INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

IN THE LAST six years, a significant development in the respiratory field has been the use of inhaled nitric oxide (NO), initially in infants with pulmonary hypertension, but more recently with adults who have respiratory distress as one manifestation of multiple-organ dysfunction syndrome (2, 6, 8). Therapeutically, inhaled NO is used as a local vasodilator in the pulmonary vasculature. However, recently, we reported that inhaled NO extends far beyond the effects of the pulmonary vasculature to affect a peripheral microvascular bed depleted of NO. NO inhalation supplements a distal microvasculature with NO so that the poor perfusion, the multistep leukocyte recruitment paradigm, and microvascular dysfunction associated with NO inhibition were all significantly abrogated. NO inhalation had absolutely no effect on the normal unperturbed microvasculature, suggesting perhaps that peripheral effects of inhaled NO will only be apparent in an NO-deficient vascular bed (4). Indeed, in a situation like ischemia-reperfusion, where numerous laboratories have shown a decrease in NO production as early as 2.5 min after reperfusion and by >90% within 60 min of reperfusion (13, 15, 21), inhaled NO was a very effective inhibitor of poor perfusion, leukocyte recruitment, and microvascular dysfunction (1). Clearly, the data to date suggest that pretreatment with inhaled NO will benefit reperfusion-type injuries of the microvasculature. However, a number of very important issues remain: 1) Can inhaled NO be applied postreperfusion injury and reverse the microvascular dysfunction associated with reperfusion? 2) Can inhaled NO leave the microvasculature and have an impact on extravascular parenchymal cells? For example, in intestinal reperfusion injury, both the vasculature and the mucosal barrier are disturbed, the latter playing a potentially important role in bacterial translocation and multiple-organ dysfunction.

Although NO reacts very quickly with heme groups, thereby allowing for rapid clearance of NO from blood, NO also undergoes auto-oxidation producing potent nitrosating agents (e.g., N₂O₃) that will nitrosate protein-bound thiol groups under physiological conditions to yield stable S-nitrosoproteins (5). These nitrosothiols may represent a stabilized form of NO in biological tissues. Indeed, Keaney et al. (7) reported that S-nitroso-albumin possessed endothelium-derived relaxing factor-like properties including vasodilation and inhibition of platelet aggregation. Although the responses were sevenfold less potent as a bolus than nitroprusside, very importantly they lasted minutes vs. seconds for nitroprusside. In addition to albumin, other smaller molecules including cysteine and glutathione were demonstrated to also have NO-carrying capacity (16, 18). These data were interpreted to mean that protein thiols can serve as an NO adduct, preserving bioactivity and increasing the half-life of NO in biological systems. A similar mechanism has been proposed by Stamler and colleagues (5) for hemoglobin. It was proposed that hemoglobin can be nitrosylated in the lung, and the NO group may be released within peripheral vascular beds (5). The major difference between red blood cell-associated S-nitrosohemoglobin vs. plasma proteins such as S-nitrosoglutathione and S-nitrosocysteine is that the hemoglobin is very unlikely to ever reach the interstitial space, whereas smaller peptides and/or proteins could cross the endothelial barrier with reasonable ease. Therefore, predictably, if S-nitrosohemoglobin is the dominant physiological NO delivery system of NO inhalation, then inhaled NO may only impact the vasculature not the extravascular space, whereas if the opposite is true, then inhaled NO should also affect parenchymal cells.

In this study, we asked three separate but related questions regarding inhaled NO. First, we asked whether inhaled NO could prevent only the vascular or also the mucosal aspects of reperfusion injury. Only the vascular dysfunction associated with reperfusion was reduced after NO inhalation. To ensure that the inhaled NO was not reaching extravascular space, we inhibited NO synthesis in the intestine and asked whether NO inhalation could supplement NO in the extravascular space. Additionally, we cannulated the major lymphatic vessel draining the intestine and measured S-nitrosothiols in lymph before and after NO inhibition with and without NO inhalation. The data revealed that inhaled NO did not supplement the extravascular space with NO. On the basis of this negative finding, the final question focused on the vascular compartment, and using intravital microscopy to visualize the microvasculature, we asked whether inhaled NO could also reverse the sequelae of reperfusion injury within single microvessels (blood flow and leukocyte recruitment).

	METHODS AND MATERIALS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

Surgery for intravital microscopy. The experimental preparation used in this study is the same as described previously (4, 11). Animal protocols were approved by the University of Calgary Animal Care Committee and met the Canadian Guidelines for Animal Research. Briefly, cats (1.2-2.4 kg) were fasted for 24 h and initially anesthetized with ketamine hydrochloride (75 mg im). The jugular vein was cannulated, and anesthesia was maintained by the administration of pentobarbital sodium. A pressure transducer (Statham P23A, Gould, Oxnard, CA) connected to a catheter in the left carotid artery monitored systemic arterial pressure. A tracheotomy was performed to support breathing by artificial ventilation. NO at 0 or 80 ppm was delivered from a certified grade NO/balance N₂ gas cylinder to the inhalation line of a Harvard ventilator via a high-accuracy Matheson flowmeter (Matheson Gas Products Canada, Edmonton, Alberta, Canada), and NO and NO₂ were measured with a Pulmonox II NO and NO₂ electrochemical analyzer (Pulmonox Research and Development, Tofield, Alberta, Canada). Throughout the experiments, exhaled NO₂ was <5 ppm and was not different among groups. This NO delivery setup was similar to the one used to deliver NO to newborn infants with respiratory distress in the neonatal intensive care unit of the Foothills Medical Centre (University of Calgary), except in our system the cats were not provided with supplemental oxygen but rather ventilated on room air.

1

Surgery for mucosal permeability. Cats were anesthetized and ventilated as for intravital microscopy. The experimental procedure has been previously described in detail (12, 17). Briefly, a 35- to 75-g segment of the small intestine was isolated from the ligament of Treitz to the ileocecal valve; blood and lymph vessels were maintained intact. The remainder of the small and large intestine was extirpated. One to three loops of small intestine ~15 cm in length were created. The intestinal segments and the mesenteric pedicle were moistened with saline-soaked gauze and covered with Saran Wrap to prevent evaporation, and body temperature was maintained at 37°C by an infrared heat lamp.

1

1

1

1

1

1

1

1

1

1

1

Lymph S-nitrosothiol assay. We used a modification of the Seville and Griess reactions for total S-nitrosothiols measurement (16, 18). Briefly, 1 ml of freshly collected lymph was mixed with 20 µl of 500 µM diethylenetriaminepentaacetic acid (Sigma). This was divided into two reactions. In reaction 1, 500 µl of the lymph were mixed with 500 µl of 1% (wt/vol) sulfanilamide in 0.5 M HCl. Reaction 2 consisted of 500 µl of lymph mixed with 500 µl of 0.2% (wt/vol) HgCl₂ in the 1% sulfanilamide from reaction 1 to liberate NO⁺ from any RSNO. (R is any molecule and S-NO is S-nitrosothiol.) Both reactions were incubated in the dark at 37°C for 10 min. Next, 500 µl of 0.2% (wt/vol) N-(1-naphthyl)ethylenediamine dihydrochloride in 0.5 M HCl were added to both reactions, and the samples were incubated for an additional 10 min at 37°C in the dark. The samples were then read at 540 nm, and total nitrosothiols were calculated as where OD is optical density.

Plasma nitrite content. Total plasma nitrite was by the Griess reaction after conversion of nitrate to nitrite with nitrate reductase.

Statistical analysis. The data were analyzed using standard statistical programs, i.e., ANOVA and Student's t-test with Bonferroni's correction for multiple comparisons where appropriate. All values are expressed as means ± SE. Statistical significance was set at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

Inhaled NO reduces ischemia/reperfusion-induced hemodynamic but not mucosal dysfunction. Intestinal blood flow under normal conditions is ~75 ml · min¹ · 100 g¹ to the small bowel in control animals (Fig. 1). After 60 min of total ischemia, a profound hyperemic episode occurs, but by 30 min, blood flow is significantly reduced to 35-40 ml · min¹ · 100 g¹ and remains at this level for the next 30 min. In animals that are made to breathe 80 ppm NO before ischemia, intestinal blood flow was the same as in animals not breathing NO (Fig. 1), suggesting that inhaled NO does not affect intestinal blood flow under normal conditions. One hour of complete ischemia also resulted in a profound hyperemia upon release of the clamp. However, the 50% reduction in blood flow postischemia was not observed in these animals. Inhalation of 80 ppm NO maintained blood flow within 20% of control levels in all animals (Fig. 1). Previous work in our laboratory had suggested that NO donors given intravenously were capable of reducing the ischemic damage to gastrointestinal epithelium (17). Figure 2 demonstrates that clearance of ⁵¹Cr-EDTA, an index of mucosal permeability, was <0.05 ml · min¹ · 100 g¹ and did not increase during the ischemia episode. However, during reperfusion, the mucosal permeability progressively increased to more than sixfold by 60 min. In animals breathing 80 ppm NO, mucosal permeability was not altered under control conditions, did not change during the ischemic episode, then increased during the 60-min reperfusion period to the same degree as in the untreated animals. These data differ dramatically from the hemodynamic results obtained in the same animals (Fig. 1) as well as the effects of NO donors on mucosal barrier function (17).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Intestinal blood flow under control, ischemia for 60 min, and reperfusion for 60 min in animals breathing room air or 80 ppm nitric oxide (NO) (n = 8). CON, control. * P < 0.05 relative to respective control.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   51Cr-EDTA clearance (as index of mucosal permeability) under control, ischemia for 60 min, and reperfusion for 60 min in animals breathing room air or 80 ppm NO (n = 8). * P < 0.05 relative to respective control.

Inhaled NO cannot reverse the increase in mucosal permeability induced by systemic L-NAME. We have previously demonstrated that inhaled NO could prevent the effects of NO inhibition in the microvasculature including vasoconstriction of arterioles and adhesion of leukocytes (4). Figure 3 is consistent with this observation; the increase in blood pressure at both the low (not shown) and high concentration of L-NAME was delayed and reduced when inhaled NO was coadministered with the NO synthesis inhibitor. In this section, we also examined whether inhaled NO could prevent the increase in mucosal permeability associated with systemic NO synthesis inhibition. Figure 4 summarizes the results of L-NAME-induced mucosal permeability alterations in the presence and absence of inhaled NO. L-NAME induced a rise in ⁵¹Cr-EDTA clearance within 10 min from 0.05 to 0.2 ml · min¹ · 100 g¹ and then to ~0.50 ml · min¹ · 100 g¹. Inhibition of NO in the intestine also induced a very similar increase in mucosal permeability with inhaled NO (Fig. 4). It should be noted that when very low concentrations of L-NAME were infused, mucosal permeability increased to only 0.15 ml · min¹ · 100 g¹ and was blunted by 50% with inhaled NO. These data clearly demonstrate an inability of sufficient NO to reach the mucosal barrier with NO inhalation, except when very low levels of L-NAME are used.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The increase () in blood pressure with NG-nitro-L-arginine methyl ester (L-NAME) infusion at 400 µg · kg1 · min1 over 60 min. Open bars are cats ventilated on room air alone; solid bars are cats inhaling 80 ppm NO in room air.  P < 0.05 relative to respective L-NAME value.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   51Cr-EDTA clearance as a function of mucosal permeability with 60 min of NO synthesis inhibition with L-NAME in animals breathing room air or inhaled NO (n = 11). * P < 0.05 relative to respective control.  P < 0.05 relative to respective L-NAME value.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Total lymph S-nitrosothiol concentrations under baseline, during NO inhalation (NO), during NO synthesis inhibition (L-NAME), and during NO synthesis inhibition and NO inhalation (L-NAME + NO). * P < 0.05 relative to baseline.

Inhaled NO reverses hemodynamic responses and leukocyte-endothelial interactions. Clearly, there was a complete lack of effect of inhaled NO on the interstitium. Therefore, we chose to further investigate the role of inhaled NO on the vasculature. We studied whether inhaled NO would have any benefit on the sequelae of ischemia if initiated after reperfusion. In this series of experiments, we used intravital microscopy and observed the microvessels directly. The shear under control conditions in a 30- to 40-µm-diameter vessel was between 500 and 600 s¹ (Fig. 6). During the reperfusion phase, the shear dropped to 300 s¹ at 60 min of reperfusion and even further at 2 h of reperfusion. In a second group of cats after 10 min of reperfusion, inhaled NO was initiated. Shear rate through the postcapillary venules was maintained at preischemic values (Fig. 6). At 60 min, inhaled NO was stopped, and we observed the microvasculature for an additional 60 min. Blood flow through the vessel did not decrease at 2 h of reperfusion and remained elevated significantly above values in animals not exposed to inhaled NO (Fig. 6).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Shear rate in vessels (an index of blood flow) under control, during ischemia (60 min), and 2 h of reperfusion. At 10 min of reperfusion, animals were made to breathe 80 ppm NO for 50 min, then the inhaled NO was terminated and values were examined 1 h later (n = 10). * P < 0.05 relative to respective control value.  P < 0.05 relative to respective untreated value.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Adhesion of leukocytes under control during 60 min of ischemia and during 2 h of reperfusion. At 10 min of reperfusion, animals were made to breathe 80 ppm NO for 50 min, then the inhaled NO was terminated and values were examined 1 h later (n = 10). * P < 0.05 relative to respective control value.  P < 0.05 relative to respective untreated value.

Nitrites are elevated during NO inhalation. Plasma nitrites were below 50 µM under normal conditions (Fig. 8). When animals were allowed to breathe NO, nitrite levels rose rapidly to 200 µM. Inhaled NO was then terminated, and interestingly, circulating nitrites remained at the same level even 1 h after inhaled NO had been discontinued. These data suggest that clearance and production of nitrites remained approximately equal over the first hour after NO inhalation was terminated.

View larger version (10K): [in this window] [in a new window]   Fig. 8.   Plasma nitrite levels are shown before (pre-NO) and for 60 min of NO inhalation, then for 60 min without inhaled NO.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

In a recent publication (4), we proposed for the first time that the commonly used approach of NO inhalation thought to only impact the lung microvasculature also has an impact on a distal microvascular bed in an NO-depleted organ. We also demonstrated that in a model of ischemia-reperfusion where NO is depleted, NO inhalation reduced the microvascular dysfunction, whereas in a model of lipopolysaccharide administration where NO is overproduced, inhaled NO had no effect on the microvascular dysfunction. The latter raises the possibility that the mechanism by which NO is delivered to the periphery appears only to have an impact on a microvasculature with low NO levels. The proposed mechanism by which inhaled NO may reach the distal microvasculature is based on the work of Loscalzo and colleagues (7, 19) and Stamler and colleagues (5) who reported that albumin, glutathione, cysteine, as well as hemoglobin could function to stabilize and transport NO around the circulation. Indeed, Takahashi et al. (20) reported an increase in nitrosylhemoglobin in sheep made to breath 60 ppm inhaled NO, consistent with the view that the formation of these molecules could have biological activity in the periphery. In this study, however, we report that the biological effects of inhaled NO could be seen in the peripheral circulation both as a pretreatment and surprisingly as a posttreatment, whereas no biological activity could be seen in the extravascular space at the mucosal barrier. Neither ischemia-reperfusion nor L-NAME-induced mucosal barrier dysfunction was reduced with 80 ppm inhaled NO, despite the well-documented reduction in mucosal dysfunction in these models with NO donors (9, 17). Moreover, we were not able to detect a rise in S-nitrosothiols in the lymph draining the interstitium of the intestine after NO inhalation, suggesting that the nitrosothiols generated during NO inhalation are not able to reach the extravascular space in functionally sufficient quantities.

The lack of effect of inhaled NO on the extravascular epithelial barrier is not due to NO being unable to reduce mucosal dysfunction. Previous work using the same identical model has demonstrated that addition of at least two different NO donors or the precursor for NO, L-arginine, reduced epithelial reperfusion injury (10, 17). Moreover, the rise in mucosal permeability due to inhibition of NO with L-NAME is definitely preventable and even reversible with NO donors (9). One could argue that insufficient amounts of inhaled NO left the lungs. However, our own data would suggest that sufficient NO reached the peripheral microvasculature to prevent the drop in total intestinal blood flow and to prevent the drop in shear rate and increase in leukocyte adhesion through single microvessels. Moreover, sufficient NO could be delivered to the periphery very rapidly because the aforementioned microvascular disturbance could also be reversed with 30-60 min by NO inhalation. Clearly, despite the elevated NO levels reaching the distal microvasculature, the same source of NO was not affecting the extravascular space.

It is possible that all of the NO is extracted by the endothelium and cells in close proximity to the circulation so that none reaches, for example, the epithelium in the intestine. However, our data reveal no increase in nitrosothiols in the lymph of NO-depleted or normal intestine during NO inhalation. In the normal intestine, an increased demand for NO is unlikely, so a lack of rise in S-nitrosothiols in lymph after NO inhalation should not be as a result of dramatic NO extraction by endothelium under unperturbed conditions. An alternative explanation may be that the inhaled NO delivered to the peripheral circulation could be predominately transported by molecules such as hemoglobin in the form of S-nitrosohemoglobin and are simply not able to transverse even an injured endothelial barrier. Indeed, we never see red blood cells in lymph even after reasonably profound ischemia-reperfusion. However, large plasma proteins such as albumin are also restricted extremely effectively by endothelium over the period of minutes to an hour so that it is unlikely that either S-nitrosoalbumin or S-nitrosohemoglobin will deliver much NO to the epithelial barrier. Smaller molecules such as glutathione and cysteine are thought to also combine with NO to form S-nitrosoglutathione and S-nitrosocysteine (16, 18). In fact, there is some evidence that NO can be transferred from S-nitrosoalbumin to the smaller molecular weight cysteine which then enhanced biological effects such as vasodilation (18). However, in that system, cysteine had to be supplemented exogenously. In light of our data, endogenous cysteine may simply not be in sufficient concentrations to obtain NO from larger molecules like albumin and deliver it out of the vasculature. Nevertheless, exogenous supplementation of cysteine could be useful with inhaled NO because this small molecule will easily cross the endothelial barrier.

Although it was not within the realm of this study to elucidate the mechanism of action by which inhaled NO reduces reperfusion-induced vascular dysfunction, a number of possibilities exist. First, Lefer and colleagues (1, 14) have clearly documented impaired endothelial NO production in postischemic arteries leading to an inability to respond to NO-dependent vasodilators (ACh) but not NO donors. Therefore, it is quite likely that inhaled NO may function to deliver NO to impaired vessels to improve blood flow. The ability of inhaled NO to reduce leukocyte adhesion is quite likely through an endothelial-dependent mechanism. We previously reported that leukocytes from an animal inhaling NO interacted with adhesion molecules immobilized to plastic as effectively as leukocytes from an animal not breathing NO (4). These data suggest that the inhaled NO does not directly affect the leukocyte. It is our view that the inhaled NO reaches the postischemic periphery and affects adhesion molecule expression and/or oxidants produced by the endothelium.

It should be noted that in this study we used 80 ppm NO vs. the many clinical studies that generally use <40 ppm inhaled NO. We do not believe that 80 ppm NO reaches levels that are toxic; methemoglobin and NO₂ levels were continuously read on-line and never rose under these conditions. In fact, the levels never exceeded the permissible 5 ppm, whereas in preliminary work from our laboratory, 300 ppm NO caused NO₂ levels to rise above this value and cardiovascular complications were noted. We feel certain that clinically safe levels of NO were employed, because 80 ppm has been used in human neonate studies for up to 10 h without any detrimental effects (22). We chose not to use lower concentrations of inhaled NO because 20 ppm was not sufficient to impact the distal microvasculature (4). It should be noted that all previous studies have focused on the effects of inhaled NO at the level of the lung, so 80 ppm inhaled NO was not necessary. We are proposing that 80 ppm will need to be used to have an impact on the peripheral vasculature to limit endothelial injury in, for example, stroke, myocardial infarction, and other pathological conditions wherein ischemia-reperfusion could conceivably play an inappropriate role. However, most importantly, the inhaled NO could be started after reperfusion and still may improve the vascular dysfunction.

In conclusion, our data have demonstrated that inhaled NO can impact very significantly the postischemic microvasculature but more importantly from a clinical standpoint can interrupt and reverse the endothelial dysfunction and leukocyte recruitment induced by an ischemic episode. Importantly, however, there are limitations to inhaled NO therapy because this approach had absolutely no impact on extravascular events such as reperfusion-induced epithelial barrier dysfunction, possibly because the carrier molecules of NO were simply unable to cross the endothelial barrier.