eNOS. Therefore, it is not unreasonable to speculate that endothelial NO has the function to regulate capillary perfusion, because of the compartmentalization of NO mainly in capillaries. Many studies have confirmed that an elevation of NO increases the fluidity of the membranes, whereas a low SS associated with excessive ROS production destabilizes the capillary endothelial cell membrane (21). Moreover, the reduction in NOx levels by IH, such as that shown by our findings, may protect capillary perfusion from excessive oxidative stress. In addition, deficient NO production by the endothelial cells may result in increased membrane rigidity, thus leading to the deceleration and trapping of formed elements in the capillary circulation, increased peripheral resistance to blood flow. Therefore, IH may preserve capillary perfusion during I/R injury by reducing oxidative stress and increasing NO levels.

In conclusion, we demonstrated that preconditioning with IH can greatly reduce oxidative stress and stimulate NO-induced vasodilation during I/R injury, thus controlling capillary perfusion. The increase in the hematocrit would maintain an increased SS that induces eNOS expression and improves endothelium-dependent dilation in I/R by increasing NO production. The ability of the inhibition of iNOS to attenuate the I/R damage without completely blocking NO formation suggests that IH protection is also associated with the reduction of iNOS levels. Although further investigations are needed to determine the possible clinical use of IH, understanding the mechanisms of IH adaptation might form the basis for effective therapeutic strategies to prevent and treat I/R damage.


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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported by the National Council of Research, Italy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Bertuglia, Univ. of Pisa, Via Trieste 41, 56100 Pisa, Italy (e-mail: sibert{at}ifc.cnr.it)

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


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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