INTRODUCTION

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Ischemia-reperfusion (I-R) lung injury plays a significant role in clinical situations such as lung transplantation (1), pulmonary thromboendarterectomy (2), reexpansion of collapsed lungs (3), and fibrinolysis after lung embolism (4). Lung failure associated with I-R is characterized by increased microvascular permeability and pulmonary vascular resistance with subsequent edema formation and impairment of gas exchange. Interestingly, the development of this syndrome is significantly influenced by variables of ventilation during lung ischemia: as a special feature of the lung, oxygen supply to pulmonary tissues can ensue from both the intravascular and the alveolar space. As a consequence, pulmonary ischemia does not necessarily result in hypoxia of the whole organ when ventilation is maintained, which may be followed by attenuation of I-R. On the other hand, alveolar oxygen represents a cofactor for the generation of free radicals, and continuous oxygen supply might thus enhance I-R injury. Against this background, lung models addressing mechanisms of I-R injury are difficult to compare. In previous studies, ischemia was induced while ventilation was stopped, with lungs being inflated (5) as well as deflated (8, 9). In other investigations with ongoing ventilation during ischemia, the role of alveolar oxygen tension was controversial: In some studies, I-R injury as found to increase in dependency on alveolar oxygen during ischemia (10), whereas other studies provided evidence for a protective (13) or missing effect of oxygen (14, 15). In addition, the alveolar distension per se as mediated by ventilation or inflation, independent of oxygen supply, may be important for the prevention of lung reperfusion injury. Several early studies reported a significant benefit of lung inflation during preservation for transplantation (16). More recent investigations have demonstrated that maintenance of ventilation during ischemia may decrease injury in vivo (13) and in ex vivo perfused-lung models (15, 19). These results suggest a significant role of biophysical factors in I-R lung injury.

Mechanical effects on the lung microvasculature may, however, not only be transferred via alveolar distension but also via vascular distension. In pulmonary artery endothelial monolayers, the establishment of a positive baseline hydrostatic pressure results in an impressive "sealing" of the monolayer, i.e., a reduction of hydraulic conductivity and a decrease in protein permeability (20). Moreover, pilot experiments performed when establishing the ischemia/reperfusion model in the rabbit lungs signaled that the baseline intravascular (hydrostatic) pressure during ischemia was to be considered as an independent and possibly important variable. The current study was thus designed to address this issue and to analyze the impact of vascular and alveolar distension, with and without oxygen supply during ischemia, on lung I-R injury. We employed warm ischemia in isolated perfused rabbit lungs as this model permits independent manipulation of perfusion and ventilation parameters over a wide range without invoking systemic influences, and it allows differentiation of hydrostatic and permeability factors contributing to lung edema. When applying a positive intravascular pressure, its height corresponded to that of the lung microvascular pressure under conditions of pulsatile flow ( 4 mm Hg, slowly declining during the ischemic period). Noteworthy, we found that significant protection against I-R-induced microvascular leakage can be conferred by (1) continued ventilation, (2) presence of alveolar oxygen and, most strikingly, (3) maintenance of a positive intravascular pressure during lung ischemia. The finding of a crucial role of vascular distension versus collapse for the development of reperfusion injury may have major implication for preservation strategies in organ transplantation.

	METHODS

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Reagents

Sterile Krebs-Henseleit hydroxyethylamylopectine buffer (KHHB) was obtained from Serag-Wiessner (Naila, Germany). The buffer contained 120 mM NaCl, 4.3 mM KCl, 1.1 mM KH₂PO₄, 24 mM NaHCO₃, 2.4 mM CaCl₂, 1.3 mM MgPO₄, and 2.4 g/L glucose, as well as 5% (wt/ vol) hydroxyethylamylopectine (MW, 200,000) as oncotic agent. Gas mixtures for ventilation containing 21%O₂/5%CO₂/79%N₂, 95%O₂/ 5%CO₂, or 95%N₂/5%CO₂ were obtained from Messer Griesheim (Herborn, Germany).

Lung Model

The technique of isolated rabbit lung perfusion has been described in previous investigations (overview in reference 21). Briefly, rabbits of either sex weighing 2.6 to 2.9 kg were anticoagulated with heparin and deeply anesthetized with a mixture of ketamine and xylazine. Tracheostomy was performed, and the animals were ventilated with room air, using a Harvard respirator (tidal volume, 30 ml; frequency, 30 breaths/ min; positive end-expiratory pressure, 1 cm H₂O [Hugo Sachs Elektronik, March-Hugstetten, Germany]). After midsternal thoracotomy, catheters were placed into the pulmonary artery and the left atrium, and perfusion with sterile KHHB was started. Sterilized perfusion circuit tubing was used throughout. In parallel with the onset of artificial perfusion, room air ventilation was changed to a gas mixture containing 21% O₂ and 5% CO₂, with a balance of N₂. After extensive rinsing of the lung vasculature, the lungs were recirculatingly perfused with a constant pulsatile flow of 100 ml/min (total recirculating volume, 300 ml). Left atrial pressure was set at 2 mm Hg (referenced at the hilus), and the whole perfusion system was equilibrated at 37° C. Additionally, the inspiration loop of the ventilation system was connected to a humidifier and heated at 37° C by a thermostat. Lungs were placed in a humidified, warmed chamber to avoid desiccation and suspended from a force transducer for continuous registration of organ weight.

The capillary filtration coefficient (Kfc) and the total vascular compliance were determined gravimetrically from the slope of the lung weight gain curve induced by a 7.5-mm Hg step elevation of the venous pressure for 8 min. The application of this method to the present model and the use of time zero extrapolation of the slope of weight gain for the calculation of Kfc have been described in detail (22). Lung weight gain was calculated as the difference in organ weight measured directly before and 5 min after each of these pressure elevation maneuvers. Vascular compliance was calculated from the initial steep increase in lung weight upon step change in microvascular pressure. Pulmonary arterial and venous pressures were monitored by pressure transducers. The microvascular pressure (Ppc) was determined by the arterial and venous double occlusion technique. Occlusion maneuvers were performed in end-expiration by means of electromagnetic tube-clamping devices, synchronized by a D/A-A/D converter connected to a personal computer. This converter allowed parallel data collection at a rate of 20 Hz, which was processed by a spread-sheet program (Microsoft Excel^TM) for calculation of microvascular pressures.

Lungs included in the study (1) had a homogeneous white appearance with no signs of hemostasis, edema, or atelectasis; (2) had initial pulmonary artery and ventilation pressures in the normal range; and (3) were isogravimetric during an initial steady-state period of at least 30 min.

Experimental Protocols

After termination of the initial steady-state period and performance of a control hydrostatic challenge, time was set at zero, and lungs were exposed to ischemia by stopping the perfusion. In the course of the ischemic period, interventions were performed according to one of the following protocols. A total of 98 lungs was investigated.

FI_O2 = 0.21/IP⁺/vent. Lungs were exposed to normoxic ventilation during 60, 120, 180 and 240 min of ischemia. Upon stopping the perfusion, the arterial and venous catheters were both clamped for maintenance of a positive intravascular pressure (IP⁺) during ischemia.

FI_O2 = 0.21/IP/vent. Lungs were exposed to normoxic ventilation during different periods of ischemia. In these experiments, clamping of the arterial and venous catheter was omitted, resulting in an intravascular pressure of zero (IP) during ischemia.

FI_O2 = 0/IP⁺/vent. Lungs were exposed to anoxic ventilation during different periods of ischemia, and a positive intravascular pressure (IP⁺) during ischemia was maintained.

FI_O2 = 0/IP/vent. Lungs were exposed to anoxic ventilation during different periods of ischemia. Clamping of the arterial and venous catheter was omitted, resulting in an intravascular pressure of zero (IP) during ischemia.

FI_O2 = 0/IP⁺/collapse. Lungs were deflated during different periods of ischemia after the remaining dead-space oxygen had been washed out by short-time ventilation with an oxygen-free gas mixture (95% N₂, 5% CO₂). Upon stopping the perfusion, a positive intravascular pressure (IP⁺) was maintained.

FI_O2 = 0/IP⁺/static. Lungs were statically inflated during 180 min of ischemia after remaining dead-space oxygen had been washed out by short-time ventilation with an oxygen-free gas mixture (95% N₂, 5% CO₂). The inflation pressure was maintained at 6 mm Hg because this pressure level corresponds to the normal mean ventilation pressure in the isolated lung model. A positive intravascular pressure (IP⁺) during ischemia was again maintained by clamping of the arterial and venous catheter.

FI_O2 = 0.95/IP⁺/vent. Lungs were exposed to hyperoxic ventilation for 180 min of ischemia, and a positive intravascular pressure (IP⁺) during ischemia was maintained.

Each group encompassed four to seven independent experiments (Table 1).

At the end of ischemia, ventilation was restored or changed to normoxia (FI_O2 = 0.21), respectively (except the FI_O2 = 0.95/IP⁺/vent lungs, which were ventilated with 95% oxygen even during reperfusion), and perfusion was reestablished with gradual increase in flow over 30 s. Hydrostatic challenges were performed at 30, 60, and 90 min after onset of reperfusion. Double occlusion maneuvers (8 s each) for assessment of microvascular pressure were performed before ischemia as well as 3, 30, 60, and 90 min after reperfusion.

Control lungs were perfused and ventilated (FI_O2 = 0.21) without interruption of flow, and Kfc as well as microvascular pressure measurements were undertaken at time points corresponding to the ischemia experiments.

Data Analysis

Data were expressed as mean ± SEM. Differences were analyzed by analysis of variance; p values of less than 0.05 were considered to represent a significant difference.

	RESULTS

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Pulmonary Arterial Pressure

In control experiments, pulmonary arterial pressures were constant throughout the entire experimental period. After 60 min of ischemia, all lungs with an intravascular pressure of zero (IP) during ischemia displayed a significant Ppa increase upon reperfusion (Figure 1). Only minor Ppa changes were noted in response to 60 min of ischemia when the intravascular pressure was maintained positive (IP⁺). When the ischemic period was extended to 120 min, the pressor response in IP lungs was even more prominent (approximately doubling of baseline pressure), whereas in IP⁺ lungs again a significant attenuation of the Ppa increase was noted. These effects were independent of the presence or absence of alveolar oxygen during ischemia. After 180 min of ischemia, which was only performed in IP⁺ lungs, all experimental conditions with alveolar oxygen absent resulted in a more impressive rise of Ppa than those with alveolar oxygen present during ischemia (Figure 2). No difference was noted between FI_O2 = 0.21 and FI_O2 = 0.95. After 240 min of ischemia, again performed only in IP⁺ lungs, the Ppa increased similarly under both normoxic and anoxic conditions. The maximal Ppa rise was, however, lower than in the lungs with 120 min ischemia under IP conditions.

View larger version (24K): [in this window] [in a new window]   Figure 1.   Impact of vascular distension, alveolar distension, and alveolar oxygen (FIO2) during ischemia on pulmonary artery pressure (PAP) in reperfused rabbit lungs. At time zero, lungs were rendered ischemic for 60 min (upper panel ) or 120 min (lower panel ). Values are given as mean ± SEM. IP = intravascular pressure. Both IP groups were different from the corresponding IP+ groups, with p < 0.01. After 120 min of ischemia, all IP+ groups were different from control groups, with p < 0.02.

View larger version (23K): [in this window] [in a new window]   Figure 2.   Impact of alveolar distension and alveolar oxygen (FIO2) during ischemia on pulmonary artery pressure (PAP) in reperfused rabbit lungs. At time zero, lungs were rendered ischemic for 180 min (upper panel ) or 240 min (lower panel ). Values are given as mean ± SEM. After 180 min of ischemia, all FIO2 = 0 lungs were different from FIO2  0.21 lungs, with p < 0.01.

Microvascular Pressure and Vascular Compliance

In control lungs, microvascular pressures did not change during the entire experimental time period (Table 1). Similarly, microvascular pressures were largely constant in all ischemic lungs, with pressure changes never surpassing 2 mm Hg after reperfusion as compared with preischemic values (Table 1). No significant changes between the different experimental groups were noted. These data indicate that changes in hydrostatic forces may only negligibly contribute to any enhancement of capillary filtration and edema formation in the course of reperfusion. Values of vascular compliance did not differ between control lungs and the different experimental groups, and they did not significantly change in the reperfusion period as compared with the preischemic period (data not given in detail).

Kfc and Weight Gain

After 60 min of ischemia, lungs with a positive intravascular pressure during ischemia displayed identical Kfc values as compared with nonischemic lungs. In IP lungs, a slight tendency toward increased permeability was observed (Figure 3, and Table 1). As obvious in Table 1, the intravascular pressure measured at the end of ischemia in the IP⁺ lungs slowly dropped with the duration of the ischemic period, but it was still in the range of 1 to 2 mm Hg even after 240 min.

View larger version (20K): [in this window] [in a new window]   Figure 3.   Impact of vascular distension, alveolar distension, and alveolar oxygen (FIO2) during ischemia on the capillary filtration coefficient (Kfc) in reperfused rabbit lungs. At time zero, lungs were rendered ischemic for 60 min (upper panel ) or 120 min (lower panel ). Hydrostatic challenges were performed before ischemia as well as 30, 60, and 90 min after reperfusion. Values are given as mean ± SEM. IP = intravascular pressure. **p < 0.01 versus all IP+ groups and controls.

After 120 min of ischemia, these differences became striking: Kfc values dramatically increased in IP lungs as compared with the corresponding IP⁺ experiments, which was accompanied by severe edema formation. This effect was observed in both normoxic and anoxic ventilated lungs. Within the group of IP⁺ lungs, organs maintained in the absence of alveolar oxygen during the 120-min period of ischemia displayed a slight tendency for Kfc increase as compared with the control lungs and the lungs ventilated with normoxic gas.

After 180 min of ischemia, lungs rendered anoxic by deflation, nitrogen ventilation, or static inflation all showed markedly increased microvascular permeability (Figure 4), accompanied by a weight increase of corresponding severity. This leakage response was, however, significantly less prominent in lungs being continuously ventilated with nitrogen during ischemia, as compared with alveolar collapse or static inflation with anoxic gas. These findings indicate protective effects by (1) oxygen and (2) oxygen-independent ventilatory movements in IP⁺ lungs. Lungs ventilated with either 21 or 95% oxygen during ischemia did not differ from control lungs in terms of Kfc or organ weight.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Impact of alveolar distension and alveolar oxygen (FIO2) during ischemia on the capillary filtration coefficient (Kfc) in reperfused rabbit lungs. At time zero, lungs were rendered ischemic for 180 min (upper panel ) or 240 min (lower panel ). Hydrostatic challenges were performed before ischemia as well 30, 60, and 90 min after reperfusion. Values are given as mean ± SEM. IP = intravascular pressure. *p < 0.05, **p < 0.01, ***p < 0.001 versus control values. In addition, the FIO2 = 0/IP+/vent lungs differ from the nonventilated groups (static or collapse) at p < 0.05.

When lungs were rendered ischemic for 240 min, the Kfc rise in FI_O2 = 0 organs was further enhanced, even in the presence of continued ventilation during ischemia. Under the "optimal" regimen characterized in this study, i.e., positive intravascular pressure and permanent ventilation with normoxic gas during the ischemic period, even the 240 min ischemia provocation caused only some intermediate Kfc increase and edema formation.

	DISCUSSION

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The present study centers on the impact of biophysical factors on the pathogenesis of lung injury in a well reproducible model of warm ischemia and reperfusion. As compared with the routine approach with interruption of perfusion and ventilation, both continuous alveolar oxygen supply during ischemia and ventilatory movements of the alveolar compartment during this period were noted to attenuate the leakage response upon reperfusion. The most impressive reduction of lung injury was, however, achieved by vascular distension through maintenance of a small positive intravascular pressure during the period of ischemia. This finding may be of interest for both the understanding of the pathogenetic events in ischemia/reperfusion and the development of organ protective strategies in lung transplantation.

As anticipated, warm I-R provoked a transient increase in Ppa upon onset of reperfusion. The pressure elevation peaked within the first few minutes after reestablishment of perfusion. It never surpassed 12 mm Hg with rapid subsequent decline of Ppa, and the nearly unchanged values of microvascular pressure reflect that it is mostly due to a transient increase in precapillary vascular resistance. Previous studies concerned with warm ischemia in rabbit lungs suggested that thromboxane is a major contributor to this initial hypertensive response (23). Although prostanoid generation was not assessed in the present study, experiments with the thromboxane receptor antagonist BM 13505, which suppressed the Ppa increase upon reperfusion by > 70% (data not given), support this notion. Several lines of evidence do, however, clearly demonstrate that the transient pressure elevation is not the driving force for the subsequent development of the capillary leakage response: (1) when the postischemic Kfc values were assessed (30 min after onset of reperfusion), the Ppa increase returned to near baseline levels in the majority of experiments; (2) the microvascular pressure levels were only marginally affected and did not significantly differ between the preischemic and postischemic hydrostatic challenge maneuvers; and (3) the leakage response was not attenuated by preapplication of a thromboxane receptor antagonist with nearly complete suppression of the initial transient hypertension (data not given). The manifold increase in Kfc values must therefore be ascribed to a dramatic increase in capillary endothelial permeability, as the microvasculature is the predominant site of lung fluid filtration, and there is no reason for assuming a manifold enlargement of the capillary filtration area in the postischemic period.

Compared with the routine approach with interruption of both perfusion and ventilation in the absence of alveolar and vascular distension, three types of "biophysical intervention" turned out to clearly affect the development of the leakage response.

Alveolar Distension

Beneficial effects of lung ventilation during preservation for transplantation were described in early studies addressing this subject (16) and were also noted in an isolated perfused lung model (19). Nevertheless, during current lung transplantation routine, lungs are stored in an atelectatic state or some state of semi-inflation. Protection by ventilation may be exerted via the continuous alveolar supply with oxygen (see below) or via oxygen-independent mechanical events such as prevention of alveolar collapse and/or cyclic "stretch" of the alveolar structures. Our results suggest that both aspects contribute to the protective effect of lung ventilation during ischemia. The role of the mechanical impact of ventilation is supported by the finding thatmost obvious for the experiments with 180 min of ischemiathe leakage response was significantly reduced in the lungs undergoing continuous ventilation with an anoxic gas as compared with collapsed lungs or those being statically inflated. These observations are in line with a study in an in vivo canine model of ischemia, showing that in the absence of O₂, ventilation per se, but not static inflation, conferred protection to the ischemic lung (13). What is the link between ventilation-dependent, dynamic mechanical forces and attenuation of lung injury? Previous studies showed an impairment of surfactant function after lung transplantation (24), which might be prevented by ongoing ventilation. Wirtz and Dobbs (25) demonstrated that even a single stretch of alveolar type II cells is a most potent stimulus of surfactant secretion. Moreover, surfactant treatment before reperfusion was recently demonstrated to improve the immediate function of lung transplants in rats (26). Altogether, these data suggest that cyclic alveolar distension might be operative via enhanced surfactant secretion, and an integer alveolar surfactant system has long since been implicated in the maintenance of the lung endoepithelial barrier function (27). This reasoning does not, of course, exclude alternative mechanisms such as stretch-related stimulation of lung parenchymal NO and prostacyclin synthesis by which anoxic ventilatory movements may be operative in limiting the leakage response in the current I-R model.

Alveolar Oxygen Supply

Previous studies addressing the impact of alveolar oxygen supply during ischemia on the development of postischemic lung injury forwarded inconsistent results. Fisher and colleagues (14) noted more or less independence of reperfusion-associated edema formation of the alveolar oxygen tension and the type of ventilation during 90 min of ischemia in rabbit lungs. Hamvas and coworkers (13) found that a combination of alveolar hypoxia (but not hyperoxia) and absence of ventilation resulted in lung injury, which provides evidence for a protective role of alveolar oxygen. Beneficial effects of oxygen were also noted in a recent study using nonperfused rat cadaver lungs, showing that O₂ ventilation preserved adenine nucleotides, which was correlated with increased pulmonary parenchymal cell viability (28). Akashi and colleagues (29) observed attenuation of ischemic rat lung injury by inflation with room air as compared with inflation with nitrogen. On the other hand, the presence of alveolar oxygen during ischemia resulted in enhanced lung injury upon reperfusion in isolated rat (10, 12) and dog lungs (11), which was ascribed to the generation of oxygen radicals during ischemia. The reasons for these apparently inconsistent results are not clear, but they were related to the dual role of oxygen, being metabolic substrate, on the one hand, and enhancing oxygen radical formation, on the other hand. In none of these studies was a possible role of vascular distension during ischemia appreciated. Interestingly, in the lungs undergoing ischemia without maintaining a positive intravascular pressure in the current study, no significant protective effect of alveolar oxygen was noted. In contrast, a mitigation of postischemic injury was observed in normoxic and even in hyperoxic ventilated lungs as compared with anoxic ventilated organs when a positive intravascular pressure was maintained. It is conceivable that under conditions of vascular distension instead of vascular collapse, oxygen provided via the alveolar space may be more easily distributed to sites not located in the vicinity of the alveolar compartment, enhanced by ventilation-induced cyclic changes of local intravascular volume and thus some limited kind of regional flow. Such a mechanism might help to maintain the metabolic function of pulmonary cells, thereby overriding putative negative effects of enhanced oxygen radical formation.

Vascular Distension

The most impressive finding of the present study was the marked protective effect of maintaining a positive intravascular pressure on the development of postischemic lung injury. In IP lungs, regardless of the presence or absence of alveolar oxygen supply, 120 min of warm ischemia resulted in immediate, severe capillary leakage, whereas even 240 min of ischemia caused only a moderate leakage response in normoxic ventilated lungs in which a positive intravascular pressure was maintained throughout the ischemic period. Several mechanisms may be considered to underly this notable finding.

(1) Prevention of vascular collapse and reopening phenomena. Because the lungs were perfused in an upright position with arterial and venous pressures referenced at the hilus, an intravascular pressure of zero would result in collapsed vasculature in large parts of the lung. This may result in physical contact of opposing endothelial surfaces, demanding an initial pressure step to overcome the vessel occlusion, and possibly contributing to endothelial injury in the postischemic lungs. In line with this reasoning, the initial transient hypertension was higher in the IP than in the IP⁺ lungs. However, this pressure peak is still largely suppressed by a thromboxane receptor antagonist (data not given), suggesting thromboxane-related vasoconstriction rather than "physical" vessel occlusion as the main underlying event of the initial Ppa elevation.

(2) Dampening of neutrophil-related inflammatory events. The lung microvasculature is known to harbor a large number of neutrophils: even under conditions of prolonged ex vivo buffer perfusion, this is as large as 1.6-fold of the respective circulating leukocyte population in rabbits, as suggested by morphometric studies (30). Neutrophils have long been implicated in the pathogenesis of postischemic lung injury (31, 32). At reduced vessel diameters caused by (partial) collapse of the lung vasculature, neutrophil-endothelial interactions and related injury might be intensified.

(3) Sealing. Hydrostatic forces acting on endothelial cells strongly affect their permeability characteristics. In a previous study concerned with porcine pulmonary artery endothelial monolayers from our group, the establishment of a positive baseline hydrostatic pressure resulted in a marked reduction of hydraulic conductivity, alongside an increase in the albumin reflection coefficient, a phenomenon called "sealing" (20). Reorganization of endothelial stress fibers has been suggested to contribute to this process of sealing, but the underlying events are still not fully elucidated. Although there is hitherto no proof that this phenomenon is also operative under in vivo conditions, it is tempting to speculate that such a mechanism of endothelial sealing might contribute to the marked differences in the leakage response between IP and IP⁺ lungs.

(4) Shear-stress-induced mediator release. Becker and colleagues (33) observed protective effects of maintained intravascular volume on injury in ischemic (not reperfused) ventilated ferret lungs, which were prevented by L-NAME likely through blocking nitric oxide generation (34). These observations may favor a protective role of vascular distension-evoked NO synthesis; however, a corresponding increase in cyclic nucleotide levels was recently reported to be missed (35). A detailed analysis of the impact of vascular distension on ischemia- and reperfusion-related vasoactive mediator generation is hitherto not available.

In conclusion, the present study has demonstrated the importance of biophysical factors for the severity of pulmonary ischemia-reperfusion injury. Vascular distension by maintenance of a low positive intravascular pressure during lung ischemia was identified as a powerful strategy for decreasing the leakage response and associated lung edema formation in the postischemic period. This protective effect was further enhanced by continued ventilation as well as supplementation of alveolar oxygen during lung ischemia. These results may have important implications for organ preservation in lung transplantation. Further elucidation of the mechanisms involved in the protection conferred by vascular distension will be a goal of future investigations.