INTRODUCTION

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Patients with acute respiratory distress syndrome (ARDS) are frequently managed with mechanical ventilation, which although life sustaining, can cause or worsen lung injury (1). Current ventilator strategies to limit this injury emphasize minimization of lung stretch, which corresponds in clinical terms, to reduction of tidal volume. This approach can result in relative alveolar hypoventilation and the development of an associated hypercapnic acidosis. Utilization of this "permissive hypercapnia" strategy is associated with improved outcome in several studies of critically ill pediatric (2) and adult (3) patients. Current concepts of permissive hypercapnia assume that the beneficial effects occur solely as a result of reduced lung stretch (4). In fact, the elevated Pa_CO2 is not conventionally assumed to play a protective role, and current management emphasizes tolerance, or even buffering, of hypercapnic acidosis in critically ill patients (5). However, acidosis has been shown to be protective in the context of ischemia- reperfusion (IR)-induced lung injury (6). In addition, elevated CO₂ has been shown to be protective in ex vivo lung injury (7- 9), therefore it is possible that protection associated with permissive hypercapnia is due to elevated CO₂ as well as reduced lung stretch.

We have previously reported that elevating the fraction of inspired CO₂ FI_CO2, without alterations in ventilator strategy, can significantly attenuate ex vivo pulmonary IR injury (7, 8). We have termed this approach "therapeutic hypercapnia" (TH) (9). There are multiple potential mechanisms to explain the protection afforded by hypercapnic acidosis in tissue injury, and these have been reviewed (9, 10). Nitric oxide (NO) is also important in lung injury because it is produced endogenously, in association with O₂, by activated alveolar macrophages and type II cells (11), it appears important in modulating tissue injury (12), and, it is utilized therapeutically for a variety of acute pulmonary conditions. Because CO₂ may be an important regulator of NO-dependent processes (13), the potential exists for effects on tissue and cellular inflammatory systems.

In critically ill patients, lung injury is seldom an isolated event. This suggestion is corroborated by data suggesting that death in patients with ARDS results from systemic effects multisystem organ failureand not from hypoxemia per se (14). The systemic implications of lung management strategies are increasingly recognized, and a recent human study of ARDS has demonstrated that utilization of a protective lung ventilation strategy was associated with an improved systemic cytokine profile (15). Therefore, the therapeutic potential of lung-targeted approaches in acute lung injury should incorporate assessment of systemic, as well as pulmonary, effects.

Laboratory studies have demonstrated that hypercapnia can be protective in specific organ systems including myocardial (16) and central nervous system (CNS) (17) ischemia. Hypercapnic acidosis also effectively suppresses systemic lactate elevation in the context of global hypoxemia (18). This has been hypothesized as a protective feedback suppressive effect of acidemia on endogenous organic acid production (19). Because lung injury, systemic organ injury, and lactic acidosis are crucial concerns in critically ill patients, these reports led us to hypothesize previously that TH, that is, deliberate elevation of inspired CO₂, might be a beneficial intervention in a variety of disease states in critically ill patients (9). However, there are no published in vivo data that address pulmonary or systemic effects of TH in the context of acute lung injury.

Because of these issues, we wished to study the effects of TH in a clinically relevant whole animal model of ischemia- reperfusion-induced acute lung injury, in order to determine whether TH might exert protective pulmonary or systemic effects. We hypothesized that in the context of comparable ventilatory strategy, TH would attenuate pulmonary and systemic injury following lung ischemia-reperfusion in an in vivo rabbit model. We further hypothesized that this protection would be associated with attenuation of pulmonary cytokine elevation and free radical-mediated injury.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Male New Zealand white rabbits weighing 2.5 to 3.0 kg were used. All experimental work conformed to the guidelines of the Canadian Committee for Animal Care, and was approved by the Animal Care Committee at the Research Institute, Hospital for Sick Children.

Anesthesia and Dissection

The experimental model was based on that previously reported, with several modifications (20). Premedication was with intramuscular ketamine (85 mg kg¹). Anesthesia was induced with intravenous pentobarbital sodium (15 to 25 mg kg¹). Incremental bolus doses of pentobarbital (5 mg kg¹) were administered as required. Sterile technique was utilized during all manipulations. A tracheotomy was performed, and an endotracheal tube (ETT; 4-mm internal diameter) was inserted to a depth of 1 cm, and secured in place. Pancuronium (1 mg intravenously) was administered after depth of anesthesia was confirmed by absence of response to paw compression. The lungs were ventilated using a small animal ventilator (Model 683; Harvard Apparatus, MA) with fraction of inspired oxygen (FI_O2) 1.0, rate 30 min¹, tidal volume (VT) 7.5 ml kg¹ and 1 cm H₂O positive end-expiratory pressure (PEEP). Under sterile conditions the right carotid artery was cannulated for arterial pressure measurement and blood sampling. Anesthesia was maintained with pentobarbital (3 to 6 mg kg¹ h¹ as per arterial pressure), and muscle relaxation with pancuronium (0.1 to 0.2 mg kg¹ h¹). Lactated Ringer's crystalloid (15 ml kg¹ h¹) was administered by continuous infusion administered via marginal ear vein. Body temperature was maintained (37.5-39.0° C) by use of a heating pack, and confirmed with an indwelling esophageal temperature probe. After median sternotomy, the lungs were dissected free from the inferior vena cava, esophagus, and diaphragm. The pericardium was left intact and retracted as necessary to permit visualization of the lung hila. Elasticated vascular ligatures (Medservice Surgical Loops; Berkley Medservices Inc., Berkley, CA) were then passed around both pulmonary hila. Thereafter, baseline stability was confirmed for 10 min. Baseline arterial blood samples were then taken for blood gas measurement (Radiometer ABL 300; Copenhagen), and blood samples were taken for cytokine assay, centrifuged, and the supernatant stored at 80° C.

Randomization to Group Allocation

After confirmation of baseline stability, and provided none of the exclusion criteria (see below) were met, randomization to group allocation, that is, control (CON) versus TH was achieved by means of choosing from sealed envelopes. Immediately following randomization, the inspired gas concentrations were adjusted as follows: CON (FI_CO2 0.00, FI_O2 0.75, balance N₂) and TH (FI_CO2 0.12, FI_O2 0.75, balance N₂). Arterial blood gases were allowed to equilibrate over a 10-min period. These inspired gas concentrations were continued for the duration of the experiment.

Ventilatory Strategy

The ventilatory strategy was determined during preliminary pilot experiments (n = 10) to produce normocapnia in the control group throughout both the baseline and left lung occlusion phases of the experiment. This ventilatory strategy was then utilized in all animals. During two lung ventilation, the ventilatory settings were VT = 7.5 ml kg¹, PEEP = 1 cm H₂O and a rate of 30 min¹; during one lung ventilation the settings were VT = 6 ml kg¹, PEEP = 1 cm H₂O and a rate of 41 min¹.

Timeline of Events

The following timeline was followed: baseline conditions established, baseline measurements, randomization to group allocation and appropriate FI_CO2, left lung ischemia (75 min), left lung reperfusion and concomitant right hilar occlusion, monitoring/observation (90 min), and final measurements.

Left Lung Ischemia-Reperfusion Protocol

After determination of baseline left lung compliance, and establishment of study conditions (FI_CO2 0.00 versus 0.12), mechanical ventilation settings were altered to one lung settings, and heparin 400 IU kg¹ was intravenously administered. The left lung was then rendered ischemic by clamping the tie around the left pulmonary hilum at end inspiration. After ischemia for 75 min, perfusion to the left lung was reestablished by simultaneously releasing the left hilum while irreversibly rendering the right lung ischemic by ligating the right hilum. This resulted in the entire cardiac output being diverted through the left lung, in order to ensure maximal reperfusion (20, 21). Reperfusion of the left lung was continued for a period of 90 min. At 90 min following reperfusion, the inspired gas was altered to 100% O₂ for final Pa_O2 assessment. Animals were then sacrificed by anesthetic overdose (pentobarbital 100 mg kg¹ intravenously).

Exclusion Criteria

Prior to randomization, the following values were required for continuation with the protocol: Pa_O2 > 250 mm Hg, Pa_CO2 30-40 mm Hg, HCO₃ > 20 mmol L¹, Hb > 12 g/dl, and temperature > 36.5° C. Where such criteria were not fulfilled, parameters were reassessed following an additional 10 min, and failure to meet the criteria at this point mandated exclusion from the protocol. Prior to reperfusion of the left lung, an additional set of criteria was applied, as follows: Hb > 10 g/dl, mean arterial pressure (MAP) > 60 mm Hg, and blood loss < 25 ml. Where these criteria were not fulfilled, animals were excluded from the protocol, and their group allocation returned to the randomization pool such that there were 10 animals per group in the final analysis.

Physiologic Variables

Systemic mean arterial pressure, peak airway pressures, and esophageal temperature were measured before occlusion, at 30 and 75 min during left lung occlusion, and every 15 min following reperfusion.

Blood Sampling Protocol

Arterial blood samples were drawn before occlusion, at 30 and 75 min during left lung occlusion, and, every 15 min following reperfusion. At the end of reperfusion FI_O2 was changed to 1.0 and Pa_O2 was measured.

Lung Mechanics

Static inflation and dynamic deflation left lung compliance were measured prior to left lung ischemia and at 90 min postreperfusion. Static inflation compliance was determined by injection of incremental volumes of 100% O₂ and measurement of pressure attained 3 s after each injection until a total volume of 20 ml was injected. Airway pressure was recorded using a pressure transducer (MP45; Validyne Engineering Corporation, Northridge, CA) connected to the side port of the endotracheal tube adapter. Both pressure and flow signals were recorded by an online PC IBM compatible computer (IPC, pentium microprocessor, 75 Hz, MS DOS 6.0), using analog-to-digital conversion at a sampling rate of 250 Hz (DT2802A; Data Translocation, Marlboro, MA) and the ANADAT/LABDAT software package (McGill University, Montreal, PQ, Canada). An inspiratory pressure-volume curve was then constructed. Dynamic expiratory compliance and expiratory resistance were determined by measurement of the exhaled lung volume following continuous positive airway pressure (CPAP) of 20 mm Hg applied for 30 s. A low deadspace (1.3 ml) thermistor pneumotachograph (NVM-1; Bear Medical Systems, Riverside, CA) was inserted between the endotracheal tube and the ventilator circuit. The flow signal was digitally integrated to volume. The dynamic compliance was calculated from the flow-volume curve as the slope of the pressure volume relationship using linear regression by means of the software package HP VEE (Hewlett-Packard, Upper Saddle River, NJ).

Postmortem Lung Sampling

Immediately postmortem, the heart-lung block was dissected from the thorax, and the left lung was isolated. The left upper lobe was excised and samples of lung tissue of approximately 100 mg were taken. These were snap frozen in liquid nitrogen and stored at 70° C for determination of lung myeloperoxidase and 8-isoprostane levels.

Bronchoalveolar Lavage (BAL)

The left lower lobe bronchus of nine control and TH preparations was cannulated via the ETT tube and lavaged with 10 ml of saline (0.9%), and 3 ml of fluid (BALF) was collected. This fluid was divided into two 1.5-ml aliquots and centrifuged. One aliquot was snap frozen in liquid nitrogen and stored at 70° C, for subsequent assay for tumor necrosis factor- (TNF)- analysis. The remaining aliquot was frozen at 20° C for subsequent measurement of total protein (22).

Wet-to-Dry Weight

The remainder of the left upper lobe lung was utilized for determination of lung wet:dry weights ratio, after sequential weighs demonstrated maximal dehydration in a drying oven.

Biochemical Analysis

TNF- assay. Analysis of serum and BALF TNF- was carried out in duplicate and in a blinded fashion with a commercially available polyclonal sandwich antibody enzyme-linked immunosorbent assay (ELISA) kit (PharMingen, Mississauga, ON, Canada), using standardized methodology (23).

Lung tissue myeloperoxidase. Analysis of lung tissue myeloperoxidase was carried out in triplicate on thawed homogenized lung samples in a blinded fashion with a commercially available assay, using standardized methodology (21). Results were standardized for tissue protein concentration.

8-Isoprostane assay. Lung tissue 8-isoprostane levels, standardized for tissue protein concentration, were assayed in duplicate in a blinded fashion (n = 4 per group) with a commercially available ELISA kit, using standardized methodology (24). Briefly, lung homogenates, were spiked with 5,000 cpm of tritium-labeled prostaglandin F₂ to quantify recovery during purification. Protein was precipitated with ethanol and removed by centifugation. To determine the total (free and esterified) 8-isoprostane, the supernatant was incubated with equal volume of 15% (wt/vol) KOH at 40° C for 1 h for alkaline hydrolysis. The developed plate was read by a microplate reader (Diamed, Mississauga, ON, Canada) at 405 nm. The concentration was calculated by computer using Cayman EIA software. Recovery from the purification step was analyzed by liquid scintillation counting of the extract and factored into the calculation.

BALF total protein assay. Analysis of lung lavage total protein concentration was carried out in duplicate and in a blinded fashion (22). Bovine serum albumin was used to construct the standard curve.

Western blot analysis. Lung tissue was assessed for the presence of nitrotyrosine using a modification of a previously described method (25). Lung tissues were homogenized with five volumes of lysis buffer containing 1% (vol/vol) NP-40, 0.5% (wt/vol) sodium deoxycholate, and 0.1% (wt/vol) sodium dodecyl sulfate (SDS) in phosphate-buffered saline (PBS), to which a protein inhibitor, phenylmethylsulfonyl fluoride, 100 µg/ml, was added. The homogenate was left on ice for 30 min before centrifugation at 10,000 × g. Protein content was estimated (22), and aliquots of 30 µg of homogenate protein were diluted with sample buffer containing 125 mM Tris, 20% (wt/vol) glycerol, and 4% (wt/vol) SDS and separated on 10% (wt/vol) polyacrylamide gels with 0.1% (vol/vol) SDS. Proteins were subsequently transferred to a nitrocellulose membrane. Nonspecific binding was blocked by incubation with 3% (wt/vol) lung fat dry milk in PBS for 1 h at room temperature with shaking, followed by overnight incubation with a 2 µg/ ml of primary antibody of antinitrotyrosine (rabbit immunoaffinity purified immunoglobulin G [IgG]: Upstate Biotechnology, Lake Placid, NY). The membrane was subsequently incubated with 1:25,000 (vol/ vol) dilution of goat anti-rabbit IgG conjugated with peroxidase for 1 h at room temperature. The peroxidase reaction was carried out using ECL Western blotting detection reagents.

Assay for apoptosis. Apoptosis was assessed in one lung from each group using a TdT-mediated dUTP nick end-labeling (TUNEL) assay. This was performed using an in situ cell death detection kit, Fluorescein (Boehringer Mannheim, Germany), according to previously described methodology (26), on formaldehyde-fixed lung sections. Briefly, tissue samples were dewaxed and rehydrated with serial ethanol concentrations, and incubated with Proteinase K (20 µg/ml). Of the TUNEL mixture 50 µl was then added to the samples, and incubated in humidified chambers for 60 min at 37° C in darkness. The samples were rinsed, and slides were embedded with antifade prior to fluorescence microscopic visualization.

Statistical analysis. Data were analyzed using SigmaStat (Version 2.0; Jandel Corporation, San Rafael, CA). Group comparisons were performed with 2-way repeated measures ANOVA. Subsequent one-way ANOVA was followed by Student-Newman-Keuls or t tests. Significance was set at p < 0.05. Results are expressed as mean ± standard error of the mean (SEM).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Ten animals per group completed the protocol. Fourteen rabbits were excluded prior to randomization, in accordance with exclusion criteria. In addition, four rabbits were excluded after randomization but before reperfusion because of hypotension (one from each group) and excessive blood loss (one from each group).

Demographic Data

There was no difference in the animal weight (2.7 ± 0.04 versus 2.7 ± 0.4 kg; p = 0.3) in the TH versus CON groups. Baseline values for Pa_CO2, systemic pH, Pa_O2, MAP, hemoglobin, serum lactate, Paw or pulmonary mechanics were similar in both groups (Figures 1 and 2).

View larger version (18K): [in this window] [in a new window]   Figure 1.   (A) Arterial CO2 Tension: PaCO2 was comparable at baseline. Prior to reperfusion, arterial PCO2 was significantly lower in CON versus TH. Following reperfusion, arterial PCO2 increased progressively in both groups, and was greater in TH versus CON (*p < 0.05). (B) Arterial pH: pH was comparable at baseline. Prior to reperfusion, arterial pH was significantly higher in CON versus TH. Following reperfusion, pH decreased progressively in both groups, and was lower in TH versus CON (*p < 0.05). (C ) Arterial oxygenation. PaO2 was comparable at baseline; decreased significantly in both groups following reperfusion; and was significantly greater in the TH versus CON following reperfusion (*p < 0.05). (D) Lactate. Plasma lactate concentration increased significantly during lung reperfusion in both groups. However, the magnitude of the increase was significantly less in the TH versus CON (*p < 0.05).

View larger version (20K): [in this window] [in a new window]   Figure 2.   (A) Peak airway pressure (Paw). Paw was comparable in both groups at baseline; increased significantly in both groups following reperfusion; and was significantly lower in TH versus CON (*p < 0.05). (B) Static Inflation compliance. Compliance was comparable in both groups at baseline, and decreased significantly in both groups following IR. The final static lung compliance value was significantly greater in TH versus CON (*p < 0.05 versus baseline in both groups, and p < 0.05 TH versus CON). (C ) Dynamic expiratory compliance. Dynamic expiratory compliance was comparable in both groups at baseline and decreased significantly in both groups following reperfusion. The final value of dynamic lung compliance was significantly greater (and the decrement smaller) in TH versus CON (*p < 0.05). (D) Lung wet:dry ratio. The wet: dry ratio was significantly lower in TH versus CON (*p < 0.05).

Arterial Blood Gases and Acid Base Data

Pa_CO2 and systemic pH were comparable in both groups at baseline (Figure 1). Prior to reperfusion, Pa_CO2 was significantly lower (and arterial pH was significantly higher) in CON versus TH (Figure 1). Following reperfusion, Pa_CO2 increased (and pH decreased) progressively for the duration of the experiment in the CON group (Figure 1). Pa_O2 was comparable in both groups at baseline, decreased significantly in both groups following reperfusion, and was significantly greater in the TH versus CON group following reperfusion (Figure 1). Following completion of the experiment, the alveolar-arterial O₂ gradient (Aa_O2, measured while FI_O2 = 1.0) was significantly lower in the TH versus CON (387 ± 42 versus 500 ± 33 mm Hg, p < 0.001). Plasma lactate concentration increased significantly during lung reperfusion in both groups (Figure 1). However, the magnitude of this lactate increase (final level  baseline level) was significantly less in the TH versus CON (3.2 ± 1.2 versus 7.6 ± 2.0 mmol/L, p < 0.001), despite the mean Pa_O2 never reaching hypoxic levels in either group (Figure 1).

Pulmonary Mechanics

Peak airway pressure (Paw) was comparable in both groups at baseline, was increased significantly in both groups following reperfusion, and was significantly lower in the TH versus CON by the end of the experiment (Figure 2). Left lung static inspiratory compliance was comparable in both groups at baseline, and decreased significantly in both groups following reperfusion (Figure 2). At the end of the experiment the static lung compliance value was greater in TH versus CON (Figure 2). Left lung dynamic expiratory compliance was comparable in both groups at baseline and decreased significantly in both groups following reperfusion (Figure 2). The final value of dynamic lung compliance was significantly greater (and the decrement smaller) in the TH group at the end of the experiment (Figure 2). Expiratory airway resistance was comparable at baseline, and was not significantly altered in either group following IR.

Lung Wet:Dry Ratio and BALF Protein

The wet:dry ratio (Figure 2) and BALF protein concentration (Figure 3) were significantly lower in TH versus CON.

View larger version (16K): [in this window] [in a new window]   Figure 3.   (A) BALF protein. The protein concentration in the BAL fluid was significantly lower in TH versus CON groups (*p < 0.05). (B) BALF TNF-. BALF TNF- was significantly lower in the TH versus CON groups (*p < 0.05). (C ) Lung tissue 8-isoprostane levels. The lung tissue 8-Isoprostane concentration was lower in TH versus CON (*p < 0.05). (D) Lung tissue myeloperoxidase levels. Lung tissue myeloperoxidase levels were similar in both groups.

Pulmonary Inflammation

BALF TNF- and lung tissue 8-isoprostane concentrations were lower in the TH versus CON group (Figure 3). Western blot analysis of nitrotyrosine residues demonstrates a prominent band of nitrotyrosylated protein at approximately 60 kD (Figure 4A). The intensity of the bands is less in the TH versus CON group, indicating less nitrotyrosine. The lung tissue myeloperoxidase levels were similar in both groups (Figure 3), suggesting comparable degrees of pulmonary neutrophil content, and examples of apoptosis, demonstrated by TUNEL assay, are presented (Figures 4B-4E).

View larger version (87K): [in this window] [in a new window]   Figure 4.   (Panel A) Western blot analysis of nitrotyrosine residues from three specimens from each group. A prominent band of nitrotyrosylated protein is demonstrated at approximately 60 kD. The intensity of the bands is greater in the CON (bands 1-3) versus TH (bands 4-6) group. (Panels B-E ) Examples of apoptosis, demonstrated by TUNEL assay. Fluorescence is greater in CON versus TH (CON = panel B; TH = panel C ), indicating more apoptosis. Comparable tissue DNA profile is demonstrated in the presence of DAPI (CON = panel D; TH = panel E ).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

These findings confirm that therapeutic hypercapnia (TH) is associated with significant protection against the pulmonary and systemic effects of lung ischemia-reperfusion (IR) injury.

Pulmonary IR is directly relevant to critical illness because IR directly mimics several important entities (e.g., transplantation, cardiopulmonary bypass, vascular anastomosis, thrombolysis, or embolectomy) that are common in critically ill patients, and IR is a constitutive component of multisystem organ failure, characterized by interruptions of tissue perfusion and cellular dysoxia (27). In addition, many of the inflammatory elements observed in IR, such as mediator release, leukocyte adhesion, and oxidative injury, are prominent features of the systemic inflammatory response syndrome that underlies multiple organ failure (28).

Rationale for Therapeutic Hypercapnia

Three lines of reasoning account for the increasing importance of CO₂ tension in critically ill patients with respiratory failure (5, 9, 10). First, ventilator strategies associated with the development of elevated Pa_CO2 have been associated with improved clinical outcome (2). Such "protective" ventilator strategies, which necessitate use of smaller tidal volumes, have been designed with the idea that reduction of pulmonary stretch would reduce mechanical biotrauma, thereby reducing lung injury. The ensuing hypercapnia has been clinically tolerated, with the notion that in the absence of contraindications and with good understanding of the physiological effects, it would not cause patient harm (5). However, in addition to the clear beneficial effects of reduced lung stretch, permissive hypercapnia might confer protection via pulmonary and/or systemic effects of raised CO₂. In any case, permissive hypercapnia, where elevated Pa_CO2 is considered a bystander, has found increasing acceptance in clinical practice. Second, increasing evidence supports the notion that elevated levels of Pa_CO2 exert independent protective effects in experimental models of pulmonary (7, 8) and systemic organ (17) injury. Third, laboratory evidence indicates that alveolar hypocapnia causes (29, 30) and worsens (29) pulmonary injury, which may be ameliorated by normalization of alveolar PCO₂ with increasing FI_CO2 (30). There are several additional adverse effects of hypocapnia, including bronchospasm, increased airway microvascular permeability, altered compliance, increased pulmonary venous admixture, and impaired end-organ oxygenation (9). These mechanisms, and possibly others, may contribute to the suggested adverse clinical outcome associated with hyperventilation in respiratory failure (3), and following head injury (31). Taken together, these data have prompted us to hypothesize previously that TH might be protective in the setting of acute lung injury and other forms of critical illness (7, 9).

Pulmonary Effects

TH significantly attenuated several adverse alterations, including static compliance, dynamic expiratory compliance, peak airway pressure, and alveolar-arterial oxygen gradient. The mechanisms of TH-mediated effects on Pa_O2 are complex. Several properties of hypercapnic acidosis, including improvement of / matching (32), reduction of tissue metabolism (19), increased cardiac output, and altered Hb-O₂ dissociation kinetics may contribute (34), in addition to the potential for TH to reduce lung injury. However the effects of TH on lung mechanics, and the alveolar-arterial oxygen gradient, measured postreperfusion after discontinuation of TH, clearly point to a protective effect of TH on acute lung injury. In addition, the effect of TH on pulmonary compliance postreperfusion may be partially explained by the known effect of acidosis to improve surfactant's surface tension lowering properties (35). This protective effect of TH occurred in the context of an unchanged ventilatory strategy. In fact, because Pa_CO2 was permitted to rise in the control group during the reperfusion period without adjustment of minute ventilation, "permissive hypercapnia" was effectively employed. This accentuates the impact of therapeutic hypercapnia, and demonstrates its potential effectiveness versus permissive hypercapnia. Furthermore, it is highly likely that attempts to normalize Pa_CO2 by increasing minute ventilation in the control group would, because of increased lung stretch, have resulted in additional degrees of lung injury. Finally, although not specifically addressed in the current study, alteration of CO₂ tension has been reported to impact on surfactant production (36). It seems possible that surfactant dysfunction occurred in association with IR injury, more so in the CON group, and that this was ameliorated through application of TH.

Mechanisms of Lung Injury

The current data confirm our previous reports that TH protects against ex vivo lung injury (7, 8). Additional mechanistic insights include documentation of attenuation of inflammatory processes including neutrophil and macrophage sequestration, cytokine release, lipid peroxidation, and tyrosine nitration.

Pulmonary neutrophil sequestration was similar in both groups. Whereas some forms of acute lung injury, for example, ventilator-induced lung injury, require PNM sequestration and activation as key elements in pathogenesis (37), this is not the case with pulmonary IR. Rather, protective strategies against lung injury following IR result in comparable degrees of lung neutrophil sequestration (21), but attenuation of indices of neutrophil activation (38).

Bronchoalveolar lavage TNF- levels were decreased in the TH group compared with control. TNF- was used here as a marker of cytokine responses to injury, because alterations in BALF TNF- reflect changes in other inflammatory cytokines across several species (23, 39), including humans (15). Because elevation in TNF- has been reported in lung injury (15, 23, 39), and because this injury can be attenuated by antibodies to TNF- (41), it is possible that reduction of TNF- represents a therapeutic mode of action of TH.

The suggestion that TH attenuates oxidant-induced injury is supported by the finding that lung tissue 8-isoprostane levels were significantly lower in TH versus CON. This is consistent with reduction of lipid peroxidation by hypercapnia in homogenized tissue (42), and may be important because of the ubiquitous distribution of oxidant-induced organ injury in critical illness.

The finding that tyrosine nitration was reduced with TH suggests a further possible mechanism, because NO and O₂, both produced in reperfusion lung injury, combine to form peroxynitrite (ONOO), which is capable of nitrating tyrosine residues and modifying regulatory proteins (43). Such nitrotyrosine formation has been demonstrated in lung tissue from patients with ARDS (11), and because CO₂ alters the rate of ONOO reactions (13) and can inactivate ONOO (44), TH may protect by prevention of tissue nitration.

Reduced BALF protein and lung wet:dry ratio in the TH group is consistent with ex vivo findings that TH attenuated increases in pulmonary microvascular permeability (7). Furthermore, because of the inhibitory effects of protein on surfactant function (45), the elevated BALF protein concentration may have additional functional significance through surfactant impairment.

The finding that apoptosis was reduced in a representative tissue sample raises an additional potential mechanism of protection. The TUNEL assay detects DNA breaks, but can distinguish between DNA fragmentation from apoptosis versus alternative causes (26). Hypercapnic acidosis could attenuate apoptosis by either a pH-mediated reduction in apotosis (46), or through inactivation of peroxynitrite, which causes tissue nitration and apoptosis (47).

Systemic Effects

TH may favorably impact on tissue/organ supply:demand balance by multiple mechanisms (9, 10). Acidosis may protect against ongoing tissue production of further organic acids (19). Furthermore, acidosis may attenuate several aspects of the inflammatory response (9). In the current study, the changes in lactate reflected the time course in bicarbonate and base deficit. Although the elevations in lactate occurred with lowering Pa_O2 (control group), systemic hypoxemia did not occur. Therefore, protection against increased systemic lactate was not because of prevention of arterial hypoxemia, and instead may have been due to prevention of local cellular dysoxia (48) or inhibition of organic acid production (19).

Use of TH may have additional systemic, that is, extrapulmonary, implications for two principal reasons. First, lung injury has been demonstrated to result in loss of compartmentalization and systemic release of lung-derived cytokines (49), and this has been proposed as a mechanism of systemic injury (39, 50). In addition, protective ventilatory strategies have been shown to attenuate the elevation in systemic cytokines (15, 39) as well as altering indices of circulating neutrophil activation (37). Second, elevation of Pa_CO2 results in higher CO₂ tensions throughout all body compartments. Therefore, unlike alternative therapeutic modalities delivered via the pulmonary route, for example, inhaled NO or nebulized bronchodilators, the systemic effects of TH are likely to be more prominent and predictable because increases in Pa_CO2 are easily attained and controlled. Furthermore, application of conventional antioxidant therapeutics, although successful in experimental settings, is limited due to the polarity profile of specific pharmacological agents. This has led to the development of antioxidants with amphiphilic characteristics (51) that can exert effects in polar and nonpolar environments, and may be less restricted to specific physiological compartments. CO₂ shares features of such a compound, and although physiological gradients exist, it is freely permeant through lipid bilayers, allowing rapid equilibration, access, and potential effect. It is becoming clear that successful biotherapeutic approaches to reperfusion injury will require strategies that are effective and act across a broad range of biological sites of action (52). CO₂, increased through the application of TH, may fulfill many of these criteria.

Limitations of Current Findings

There are several limitations of the current study that prevent immediate extrapolation to the clinical scenario. We have not demonstrated whether the in vivo protective effects of TH are a function of the acidosis or hypercapnia per se. However, we have demonstrated previously in an ex vivo lung preparation that the protection is reflected, in part, by the degree of extracellular acidosis (8). Furthermore, buffering hypercapnic acidosis in ex vivo-perfused lungs is associated with loss of protection (8). The possibility exists that right lung injury may have developed during the period of left lung ischemia. This is unlikely, because serial mechanics were unchanged in the right lung during left lung ischemia, and systemic oxygenation was not impaired in pilot studies of prolonged left lung ischemia (120 min). It is possible that significant species variability exists, for example, distribution of pulmonary xanthine oxidase content (53), or species susceptibility to capillary stress failure (54). Caution must therefore be exercised with extrapolation to other species or models. The current study does not describe dose-response characteristics, or the issue of optimal timing of application. We believed following pilot studies that the critical issues were to study the protective effects and mechanisms of TH, rather than dissect a comprehensive dose- response tabulation. The importance of a dose-response profile is underscored by the findings that increasing levels of Pa_CO2 were associated with improved, but at higher doses worsened, neurological outcome in a neonatal rabbit stroke model (17). The current study does not characterize in detail the specific effects of TH on pulmonary and systemic hemodynamic parameters. The model as described does not lend itself easily to determination of pulmonary artery pressure because of the need for sequential hilar ligation. However the effects of hypercapnic acidosis (HCA) on pulmonary and systemic hemodynamics are well described (55). We have previously determined, in the isolated perfused rabbit lung, that HCA is a pulmonary vasoconstrictor, elevating pulmonary arterial pressure (Ppa) and pulmonary vascular resistance (PVR), while isohydric hypercapnia is a potent pulmonary vasodilator (8).

Finally, the issue of duration of effect is of critical importance, because many in vivo experimental models utilize a duration that is far shorter than that of a clinical illness. This may be important with homeostatic processes such acid-base buffering.

Significance and Application

The current data provide comprehensive evidence of in vivo protection in a clinically relevant model. Because these data are consistent with a large body of evolving literature, this suggests that TH might be applicable across a broad spectrum of pathological entities. Finally, the physiological effects of elevated Pa_CO2 are well known to clinicians, and there is a growing acceptance of elevated Pa_CO2 in practice. Therefore, if the current findings are confirmed in additional models, testing of TH may be an option in critically ill patients.