ORIGINAL ARTICLE
High-altitude pulmonary edema can be prevented by heat shock protein 70Ymediated hyperbaric oxygen preconditioning Ming-Che Tsai, MD, Hung-Jung Lin, MD, Mao-Tsun Lin, DDS, PhD, Ko-Chi Niu, MD, PhD, Ching-Ping Chang, PhD, and Thomas Chang-Yao Tsao, MD, Taichung, Taiwan
BACKGROUND: The primary goal of this study was to test whether high-altitude exposure (HAE of 9.7% O2 at 0.47 absolute atmosphere [ATA] for 3 days) was capable of increasing lung edema, neutrophil, and hemorrhage scores as well as decreasing lung levels of both aquaporin 1 (AQP1) and AQP5 proteins and messenger RNA (mRNA) expression in rats, with a secondary goal to test whether a preinduction of heat shock protein 70 (HSP70) by hyperbaric oxygen preconditioning (HBO2P of 100% O2 at 2.0 ATA for 1 hour per day for 5 consecutive days) attenuated the HAE-induced increased lung injury scores and decreased lung AQP1 and AQP5 protein and mRNA expressions. METHODS: Rats were assigned to (1) non-HBO2P (21% O2 at 1.0 ATA) + non-HAE (21% O2 at 1.0 ATA) group; (2) non-HBO2P + HAE group; (3) HBO2P + HAE group; and HBO2P + HSP70 antibodies (Ab) + HAE group. For the HSP70 Ab group, a neutralizing HSP70 Ab was injected intravenously at 24 hours before HAE. All the physiologic and biochemical parameters were obtained at the end of HAE or the equivalent period of non-HAE. The cardiovascular and blood gas parameters were monitored for all experiments. Bronchoalveolar lavage (BAL) was performed to determine proinflammatory cytokines (interleukin 6, interleukin 1A, and tumor necrosis factor >). Parts of the lung were excised for myeloperoxidase activity measurement, whereas the rest was collected for lung damage score assessments. AQP1 and AQP5 protein and mRAN expressions were also determined in the lung tissues. RESULTS: In the non-HBO2P + HAE group, the animals displayed higher values of lung myeloperoxidase activity, BAL proinflammatory cytokines, lung water weight, and acute lung injury scores compared with those of the non-HBO2P + non-HAE controls. In contrast, the non-HBO2P + HAE group rats had lower values of lung AQP1 and AQP5 protein and mRNA expressions, mean arterial pressure, heart rate, SO2, PaCO2, HCO3j, and pH compared with those of non-HBO2P + non-HAE group rats. The increased acute lung edema, neutrophil, and hemorrhage scores; increased BAL levels of proinflammatory cytokines; decreased lung AQP1 and AQP5 protein and mRNA expressions; and hypotension, bradycardia, hypoxia, and acidosis caused by HAE were all significantly attenuated by HBO2P. CONCLUSION: Our data indicate that HBO2P may attenuate high-altitude acute lung injury by a preinduction of lung HSP70 in rats. (J Trauma Acute Care Surg. 2014;77: 585Y591. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: High-altitude pulmonary edema; hyperbaric oxygen preconditioning; lung injury score; aquaporin 1; aquaporin 5.
A
t high-altitude exposure (HAE) conditions, hypoxia induces pulmonary hypertension as well as impairment of the respiratory transepithelial sodium transport and results in alveolar fluid accumulation as well as alveolar fluid clearance deficits, which facilitate pulmonary edema.1,2 Evidence has also accumulated to indicate that decreases in lung expression of both aquaporin 1 (AQP1) and AQP5 may play an important role in pulmonary edema.3Y7
Submitted: April 29, 2014, Revised: June 3, 2014, Accepted: June 4, 2014. From the Institute of Medicine and School of Medicine (M.-C.T.), Chung Shan Medical University; and Division of Thoracic Medicine (T.C.-Y.T.), Chung Shan Medical University Hospital and Chung Shan Medical University, Taichung; and Departments of Emergency Medicine (H.-J.L.), Medical Research (M.-T.L.), and Hyperbaric Oxygen (K.-C.N.), Chi Mei Medical Center; and Department of Biotechnology (M.-T.L., C.-P.C.), Southern Taiwan University of Science and Technology, Tainan, Taiwan. Address for reprints: Ching-Ping Chang, PhD, Department of Biotechnology, Southern Taiwan University of Science and Technology, 1 Nan-Tai Road, Tainan 710, Taiwan; email: [email protected]; Thomas Chang-Yao Tsao, MD, Division of Thoracic Medicine, Chung Shan Medical University Hospital and Chung Shan Medical University, No.110, Sec.1, Jianguo N.Rd., Taichung City 40201, Taiwan; email: [email protected]. DOI: 10.1097/TA.0000000000000408
The 70-kD family of heat shock protein (HSP), HSP70, is up-regulated by exercise preconditioning in cell protection and survival in stroke.8 Preinduction of lung HSP70 protein with a sublethal dose of HAE (e.g., hypobaric hypoxia preconditioning, 18.3% O2 at 0.66 absolute atmosphere [ATA]) were able to protect against a subsequent lethal damage exerted by a lethal dose of HAE (9.7% O2 at 0.47 ATA).9 Preinduction of HSP70 by hypobaric hypoxia also promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain.10 Hyperbaric oxygen preconditioning (HBO2P), like hypobaric hypoxia preconditioning, attenuates brain edema caused by hypobaric hypoxia in rats.11 However, it is not known whether the increased lung injury scores and the decreased levels of both AQP1 and AQP5 proteins and messenger RNA (mRNA) expression in the lung caused by HAE12 can be affected by HSP70-mediated HBO2P in rats. The primary purpose of this study was to investigate whether HAE was capable of increasing lung edema, neutrophil, and hemorrhage scores as well as decreasing lung levels of both AQP1 and AQP5 protein and mRNA expression in rats, with a secondary goal to test whether a preinduction of HSP70 by HBO2P attenuated the HAE-induced acute lung injury scores as well as decreased lung AQP1 and AQP5 protein and
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Figure 1. Experimental design. The HBO2P group was treated with 100% O2 at 2.0 ATA for 1 hour per day for 5 consecutive days. The NBA (or non-HBO2P) + HAE (simulated high-altitude exposure) group, the HBO2P + HAE group, and the HBO2P + HSP70 Ab + HAE group were all subjected to HAE (9.7% at 0.47 ATA) for 3 days. HSP70 Ab was administered 1 day before the onset of HE. The NBA (or non-HBO2P) and non-HAE groups were treated with 21% O2 at 1.0 ATA.
mRNA expression in rats. Our expected results would verify the hypothesis that HBO2P attenuates HAE-induced pulmonary edema by preinduction of lung HSP70 in rats.
MATERIALS AND METHODS
and HBO2P + HSP70 Ab group rats before the start of HAE (n = 8 per group, Fig. 2). In Experiment 2, acute lung injury scores were assessed in all four groups of rats 3 days after HAE or NBA (n = 8 per group, Fig. 2). In Experiment 3, lung weights were determined in all four groups of rats 0 hour and 3 days after HAE or NBA (n = 8 per group, Fig. 3). In Experiment 4, Western blot analysis of AQP1 and AQP5 in the lungs for all four groups of rats were assessed 3 days after HAE or NBA (n = 8 per group, Fig. 4). In Experiment 5, real-time polymerase chain reaction (RT-PCR) analysis of AQP1 mRNA and AQP5 mRNA in the lungs for all four groups of rats were performed 3 days after HAE or NBA (n = 8 per group, Fig. 5). In Experiment 6, mean arterial pressure (MAP); heart rate (HR); blood levels of SO2, PaCO2, HCO3j, and pH; bronchoalveolar lavage (BAL) fluid levels of proinflammatory cytokines; and lung levels of myeloperoxidase (MPO) activity for all four groups of rats were determined 3 days after HAE or NBA (n = 8 per group; Results).
Lung Morphology At the end of the experiments, the animals were killed and thoroughly exsanguinated before their lungs were excised en bloc. Lung tissues were fixed in 10% buffered formalin for
Animals All the experiments were in accordance with the ethical guidelines of the Committee for the Control and Supervision of Experiments on Animals, Chi Mei Medical Center (Tainan, Taiwan). Adult male Wistar rats (235Y256 g at the beginning of the study) were used. The animals were maintained at 24-C (1.0-C) with a 12:12-hour light-to-dark cycle and given food and water ad libitum.
Study Groups The rats were randomly divided into four groups of eight: the nonhyperbaric oxygen preconditioned plus nonYhigh-altitude exposed (non-HBO2P + non-HAE) group, the non-HBO2P + HAE group, the HBO2P + HAE group, and the HBO2P + HSP70 antibody (Ab) + HAE group (Fig. 1). The HBO2P groups were given 100% O2 at 2.0 ATA for 1 hour per day for 5 consecutive days. The HAE groups were exposed to simulated HAE (9.7% O2 at 0.47 ATA of 6,000 m) in a hypobaric chamber for 3 days. The protective role of HSP70 in the rat lung9 has been studied previously by using anti-HSP70 Ab. A neutralizing polyclonal rabbit antimouse HSP70 Ab (0.2 mg per kilogram of body weight; ADI-SPA-812, Enzo Life Sciences, Inc., Farmingdale, NY) dissolved in nonpyrogenic sterile saline was injected intravenously at 24 hours before simulated HAE. Immediately after returning to normal (normobaric) atmosphere (NBA), the rats were overdosed with a general anesthetic, and then, their lungs were excised en bloc for water content measurements and biochemical evaluation and analysis.
Experimental Design In Experiment 1, HSP70 protein expression in lung tissue was determined in non-HBO2P group rats, HBO2P group rats, 586
Figure 2. Densitometer analysis of the relative expression of HSP70 by lung tissue samples taken from the NBA rats, HBO2P rats, and HBO2P + HSP70 Ab rats. *p G 0.05 for HBO2P group versus NBA or non-HBO2P group; +p G 0.05 for HBO2P + HSP70 Ab group versus HBO2P group. See Figure 1 legend for group abbreviations. * 2014 Lippincott Williams & Wilkins
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Figure 3. Illustrative lung microscopic picture: NBA rats (A); NBA + HAE rats (B); HBO2P + HAE rats (C ); and HBO2P + HSP70 Ab + HAE rats (D). The NBA + HAE rats displayed interstitial edema, neutrophil accumulation, and hemorrhage. The lung pathologic changes that occurred during HAE were significantly attenuated by HBO2P + HAE (n = 8 each, p G 0.05). In addition, the beneficial effects of HBO2P in reducing acute lung injury scores were significantly attenuated by HBO2P + HSP70 Ab (n = 8 each, p G 0.05).
24 hours and then embedded in paraffin and cut into 3-Kmthick sections as described previously.12 Sections were stained with hematoxylin and eosin, and images were taken with an Olympus BX51 microscope (Olympus Optical Co., LTD., Tokyo, Japan) with a 40 objective. A lung injury scoring method of Su et al.5,13,14 was modified and applied to quantify changes in lung architecture visible by light microscope. In brief, a total of three slides prepared using each lung sample were randomly screened, and the mean was taken as the representative values of the sample. For presentation, we chose typical examples that were observed in all preparations of the same treatments.
Western Blot As described previously,12 the proteins were electrophoretically transferred to polyvinylidene difluoride membranes treated previously with methanol and blocked for 1 hour at room temperature in TBS-T (Tris-buffered saline containing
RT-PCR and Protein Preparation Total RNA was isolated from frozen lung tissues, with the aid of Trizol reagent (Invitrogen, Carlsbad, CA) and RNA kit (Biomed, Beijing, China). RNA was then reverse transcribed to synthesize first-strand complementary DNA. Real-time, quantitative PCR was performed using a Line-Genesequence detector (Bioer, Gangzhou, China). AQP1, AQP5, and actin primers were listed as described previously.12 In brief, the specificity of primers was determined by solubility curve. The size of RT-PCR products was confirmed by electrophoresis on 1% agarose gels. PCR product was sequenced, and amplified production of AQP1, AQP5, and actin was 213 base pairs (bp), 186 bp, and 150 bp, respectively. Relative quantification of mRNA expression was calculated with the 2-$CT method: $CT = CT AQPs j CT actin. The procedures for protein preparation of lung tissues were described previously.12
Figure 4. Percentage change of lung weight for all four groups. Data are presented as mean (SD). *p G 0.01 for NBA + HAE group versus NBA group (n = 8); +p G 0.05 for HBO2P + HAE group (n = 8) versus NBA + HAE group (n = 8); #p G 0.05 for HBO2P + HSP70 Ab + HAE group (n = 8).
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concentrations of these cytokines in the samples were calculated from the standard curve multiplied by the dilution factor and were expressed as picogram per milliliter.
Determination of MPO Activity A spectrophotometric method15 was used to determine MPO activity in the lung tissue. The specimen was freeze thawed and sonicated three times. Homogenates were centrifuged at 15,000 G for 10 minutes at 4-C. A 100-KL aliquot of supernatant was mixed with 900 KL of 50-mmol/L phosphate buffer (pH 6.0) containing 0.167-mg/mL o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. One unit of peroxidase activity equaled the amount of enzyme decomposing 1 Kmol of hydrogen peroxide per minute at 25-C. Decomposition of hydrogen peroxide was calculated from the oxidation of o-dianisidine using an absorption coefficient of 11.3/mM/cm at 460 nm.
Physiologic Variable Monitoring
Figure 5. Immunoblot of both AQP1 and AQP5. Western blot analysis of AQP1 and AQP5 in the lungs of NBA rats (n = 8), NBA + HAE rats (n = 8), HBO2P + HAE rats (n = 8), and HBO2P + HSP70 Ab + HAE rats (n = 8). Fold-change values represent mean of eight samples (n = 8) divided by the mean of the eight controls (n = 8). Data are presented as mean (SD). *p G 0.01 for NBA + HAE group versus NBA group; +p G 0.05 for HBO2P + HAE group (n = 8) versus NBA + HAE group; #p G 0.01 for HBO2P + HSP70 Ab + HAE group versus HBO2P + HAE group.
0.1% Ten 20) containing 5% nonfat dry milk and incubated overnight at 4-C with anti-AQP1 Ab (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-AQP5 Ab (1:200, Santa Cruz Biotechnology, Inc.), and polyclonal Ab specific for HSP70 (SPA-812, Stressgen Biotechnology, Victoria, Canada) in TBS-T; the membranes were incubated with horseradish peroxidaseYlabeled anti-rabbit Ab (1:3,000, Santa Cruz Biotechnology, Inc.) for 2 hours to 3 hours at room temperature. The value of the relative density of AQP1 and AQP5 band was normalized to the density of actin to represent the amount of AQP1 and AQP5 protein. The ratio of control group was regarded as 100%, and the results of HBO2P + HAE and HAE groups were expressed as a percentage of the value from the control group as described previously.12
At the end of HAE for the HAE group or the equivalent period for the NBA group, the animals were anesthetized and their bilateral femoral artery were cannulated using polyethylene tubes. One tube was used for continuous monitoring of MAP and HR via a pressure transducer, whereas the other was used for collecting blood samples to determine the animal’s arterial pH, PaCO2, SO2, and HCO3j levels. Both the MAP and HR were recorded using a polygraph. Core body temperature was monitored by a thermocouple inserted into the rectum. The core temperature was maintained at a constant level of approximately 37-C with a hot water circulating heating pad. After stabilization, all physiologic variables were obtained for data presentation, and then, the blood samples were obtained for biochemical determination.
Statistical Analysis Statistical analysis was performed using SPSS 15.0 (SPSS, Chicago, IL). All quantitative data were expressed as mean (SD). One-way analysis of variance was used to test the differences in HSP70, AQP1, AQP5, AQP5 mRNA, and lung injury score, and t test was used for AQP1 mRNA expression because of the unequal homogeneity of variance. p G 0.05 was considered statistically significant.
RESULTS Preinduction of HSP70 Expression by HBO2P Western blot analysis revealed that the HBO2P group rats had significantly higher lung expression of HSP70 compared with the non-HBO2P group rats (Fig. 2, p G 0.05). In addition, the HBO2P + HSP70 Ab group rats had significantly lower lung levels of HSP70 expression compared with the HBO2P group rats without HSP70 Ab ( p G 0.05, Fig. 2).
Measurement of Proinflammatory Cytokines in BAL Fluid
Increased Acute Lung Injury Scores After HAE
The concentrations of interleukin 6 (IL-6), IL-1A, and tumor necrosis factor > (TNF->) in BAL fluid were determined using double-Ab sandwich enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) according to the manufacturer’s instruction. Optical densities were read on a plate reader set at 450 nm for IL-6, IL-1A, and TNF->. The
Rats in the NBA + HAE group demonstrated symptoms of lethargy, tachycardia, and respiratory distress in the hypobaric chamber. At the opening of the chest, their lungs showed congestion, swelling, and hemorrhage. Histologic examination of the lung showed marked interstitial edema in all the sections in this group. All the injury scores including edema
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(3.46 [0.08] vs. 0.48 [0.03]), neutrophil infiltration (3.73 [0.06] vs. 0.49 [0.04]), and hemorrhage (2.27 [0.09] vs. 0.26 [0.05]) scores were significantly increased in the NBA + HAE group compared with those of the NBA group (Fig. 3). When compared with those of the NBA + HAE group, the animals of the HBO2P + HAE group had significantly lower scores (edema, 1.0 [0.07]; neutrophil, 1.22 [0.11]; hemorrhage, 1.08 [0.10]) of acute lung injury (Fig. 3). However, the beneficial effects of HBO2P were significantly attenuated in the HBO2P + HSP70 Ab group rats (edema, 3.85 [0.09]; neutrophil, 3.79 [0.07]; hemorrhage, 3.71 [0.05]) (Fig. 3). The numbers used for each group were eight animals.
Increased Lung Weight After HAE After 3 days of HAE, lung weight was 14% higher, because of edema, in the NBA + HAE group than in the NBA + NBA control group (Fig. 4). Lung weight was significantly lower in the HBO2P + HAE group than in the NBA + HAE group ( p G 0.05). Again, the beneficial effects of HBO2P treatment were significantly ( p G 0.05) attenuated in the HBO2P + HSP + 70 Ab pretreatment group (Fig. 4).
Decreased Lung Levels of Both AQP1 and AQP5 After HAE Both Western blot analysis and RT-PCR revealed expression of both AQP1 and AQP5 proteins (Fig. 5), and mRNA (Fig. 6) in the lung tissues of the NBA + HAE group significantly decreased compared with those of the NBA group ( p G 0.01). Expressions of both AQP1 and AQP5 proteins and mRNA in the lung tissues of the HBO2P + HAE group were significantly higher than those of the NBA + HAE group
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( p G 0.01). Again, the beneficial effects of HBO2P therapy were significantly ( p G 0.05) reduced in the HBO2P + HSP + HSP70 Ab pretreatment group (Figs. 5 and 6).
Decrease in MAP, HR, SO2, PaCO2, HCO3j, and pH Values After HAE The values of MAP (50 mg vs. 90 mm Hg), HR (250 beats/ min vs. 380 beats/min), SO2 (52% vs. 100%), PaCO2 (22 mm Hg vs. 45 mm Hg), HCO3j (18 mmol/L vs. 27 mmol/L), and pH (7.2 vs. 7.4) in the NBA rats were significantly lower after HAE than in the non-HAE controls. However, HBO2P significantly ( p G 0.05, n = 8 each group) reduced the HAE-induced decrease of MAP (80 mg), HR (350 beats/min), SO2 (80%), PaCO2 (40 mm Hg), HCO3j (25 mmol/L), and pH (7.3). In addition, HSP70 Ab preconditioning significantly ( p G 0.05, n = 8 each group) reduced the beneficial effects of HBO2P in HAE (MAP, 42 mm Hg; HR, 200 beats/min; SO2, 40%; PaCO2, 20 mm Hg; HCO3j, 16 mmol/L; and pH 7.18).
Increase in Lung Levels of Systemic Inflammatory Response Molecules After HAE The values of lung MPO (320-Kg/mg protein vs. 112-Kg/mg protein), and BAL fluid levels of IL-6 (11 pg/mL vs. 52 pg/mL), IL-1A (60 pg/mL vs. 39 pg/mL), and TNF-> (40 pg/mL vs. 8 pg/mL) in the NBA rats were significantly ( p G 0.05, n = 8 each group) higher after HAE than in the non-HAE controls. However, HBO2P significantly ( p G 0.05, n = 8 each group) attenuated the HAE-induced increase of lung MPO (125-Kg/mg protein) and BAL fluid IL-6 (70 pg/mL), IL-1A (41 pg/mL), and TNF-> (15 pg/mL). In addition, HSP70 Ab preconditioning significantly ( p G 0.05, n = 8 each group) attenuated the beneficial effects of HBO2P in HAE (MPO, 334-Kg/mg protein; IL-6, 118 pg/mL; IL-1A, 65 pg/mL; and TNF->, 45 pg/mL).
DISCUSSION
Figure 6. RT-PCR analysis of AQP1 mRNA and AQP5 mRNA in the lungs of NBA rats (n = 8), NBA + HAE rats (n = 8), HBO2P + HAE rats (n = 8), and HBO2P + HSP70 + HAE rats (n = 8). Data are presented as mean (SD). *p G 0.05 for NBA + HAE group versus NBA group; +p G 0.05 for HBO2P + HAE group versus NBA + HAE group; #p G 0.05 for HBO2P + HSP70 Ab + HAE group versus HBO2P + HAE group.
The reduction in barometric pressure and the consequent fall in the PaO2 at higher altitudes lead to a drop of the oxygen saturation, which lead to multiple-organ dysfunction. Indeed, as shown in the present study, 3 days after the initiation of HAE, the non-HBO2P rats had hypoxia, hypotension, bradycardia, acidosis, and tissue up-regulation of both proinflammatory cytokines including IL-1A, IL-6, and TNF-> and an indicator for polymorphonuclear leukocytes accumulation such as MPO activity. In addition, the non-HBO2P animals had higher values of lung edema, neutrophils, and hemorrhage scores and lung water as well as decreased lung levels of both AQP1 and AQP5 protein and mRNA expression. It is believed that a decrease in lung expression of both AQP1 and AQP5 protein and mRNA may play an important role in pulmonary edema.3Y7 Therefore, both the increased values of both the lung edema score and the lung water weight are caused by a decrease in both AQP1 and AQP5 protein and mRNA expression in the non-HBO2P + HAE group rats. Probably the most striking finding of present study is that the preinduction of HSP72 in the lung tissue by HBO2P significantly attenuated HAE-induced increased acute lung injury scores including edema, inflammation, and hemorrhage. The beneficial effects
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of HBO2P in preventing the occurrence of these high mountain sickness syndromes were all attenuated by the inhibition of HSP70 expression by HSP70 Ab pretreatment. Our data strongly indicate that preinduction of HSP70 in the lung tissue before onset of HAE may be one of the key issues. HSP70 is up-regulated by hypoxia and involved in cell protection and survival in acute myocardial stress,16 severe sepsis,17 and ischemic stroke.18 Preinduction of HSP70 by drugs promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain.10 Hypobaric hypoxia preconditioning such as at chromic exposure at a hypobaric hypoxia chamber, has recently been known to attenuate acute lung injury during HAE in rats via up-regulating HSP70.9,19 The data indicate that hypoxic preconditioning may be beneficial for preventing HAE acute lung injury. Indeed, the most popular preventive approach for HAE acute lung injury is gradual ascending,20 which is actually a kind of hypoxic preconditioning. Since hypoxic preconditioning has not been used clinically because of safety concerns, HBO2P seems to be more attractive and easier to be accepted ethically because it has been used safely for various disorders.21Y26 HBO2P increases tissue oxygen levels by a giving 100% oxygen at higher pressure. Upon termination of HBO2P, the body would experience a relative hypoxia because the oxygen level is returned to normal level at 21%. Therefore, repeating HBO2P may produce a cycle of hyperoxia and then hypoxia and head to hypoxia-inducing factor 1> (HIF-1>) accumulation. Several studies have demonstrated that ischemic tolerance induced by HBO2P involves the up-regulation of HIF-1> in rats.27Y29 Our present data further demonstrated that HBO2P, like hypoxia preconditioning, significantly reduces acute lung injury in rats during simulated HAE by up-regulating lung HSP70. As shown in the present study, HAE caused decreased lung expression of both AQP1 and AQP5 both at protein and at gene level accompanied by pulmonary edema in rats. Pearson correlation analysis revealed that there was an inverse correlation between lung injury scores and mRNA and protein expression of AQP1 and AQP5 in the lungs of rats.12 Lower mRNA and lower protein expression of both AQP1 and AQP5 may induce reduction of transcellular rate of excess water removal and result in pulmonary edema formation,6 which could be significantly reversed by HSP70-mediated HBO2P. AQP1 and AQP5 are mainly distributed in microvascular endothelia and in alveolar epithelia, respectively.30 It can be derived from the foregoing statements that HSP70-mediated HBO2P may attenuate HAE-induced pulmonary edema by increasing both mRNA and protein expression of AQP1 and AQP5 in the lungs of rats. The levels of IL-1A in the BAL fluid are elevated in patients with adult respiratory distress syndrome.31 Both TNF-> and IL-6 are believe to be an important mediator in the early cascade of endotoxin-induced lung inflammation.32 As demonstrated in the present results, the BAL levels of IL-1A, TNF->, and IL-6 and the lung levels of MPO are elevated by HAE. Besides lung inflammation, other parameters such as hypoxia, hypotension, bradycardia, and acidosis may be related to the occurrence of acute lung damage in HAE, which can be prevented by HSP70-mediated HBO2P. A comparison of HBO2P versus hypoxic preconditioning9 was performed in rats and found that HBO2P + HAE 590
elicited similar preconditioning efficacy in reducing pulmonary edema. However, hypoxic preconditioning has not been used clinically because of safety concerns. In contrast, HBO2P seems to be more attractive and easier to be accepted ethically because it has been used safely for various disorders.23,26 Our experiment has several limitations. First, we used very low numbers of rats. Second, the rodent model of HAE resembles the pathophysiology response of humans only to a certain point. It is difficult to extrapolate the data from our rat model to predict exactly the human response. However, our approach is promising and deserves further preclinical and clinical study to assess elements of the pathophysiology of critical illness. In summary, HBO2P, in addition to inducing lung overexpression of HSP70, significantly attenuated the HAEinduced hypotension, hypoxia, bradycardia, acidosis, increased BAL fluid levels of proinflammatory cytokines, increased lung levels of MPO (an indicator for leukocytes accumulation), increased lung water weight, decreased lung levels of both AQP1 and AQP5 protein and mRNA expression, and increased scores of lung edema, neutrophils, and hemorrhage. The beneficial effects of HBO2P in inducing overexpression of lung HSP70 as well as in preventing the HAE-induced hypotension, bradycardia, hypoxia, acidosis, lung inflammation, and increased scores of lung edema, neutrophils, and hemorrhage could be significantly reduced by HSP70 Ab preconditioning. These results suggest that high-altitude pulmonary edema can be prevented by HSP70-mediated HBO2P in rats. AUTHORSHIP M.-C.T., H.-J.L., and T.C.-Y.T. participated in the design of the study and obtained the financial support. M.-T.L., K.-C.N., and C.-P.C. made contribution to the literature search, data collection/analysis/interpretation, and paper writing. T.C.-Y.T. and C.-P.C. oversaw the study and gave final approval of the version to be published.
DISCLOSURE This study was supported by grants from the Chi Mei Medical Center (Tainan, Taiwan), the National Science Council of the Republic of China (Taipei, Taiwan), and the Department of Health of the Republic of China (Taipei, Taiwan).
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8. Liebelt B, Papapetrou P, Ali A, Guo M, Ji X, Peng C, Rogers R, Curry A, Jimenez D, Ding Y. Exercise preconditioning reduces neuronal apoptosis in stroke by up-regulating heat shock protein-70 (heat shock protein-72) and extracellular-signal-regulated-kinase 1/2. Neuroscience. 2010;166: 1091Y1100. 9. Lin HJ, Wang CT, Niu KC, Gao C, Li Z, Lin MT, Chang CP. Hypobaric hypoxia preconditioning attenuates acute lung injury during high-altitude exposure in rats via up-regulating heat-shock protein 70. Clin Sci. 2011; 121:223Y231. 10. Zhang K, Zhao T, Huang X, Liu ZH, Xiong L, Li MM, Wu LY, Zhao YQ, Zhu LL, Fan M. Preinduction of HSP70 promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain. Cell Stress Chaperones. 2009;14:407Y415. 11. Lin H, Chang CP, Lin HJ, Lin MT, Tsai CC. Attenuating brain edema, hippocampal oxidative stress, and cognitive dysfunction in rats using hyperbaric oxygen preconditioning during simulated high-altitude exposure. J Trauma Acute Care Surg. 2012;72:1220Y1227. 12. Li Z, Gao C, Wang Y, Liu F, Ma L, Deng C, Niu KC, Lin MT, Wang C. Reducing pulmonary injury by hyperbaric oxygen preconditioning during simulated high altitude exposure in rats. J Trauma. 2011;71:673Y679. 13. Chang CH, Kao CH, Chio CC, Lin CH, Lin MT, Chang CP. Attenuating heatstroke-induced acute lung inflammation, edema, and injury in rats by exercise preconditioning. J Trauma Acute Care Surg. 2013;74:1052Y1059. 14. Su X, Bai C, Hong Q, Zhu D, He L, Wu J, Ding F, Fang X, Matthay MA. Effect of continuous hemofiltration on hemodynamics, lung inflammation and pulmonary edema in a canine model of acute lung injury. Intensive Care Med. 2003;29:2034Y2042. 15. Yang HH, Hou CC, Lin MT, Chang CP. Attenuating heat-induced acute lung inflammation and injury by dextromethorphan in rats. Am J Respir Cell Mol Biol. 2012;46:407Y413. 16. Das DK, Maulik N, Moraru II. Gene expression in acute myocardial stress. Induction by hypoxia, ischemia, reperfusion, hyperthermia and oxidative stress. J Mol Cell Cardiol. 1995;27:181Y193. 17. Bruemmer-Smith S, Stu¨ber F, Schroeder S. Protective functions of intracellular heat-shock protein (HSP) 70-expression in patients with severe sepsis. Intensive Care Med. 2001;27:1835Y1841. 18. Weinstein PR, Hong S, Sharp FR. Molecular identification of the ischemic penumbra. Stroke. 2004;35:2666Y2670. 19. Lin HJ, Chang CP, Niu KC, Lin MT. Hypobaric hypoxia or hyperbaric oxygen preconditioning reduces high-altitude lung and brain injury in rats. Science. 2013;338(6113)1485:51Y52. 20. Maggiorini M. High altitude-induced pulmonary edema. Cardiovasc Res. 2006;72:41Y50.
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21. Hu SL, Hu R, Li F, Liu Z, Xia YZ, Cui GY, Feng H. Hyperbaric oxygen preconditioning protects against traumatic brain injury at high altitude. Acta Neurochir Suppl. 2008;105:191Y196. 22. Niu KC, Lin MT, Chang CP. Hyperbaric oxygen improves survival in heatstroke rats by reducing multiorgan dysfunction and brain oxidative stress. Eur J Pharmacol. 2007;569:94Y102. 23. Ostrowski RP, Graupner G, Titova E, Zhang J, Chiu J, Dach N, Corleone D, Tang J, Zhang JH. The hyperbaric oxygen preconditioning-induced brain protection is mediated by a reduction of early apoptosis after transient global cerebral ischemia. Neurobiol Dis. 2008;29:1Y13. 24. Dong H, Xiong L, Zhu Z, Chen S, Hou L, Sakabe T. Preconditioning with hyperbaric oxygen and hyperoxia induces tolerance against spinal cord ischemia in rabbits. Anesthesiology. 2002;96:907Y912. 25. Kim CH, Choi H, Chun YS, Kim GT, Park JW, Kim MS. Hyperbaric oxygenation pretreatment induces catalase and reduces infarct size in ischemic rat myocardium. Pflugers Arch. 2001;442:519Y525. 26. Yu SY, Chiu JH, Yang SD, Yu HY, Hsieh CC, Chen PJ, Lui WY, Wu CW. Preconditioned hyperbaric oxygenation protects the liver against ischemiareperfusion injury in rats. J Surg Res. 2005;128:28Y36. 27. Gu GJ, Li YP, Peng ZY, Xu JJ, Kang ZM, Xu WG, Tao HY, Ostrowski RP, Zhang JH, Sun XJ. Mechanism of ischemic tolerance induced by hyperbaric oxygen preconditioning involves upregulation of hypoxia-inducible factor-1alpha and erythropoietin in rats. J Appl Physiol. 2008;104: 1185Y1191. 28. Peng Z, Ren P, Kang Z, Du J, Lian Q, Liu Y, Zhang JH, Sun X. Upregulated HIF-1alpha is involved in the hypoxic tolerance induced by hyperbaric oxygen preconditioning. Brain Res. 2008;1212:71Y78. 29. Salhanick SD, Belikoff B, Orlow D, Holt D, Reenstra W, Buras JA. Hyperbaric oxygen reduces acetaminophen toxicity and increases HIF-1alpha expression. Acad Emerg Med. 2006;13:707Y714. 30. Ba¨rtsch P. High altitude pulmonary edema. Respiration. 1997;64:435Y443. Review. 31. Pugin J, Ricou B, Steinberg KP, Suter PM, Martin TR. Proinflammatory activity in bronchoalveolar lavage fluids from patients with ARDS, a prominent role for interleukin-1. Am J Respir Crit Care Med. 1996;153: 1850Y1856. 32. Xing Z, Jordana M, Kirpalani H, Driscoll KE, Schall TJ, Gauldie J. Cytokine expression by neutrophils and macrophages in vivo: endotoxin induces tumor necrosis factor-alpha, macrophage inflammatory protein-2, interleukin-1 beta, and interleukin-6 but not RANTES or transforming growth factor-beta 1 mRNA expression in acute lung inflammation. Am J Respir Cell Mol Biol. 1994;10:148Y153.
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High-altitude pulmonary edema can be prevented by heat shock protein 70Ymediated hyperbaric oxygen preconditioning Ming-Che Tsai, MD, Hung-Jung Lin, MD, Mao-Tsun Lin, DDS, PhD, Ko-Chi Niu, MD, PhD, Ching-Ping Chang, PhD, and Thomas Chang-Yao Tsao, MD, Taichung, Taiwan
BACKGROUND: The primary goal of this study was to test whether high-altitude exposure (HAE of 9.7% O2 at 0.47 absolute atmosphere [ATA] for 3 days) was capable of increasing lung edema, neutrophil, and hemorrhage scores as well as decreasing lung levels of both aquaporin 1 (AQP1) and AQP5 proteins and messenger RNA (mRNA) expression in rats, with a secondary goal to test whether a preinduction of heat shock protein 70 (HSP70) by hyperbaric oxygen preconditioning (HBO2P of 100% O2 at 2.0 ATA for 1 hour per day for 5 consecutive days) attenuated the HAE-induced increased lung injury scores and decreased lung AQP1 and AQP5 protein and mRNA expressions. METHODS: Rats were assigned to (1) non-HBO2P (21% O2 at 1.0 ATA) + non-HAE (21% O2 at 1.0 ATA) group; (2) non-HBO2P + HAE group; (3) HBO2P + HAE group; and HBO2P + HSP70 antibodies (Ab) + HAE group. For the HSP70 Ab group, a neutralizing HSP70 Ab was injected intravenously at 24 hours before HAE. All the physiologic and biochemical parameters were obtained at the end of HAE or the equivalent period of non-HAE. The cardiovascular and blood gas parameters were monitored for all experiments. Bronchoalveolar lavage (BAL) was performed to determine proinflammatory cytokines (interleukin 6, interleukin 1A, and tumor necrosis factor >). Parts of the lung were excised for myeloperoxidase activity measurement, whereas the rest was collected for lung damage score assessments. AQP1 and AQP5 protein and mRAN expressions were also determined in the lung tissues. RESULTS: In the non-HBO2P + HAE group, the animals displayed higher values of lung myeloperoxidase activity, BAL proinflammatory cytokines, lung water weight, and acute lung injury scores compared with those of the non-HBO2P + non-HAE controls. In contrast, the non-HBO2P + HAE group rats had lower values of lung AQP1 and AQP5 protein and mRNA expressions, mean arterial pressure, heart rate, SO2, PaCO2, HCO3j, and pH compared with those of non-HBO2P + non-HAE group rats. The increased acute lung edema, neutrophil, and hemorrhage scores; increased BAL levels of proinflammatory cytokines; decreased lung AQP1 and AQP5 protein and mRNA expressions; and hypotension, bradycardia, hypoxia, and acidosis caused by HAE were all significantly attenuated by HBO2P. CONCLUSION: Our data indicate that HBO2P may attenuate high-altitude acute lung injury by a preinduction of lung HSP70 in rats. (J Trauma Acute Care Surg. 2014;77: 585Y591. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: High-altitude pulmonary edema; hyperbaric oxygen preconditioning; lung injury score; aquaporin 1; aquaporin 5.
A
t high-altitude exposure (HAE) conditions, hypoxia induces pulmonary hypertension as well as impairment of the respiratory transepithelial sodium transport and results in alveolar fluid accumulation as well as alveolar fluid clearance deficits, which facilitate pulmonary edema.1,2 Evidence has also accumulated to indicate that decreases in lung expression of both aquaporin 1 (AQP1) and AQP5 may play an important role in pulmonary edema.3Y7
Submitted: April 29, 2014, Revised: June 3, 2014, Accepted: June 4, 2014. From the Institute of Medicine and School of Medicine (M.-C.T.), Chung Shan Medical University; and Division of Thoracic Medicine (T.C.-Y.T.), Chung Shan Medical University Hospital and Chung Shan Medical University, Taichung; and Departments of Emergency Medicine (H.-J.L.), Medical Research (M.-T.L.), and Hyperbaric Oxygen (K.-C.N.), Chi Mei Medical Center; and Department of Biotechnology (M.-T.L., C.-P.C.), Southern Taiwan University of Science and Technology, Tainan, Taiwan. Address for reprints: Ching-Ping Chang, PhD, Department of Biotechnology, Southern Taiwan University of Science and Technology, 1 Nan-Tai Road, Tainan 710, Taiwan; email: [email protected]; Thomas Chang-Yao Tsao, MD, Division of Thoracic Medicine, Chung Shan Medical University Hospital and Chung Shan Medical University, No.110, Sec.1, Jianguo N.Rd., Taichung City 40201, Taiwan; email: [email protected]. DOI: 10.1097/TA.0000000000000408
The 70-kD family of heat shock protein (HSP), HSP70, is up-regulated by exercise preconditioning in cell protection and survival in stroke.8 Preinduction of lung HSP70 protein with a sublethal dose of HAE (e.g., hypobaric hypoxia preconditioning, 18.3% O2 at 0.66 absolute atmosphere [ATA]) were able to protect against a subsequent lethal damage exerted by a lethal dose of HAE (9.7% O2 at 0.47 ATA).9 Preinduction of HSP70 by hypobaric hypoxia also promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain.10 Hyperbaric oxygen preconditioning (HBO2P), like hypobaric hypoxia preconditioning, attenuates brain edema caused by hypobaric hypoxia in rats.11 However, it is not known whether the increased lung injury scores and the decreased levels of both AQP1 and AQP5 proteins and messenger RNA (mRNA) expression in the lung caused by HAE12 can be affected by HSP70-mediated HBO2P in rats. The primary purpose of this study was to investigate whether HAE was capable of increasing lung edema, neutrophil, and hemorrhage scores as well as decreasing lung levels of both AQP1 and AQP5 protein and mRNA expression in rats, with a secondary goal to test whether a preinduction of HSP70 by HBO2P attenuated the HAE-induced acute lung injury scores as well as decreased lung AQP1 and AQP5 protein and
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Figure 1. Experimental design. The HBO2P group was treated with 100% O2 at 2.0 ATA for 1 hour per day for 5 consecutive days. The NBA (or non-HBO2P) + HAE (simulated high-altitude exposure) group, the HBO2P + HAE group, and the HBO2P + HSP70 Ab + HAE group were all subjected to HAE (9.7% at 0.47 ATA) for 3 days. HSP70 Ab was administered 1 day before the onset of HE. The NBA (or non-HBO2P) and non-HAE groups were treated with 21% O2 at 1.0 ATA.
mRNA expression in rats. Our expected results would verify the hypothesis that HBO2P attenuates HAE-induced pulmonary edema by preinduction of lung HSP70 in rats.
MATERIALS AND METHODS
and HBO2P + HSP70 Ab group rats before the start of HAE (n = 8 per group, Fig. 2). In Experiment 2, acute lung injury scores were assessed in all four groups of rats 3 days after HAE or NBA (n = 8 per group, Fig. 2). In Experiment 3, lung weights were determined in all four groups of rats 0 hour and 3 days after HAE or NBA (n = 8 per group, Fig. 3). In Experiment 4, Western blot analysis of AQP1 and AQP5 in the lungs for all four groups of rats were assessed 3 days after HAE or NBA (n = 8 per group, Fig. 4). In Experiment 5, real-time polymerase chain reaction (RT-PCR) analysis of AQP1 mRNA and AQP5 mRNA in the lungs for all four groups of rats were performed 3 days after HAE or NBA (n = 8 per group, Fig. 5). In Experiment 6, mean arterial pressure (MAP); heart rate (HR); blood levels of SO2, PaCO2, HCO3j, and pH; bronchoalveolar lavage (BAL) fluid levels of proinflammatory cytokines; and lung levels of myeloperoxidase (MPO) activity for all four groups of rats were determined 3 days after HAE or NBA (n = 8 per group; Results).
Lung Morphology At the end of the experiments, the animals were killed and thoroughly exsanguinated before their lungs were excised en bloc. Lung tissues were fixed in 10% buffered formalin for
Animals All the experiments were in accordance with the ethical guidelines of the Committee for the Control and Supervision of Experiments on Animals, Chi Mei Medical Center (Tainan, Taiwan). Adult male Wistar rats (235Y256 g at the beginning of the study) were used. The animals were maintained at 24-C (1.0-C) with a 12:12-hour light-to-dark cycle and given food and water ad libitum.
Study Groups The rats were randomly divided into four groups of eight: the nonhyperbaric oxygen preconditioned plus nonYhigh-altitude exposed (non-HBO2P + non-HAE) group, the non-HBO2P + HAE group, the HBO2P + HAE group, and the HBO2P + HSP70 antibody (Ab) + HAE group (Fig. 1). The HBO2P groups were given 100% O2 at 2.0 ATA for 1 hour per day for 5 consecutive days. The HAE groups were exposed to simulated HAE (9.7% O2 at 0.47 ATA of 6,000 m) in a hypobaric chamber for 3 days. The protective role of HSP70 in the rat lung9 has been studied previously by using anti-HSP70 Ab. A neutralizing polyclonal rabbit antimouse HSP70 Ab (0.2 mg per kilogram of body weight; ADI-SPA-812, Enzo Life Sciences, Inc., Farmingdale, NY) dissolved in nonpyrogenic sterile saline was injected intravenously at 24 hours before simulated HAE. Immediately after returning to normal (normobaric) atmosphere (NBA), the rats were overdosed with a general anesthetic, and then, their lungs were excised en bloc for water content measurements and biochemical evaluation and analysis.
Experimental Design In Experiment 1, HSP70 protein expression in lung tissue was determined in non-HBO2P group rats, HBO2P group rats, 586
Figure 2. Densitometer analysis of the relative expression of HSP70 by lung tissue samples taken from the NBA rats, HBO2P rats, and HBO2P + HSP70 Ab rats. *p G 0.05 for HBO2P group versus NBA or non-HBO2P group; +p G 0.05 for HBO2P + HSP70 Ab group versus HBO2P group. See Figure 1 legend for group abbreviations. * 2014 Lippincott Williams & Wilkins
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Figure 3. Illustrative lung microscopic picture: NBA rats (A); NBA + HAE rats (B); HBO2P + HAE rats (C ); and HBO2P + HSP70 Ab + HAE rats (D). The NBA + HAE rats displayed interstitial edema, neutrophil accumulation, and hemorrhage. The lung pathologic changes that occurred during HAE were significantly attenuated by HBO2P + HAE (n = 8 each, p G 0.05). In addition, the beneficial effects of HBO2P in reducing acute lung injury scores were significantly attenuated by HBO2P + HSP70 Ab (n = 8 each, p G 0.05).
24 hours and then embedded in paraffin and cut into 3-Kmthick sections as described previously.12 Sections were stained with hematoxylin and eosin, and images were taken with an Olympus BX51 microscope (Olympus Optical Co., LTD., Tokyo, Japan) with a 40 objective. A lung injury scoring method of Su et al.5,13,14 was modified and applied to quantify changes in lung architecture visible by light microscope. In brief, a total of three slides prepared using each lung sample were randomly screened, and the mean was taken as the representative values of the sample. For presentation, we chose typical examples that were observed in all preparations of the same treatments.
Western Blot As described previously,12 the proteins were electrophoretically transferred to polyvinylidene difluoride membranes treated previously with methanol and blocked for 1 hour at room temperature in TBS-T (Tris-buffered saline containing
RT-PCR and Protein Preparation Total RNA was isolated from frozen lung tissues, with the aid of Trizol reagent (Invitrogen, Carlsbad, CA) and RNA kit (Biomed, Beijing, China). RNA was then reverse transcribed to synthesize first-strand complementary DNA. Real-time, quantitative PCR was performed using a Line-Genesequence detector (Bioer, Gangzhou, China). AQP1, AQP5, and actin primers were listed as described previously.12 In brief, the specificity of primers was determined by solubility curve. The size of RT-PCR products was confirmed by electrophoresis on 1% agarose gels. PCR product was sequenced, and amplified production of AQP1, AQP5, and actin was 213 base pairs (bp), 186 bp, and 150 bp, respectively. Relative quantification of mRNA expression was calculated with the 2-$CT method: $CT = CT AQPs j CT actin. The procedures for protein preparation of lung tissues were described previously.12
Figure 4. Percentage change of lung weight for all four groups. Data are presented as mean (SD). *p G 0.01 for NBA + HAE group versus NBA group (n = 8); +p G 0.05 for HBO2P + HAE group (n = 8) versus NBA + HAE group (n = 8); #p G 0.05 for HBO2P + HSP70 Ab + HAE group (n = 8).
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concentrations of these cytokines in the samples were calculated from the standard curve multiplied by the dilution factor and were expressed as picogram per milliliter.
Determination of MPO Activity A spectrophotometric method15 was used to determine MPO activity in the lung tissue. The specimen was freeze thawed and sonicated three times. Homogenates were centrifuged at 15,000 G for 10 minutes at 4-C. A 100-KL aliquot of supernatant was mixed with 900 KL of 50-mmol/L phosphate buffer (pH 6.0) containing 0.167-mg/mL o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. One unit of peroxidase activity equaled the amount of enzyme decomposing 1 Kmol of hydrogen peroxide per minute at 25-C. Decomposition of hydrogen peroxide was calculated from the oxidation of o-dianisidine using an absorption coefficient of 11.3/mM/cm at 460 nm.
Physiologic Variable Monitoring
Figure 5. Immunoblot of both AQP1 and AQP5. Western blot analysis of AQP1 and AQP5 in the lungs of NBA rats (n = 8), NBA + HAE rats (n = 8), HBO2P + HAE rats (n = 8), and HBO2P + HSP70 Ab + HAE rats (n = 8). Fold-change values represent mean of eight samples (n = 8) divided by the mean of the eight controls (n = 8). Data are presented as mean (SD). *p G 0.01 for NBA + HAE group versus NBA group; +p G 0.05 for HBO2P + HAE group (n = 8) versus NBA + HAE group; #p G 0.01 for HBO2P + HSP70 Ab + HAE group versus HBO2P + HAE group.
0.1% Ten 20) containing 5% nonfat dry milk and incubated overnight at 4-C with anti-AQP1 Ab (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-AQP5 Ab (1:200, Santa Cruz Biotechnology, Inc.), and polyclonal Ab specific for HSP70 (SPA-812, Stressgen Biotechnology, Victoria, Canada) in TBS-T; the membranes were incubated with horseradish peroxidaseYlabeled anti-rabbit Ab (1:3,000, Santa Cruz Biotechnology, Inc.) for 2 hours to 3 hours at room temperature. The value of the relative density of AQP1 and AQP5 band was normalized to the density of actin to represent the amount of AQP1 and AQP5 protein. The ratio of control group was regarded as 100%, and the results of HBO2P + HAE and HAE groups were expressed as a percentage of the value from the control group as described previously.12
At the end of HAE for the HAE group or the equivalent period for the NBA group, the animals were anesthetized and their bilateral femoral artery were cannulated using polyethylene tubes. One tube was used for continuous monitoring of MAP and HR via a pressure transducer, whereas the other was used for collecting blood samples to determine the animal’s arterial pH, PaCO2, SO2, and HCO3j levels. Both the MAP and HR were recorded using a polygraph. Core body temperature was monitored by a thermocouple inserted into the rectum. The core temperature was maintained at a constant level of approximately 37-C with a hot water circulating heating pad. After stabilization, all physiologic variables were obtained for data presentation, and then, the blood samples were obtained for biochemical determination.
Statistical Analysis Statistical analysis was performed using SPSS 15.0 (SPSS, Chicago, IL). All quantitative data were expressed as mean (SD). One-way analysis of variance was used to test the differences in HSP70, AQP1, AQP5, AQP5 mRNA, and lung injury score, and t test was used for AQP1 mRNA expression because of the unequal homogeneity of variance. p G 0.05 was considered statistically significant.
RESULTS Preinduction of HSP70 Expression by HBO2P Western blot analysis revealed that the HBO2P group rats had significantly higher lung expression of HSP70 compared with the non-HBO2P group rats (Fig. 2, p G 0.05). In addition, the HBO2P + HSP70 Ab group rats had significantly lower lung levels of HSP70 expression compared with the HBO2P group rats without HSP70 Ab ( p G 0.05, Fig. 2).
Measurement of Proinflammatory Cytokines in BAL Fluid
Increased Acute Lung Injury Scores After HAE
The concentrations of interleukin 6 (IL-6), IL-1A, and tumor necrosis factor > (TNF->) in BAL fluid were determined using double-Ab sandwich enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) according to the manufacturer’s instruction. Optical densities were read on a plate reader set at 450 nm for IL-6, IL-1A, and TNF->. The
Rats in the NBA + HAE group demonstrated symptoms of lethargy, tachycardia, and respiratory distress in the hypobaric chamber. At the opening of the chest, their lungs showed congestion, swelling, and hemorrhage. Histologic examination of the lung showed marked interstitial edema in all the sections in this group. All the injury scores including edema
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(3.46 [0.08] vs. 0.48 [0.03]), neutrophil infiltration (3.73 [0.06] vs. 0.49 [0.04]), and hemorrhage (2.27 [0.09] vs. 0.26 [0.05]) scores were significantly increased in the NBA + HAE group compared with those of the NBA group (Fig. 3). When compared with those of the NBA + HAE group, the animals of the HBO2P + HAE group had significantly lower scores (edema, 1.0 [0.07]; neutrophil, 1.22 [0.11]; hemorrhage, 1.08 [0.10]) of acute lung injury (Fig. 3). However, the beneficial effects of HBO2P were significantly attenuated in the HBO2P + HSP70 Ab group rats (edema, 3.85 [0.09]; neutrophil, 3.79 [0.07]; hemorrhage, 3.71 [0.05]) (Fig. 3). The numbers used for each group were eight animals.
Increased Lung Weight After HAE After 3 days of HAE, lung weight was 14% higher, because of edema, in the NBA + HAE group than in the NBA + NBA control group (Fig. 4). Lung weight was significantly lower in the HBO2P + HAE group than in the NBA + HAE group ( p G 0.05). Again, the beneficial effects of HBO2P treatment were significantly ( p G 0.05) attenuated in the HBO2P + HSP + 70 Ab pretreatment group (Fig. 4).
Decreased Lung Levels of Both AQP1 and AQP5 After HAE Both Western blot analysis and RT-PCR revealed expression of both AQP1 and AQP5 proteins (Fig. 5), and mRNA (Fig. 6) in the lung tissues of the NBA + HAE group significantly decreased compared with those of the NBA group ( p G 0.01). Expressions of both AQP1 and AQP5 proteins and mRNA in the lung tissues of the HBO2P + HAE group were significantly higher than those of the NBA + HAE group
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( p G 0.01). Again, the beneficial effects of HBO2P therapy were significantly ( p G 0.05) reduced in the HBO2P + HSP + HSP70 Ab pretreatment group (Figs. 5 and 6).
Decrease in MAP, HR, SO2, PaCO2, HCO3j, and pH Values After HAE The values of MAP (50 mg vs. 90 mm Hg), HR (250 beats/ min vs. 380 beats/min), SO2 (52% vs. 100%), PaCO2 (22 mm Hg vs. 45 mm Hg), HCO3j (18 mmol/L vs. 27 mmol/L), and pH (7.2 vs. 7.4) in the NBA rats were significantly lower after HAE than in the non-HAE controls. However, HBO2P significantly ( p G 0.05, n = 8 each group) reduced the HAE-induced decrease of MAP (80 mg), HR (350 beats/min), SO2 (80%), PaCO2 (40 mm Hg), HCO3j (25 mmol/L), and pH (7.3). In addition, HSP70 Ab preconditioning significantly ( p G 0.05, n = 8 each group) reduced the beneficial effects of HBO2P in HAE (MAP, 42 mm Hg; HR, 200 beats/min; SO2, 40%; PaCO2, 20 mm Hg; HCO3j, 16 mmol/L; and pH 7.18).
Increase in Lung Levels of Systemic Inflammatory Response Molecules After HAE The values of lung MPO (320-Kg/mg protein vs. 112-Kg/mg protein), and BAL fluid levels of IL-6 (11 pg/mL vs. 52 pg/mL), IL-1A (60 pg/mL vs. 39 pg/mL), and TNF-> (40 pg/mL vs. 8 pg/mL) in the NBA rats were significantly ( p G 0.05, n = 8 each group) higher after HAE than in the non-HAE controls. However, HBO2P significantly ( p G 0.05, n = 8 each group) attenuated the HAE-induced increase of lung MPO (125-Kg/mg protein) and BAL fluid IL-6 (70 pg/mL), IL-1A (41 pg/mL), and TNF-> (15 pg/mL). In addition, HSP70 Ab preconditioning significantly ( p G 0.05, n = 8 each group) attenuated the beneficial effects of HBO2P in HAE (MPO, 334-Kg/mg protein; IL-6, 118 pg/mL; IL-1A, 65 pg/mL; and TNF->, 45 pg/mL).
DISCUSSION
Figure 6. RT-PCR analysis of AQP1 mRNA and AQP5 mRNA in the lungs of NBA rats (n = 8), NBA + HAE rats (n = 8), HBO2P + HAE rats (n = 8), and HBO2P + HSP70 + HAE rats (n = 8). Data are presented as mean (SD). *p G 0.05 for NBA + HAE group versus NBA group; +p G 0.05 for HBO2P + HAE group versus NBA + HAE group; #p G 0.05 for HBO2P + HSP70 Ab + HAE group versus HBO2P + HAE group.
The reduction in barometric pressure and the consequent fall in the PaO2 at higher altitudes lead to a drop of the oxygen saturation, which lead to multiple-organ dysfunction. Indeed, as shown in the present study, 3 days after the initiation of HAE, the non-HBO2P rats had hypoxia, hypotension, bradycardia, acidosis, and tissue up-regulation of both proinflammatory cytokines including IL-1A, IL-6, and TNF-> and an indicator for polymorphonuclear leukocytes accumulation such as MPO activity. In addition, the non-HBO2P animals had higher values of lung edema, neutrophils, and hemorrhage scores and lung water as well as decreased lung levels of both AQP1 and AQP5 protein and mRNA expression. It is believed that a decrease in lung expression of both AQP1 and AQP5 protein and mRNA may play an important role in pulmonary edema.3Y7 Therefore, both the increased values of both the lung edema score and the lung water weight are caused by a decrease in both AQP1 and AQP5 protein and mRNA expression in the non-HBO2P + HAE group rats. Probably the most striking finding of present study is that the preinduction of HSP72 in the lung tissue by HBO2P significantly attenuated HAE-induced increased acute lung injury scores including edema, inflammation, and hemorrhage. The beneficial effects
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of HBO2P in preventing the occurrence of these high mountain sickness syndromes were all attenuated by the inhibition of HSP70 expression by HSP70 Ab pretreatment. Our data strongly indicate that preinduction of HSP70 in the lung tissue before onset of HAE may be one of the key issues. HSP70 is up-regulated by hypoxia and involved in cell protection and survival in acute myocardial stress,16 severe sepsis,17 and ischemic stroke.18 Preinduction of HSP70 by drugs promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain.10 Hypobaric hypoxia preconditioning such as at chromic exposure at a hypobaric hypoxia chamber, has recently been known to attenuate acute lung injury during HAE in rats via up-regulating HSP70.9,19 The data indicate that hypoxic preconditioning may be beneficial for preventing HAE acute lung injury. Indeed, the most popular preventive approach for HAE acute lung injury is gradual ascending,20 which is actually a kind of hypoxic preconditioning. Since hypoxic preconditioning has not been used clinically because of safety concerns, HBO2P seems to be more attractive and easier to be accepted ethically because it has been used safely for various disorders.21Y26 HBO2P increases tissue oxygen levels by a giving 100% oxygen at higher pressure. Upon termination of HBO2P, the body would experience a relative hypoxia because the oxygen level is returned to normal level at 21%. Therefore, repeating HBO2P may produce a cycle of hyperoxia and then hypoxia and head to hypoxia-inducing factor 1> (HIF-1>) accumulation. Several studies have demonstrated that ischemic tolerance induced by HBO2P involves the up-regulation of HIF-1> in rats.27Y29 Our present data further demonstrated that HBO2P, like hypoxia preconditioning, significantly reduces acute lung injury in rats during simulated HAE by up-regulating lung HSP70. As shown in the present study, HAE caused decreased lung expression of both AQP1 and AQP5 both at protein and at gene level accompanied by pulmonary edema in rats. Pearson correlation analysis revealed that there was an inverse correlation between lung injury scores and mRNA and protein expression of AQP1 and AQP5 in the lungs of rats.12 Lower mRNA and lower protein expression of both AQP1 and AQP5 may induce reduction of transcellular rate of excess water removal and result in pulmonary edema formation,6 which could be significantly reversed by HSP70-mediated HBO2P. AQP1 and AQP5 are mainly distributed in microvascular endothelia and in alveolar epithelia, respectively.30 It can be derived from the foregoing statements that HSP70-mediated HBO2P may attenuate HAE-induced pulmonary edema by increasing both mRNA and protein expression of AQP1 and AQP5 in the lungs of rats. The levels of IL-1A in the BAL fluid are elevated in patients with adult respiratory distress syndrome.31 Both TNF-> and IL-6 are believe to be an important mediator in the early cascade of endotoxin-induced lung inflammation.32 As demonstrated in the present results, the BAL levels of IL-1A, TNF->, and IL-6 and the lung levels of MPO are elevated by HAE. Besides lung inflammation, other parameters such as hypoxia, hypotension, bradycardia, and acidosis may be related to the occurrence of acute lung damage in HAE, which can be prevented by HSP70-mediated HBO2P. A comparison of HBO2P versus hypoxic preconditioning9 was performed in rats and found that HBO2P + HAE 590
elicited similar preconditioning efficacy in reducing pulmonary edema. However, hypoxic preconditioning has not been used clinically because of safety concerns. In contrast, HBO2P seems to be more attractive and easier to be accepted ethically because it has been used safely for various disorders.23,26 Our experiment has several limitations. First, we used very low numbers of rats. Second, the rodent model of HAE resembles the pathophysiology response of humans only to a certain point. It is difficult to extrapolate the data from our rat model to predict exactly the human response. However, our approach is promising and deserves further preclinical and clinical study to assess elements of the pathophysiology of critical illness. In summary, HBO2P, in addition to inducing lung overexpression of HSP70, significantly attenuated the HAEinduced hypotension, hypoxia, bradycardia, acidosis, increased BAL fluid levels of proinflammatory cytokines, increased lung levels of MPO (an indicator for leukocytes accumulation), increased lung water weight, decreased lung levels of both AQP1 and AQP5 protein and mRNA expression, and increased scores of lung edema, neutrophils, and hemorrhage. The beneficial effects of HBO2P in inducing overexpression of lung HSP70 as well as in preventing the HAE-induced hypotension, bradycardia, hypoxia, acidosis, lung inflammation, and increased scores of lung edema, neutrophils, and hemorrhage could be significantly reduced by HSP70 Ab preconditioning. These results suggest that high-altitude pulmonary edema can be prevented by HSP70-mediated HBO2P in rats. AUTHORSHIP M.-C.T., H.-J.L., and T.C.-Y.T. participated in the design of the study and obtained the financial support. M.-T.L., K.-C.N., and C.-P.C. made contribution to the literature search, data collection/analysis/interpretation, and paper writing. T.C.-Y.T. and C.-P.C. oversaw the study and gave final approval of the version to be published.
DISCLOSURE This study was supported by grants from the Chi Mei Medical Center (Tainan, Taiwan), the National Science Council of the Republic of China (Taipei, Taiwan), and the Department of Health of the Republic of China (Taipei, Taiwan).
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