Lung tissue is one of the most vulnerable tissues subject to endotoxemia. Endotoxemia readily causes acute lung injury (ALI), which can progress to acute respiratory distress syndrome (ARDS) and subsequent multiple organ dysfunction.1 ALI is characterized by intense inflammation,2 with neutrophil accumulation, interstitial edema, disruption of endothelial and epithelial integrity, and leakage of protein into the alveolar space.3,4 Despite several decades of research, the ultimate mechanism of ALI induced by endotoxin remains to be determined, the mortality from ALI remains unacceptably high, and the therapeutic regimen requires further investigation.
Volatile anesthetics such as isoflurane are frequently used in clinical routine for anesthesia during surgical interventions. More recently, there has been increasing use of volatile anesthetics for sedation in intensive care units.5 Growing evidence indicates that isoflurane, in addition to having anesthetic features, also has antiinflammatory properties, decreasing the cytokine release from alveolar macrophages and monocytes after lipopolysaccharide (LPS) stimulation.6–12
Additionally, the preconditioning properties of volatile anesthetics, which contribute to tissue protection in cardiac surgery, have been well described in the literature and are established in clinical practice.13 In the lung, however, protective effects have not been well characterized. In isolated rat lungs, pretreatment with either isoflurane or sevoflurane diminished early reperfusion-induced lung edema, vascular permeability, and production of nitric oxide (NO) metabolites.14,15In vivo studies showed that isoflurane pretreatment protects the vasculature endothelium associated with LPS-induced inflammation and reduces endotoxin-induced polymorphonuclear leukocyte (PMN) recruitment and microvascular protein leakage.6,7 However, the effects of isoflurane pretreatment on proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and survival in severe endotoxin-induced ALI, have not been studied. Furthermore, inducible NO synthase (iNOS)-mediated NO release has been implicated in the pathogenesis of ALI and ARDS due to various causes.16–18 In this study, the effect of isoflurane preconditioning on pulmonary proinflammatory cytokine release and mortality of rats induced by severe sepsis was investigated.
Experimental Procedure and Animal Model of ALI
Male Sprague-Dawley rats (250-300 g) were purchased from the Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China) and were housed in air-filtered, temperature-controlled units with access to food and water ad libitum. The experimental protocols were approved by the institutional animal care committee and complied with National Institutes of Health guidelines for animal experimentation. The rats were allowed to stabilize and were then randomized to 1 of 4 groups (n = 8 for each group): 1) sham rats (injected intraperitoneally [IP] with saline) pretreated with vehicle (100% O2) (sham-vehicle); 2) sham rats pretreated with isoflurane (sham-ISO); 3) LPS rats (injected IP with LPS) pretreated with vehicle (vehicle-LPS); and 4) LPS rats pretreated with isoflurane (ISO-LPS). The isoflurane groups received isoflurane for 30 min immediately before LPS or saline, as described previously.6,7 Isoflurane 1.4% was delivered through the tracheostomy using a rodent ventilator (ALC-V8, Shanghai Alcott Biotech Co., Shanghai, China) at a rate of 75 breaths/min and a tidal volume of 3 mL. The end-tidal isoflurane concentration was measured with a gas analyzer (Smart Anesthesia Multi-gas Module, Wipro GE Healthcare, UK). The sham-vehicle and vehicle-LPS rats’ lungs were ventilated with 100% O2 for 30 min before saline or LPS administration. LPS (40 mg/kg, Escherichia coli, serotype 0111, B4; Sigma, St. Louis, MO) dissolved in 0.5 mL saline or saline alone was injected IP as described previously.19 We measured the LPS content in the saline by using the Limulus amoebocyte lysate test (E-Toxate, Sigma). The saline used in this study contained <15 pg LPS/mL. The experiments were conducted such that initiation of isoflurane (or 100% O2) was timepoint 0; therefore, LPS was administered at the timepoint equal to 30 min. All animals were anesthetized with sodium pentobarbital (40 mg/kg, IP), and anesthesia was maintained by additional injections (15 mg/kg, IP) hourly during hemodynamic monitoring. The rats were placed supine on a heating blanket and under a heating lamp to maintain a temperature of 37°C throughout the experiment. A polyethylene catheter was placed in the femoral artery for monitoring mean arterial blood pressure (MAP), heart rate, and blood sampling. MAP was recorded with a polygraph recorder (Dash 4000, GE Medical Systems Information Technologies, UK). The arterial catheter was infused with saline at a rate of 0.5 mL/h. Arterial blood gas analysis was performed at the end of the 6 h.
Water Content Determination and Histologic Examination
The animals were killed by decapitation, and the following variables were analyzed. To evaluate the severity of ALI, formation of endotoxin-induced pulmonary edema was determined by wet/dry weight ratios of the lungs at 6 h in the rats that were not subject to histological examination (n = 4). Lungs were removed, blotted dry, and weighed. They were then incubated at 60°C for 72 h and reweighed. The wet/dry ratio was then calculated. The morphologic alterations in the lungs were examined from individual rats at 6 h after LPS administration. The lungs were fixed with 4% paraformaldehyde and were embedded in paraffin. Paraffin sections 4 μm thick were stained with hematoxylin and eosin for examination by light microscopy. A scoring system to grade the degree of lung injury was used, based on the following histologic features: edema, hyperemia and congestion, neutrophil margination and tissue infiltration, intraalveolar hemorrhage and debris, and cellular hyperplasia. Each feature was graded as absent, mild, moderate, or severe, with a score of 0-3.20 Grading was performed by a blinded pathologist. The lung injury score was calculated by adding the individual scores for each category.
Capillary Protein Leakage
Pulmonary microvascular permeability was determined using the Evans blue dye extravasation technique at 6 h. Evans blue (30 mg/kg; Sigma) was injected IV 30 min before death. The lungs were perfused free of blood and removed, and Evans blue content in lung tissue was determined spectrophotometrically at an optical density of 620 nm.21
In separate experiments (n = 20 per group), isoflurane or vehicle was administered for 30 min using a rodent ventilator before LPS administration as described above. These groups of rats were not subject to hemodynamic monitoring or tissue sampling; they were then allowed to recover in room air and were frequently observed by dedicated research personnel to determine 10-day survival statistics.
Determination of Pulmonary Nitrite/Nitrate, TNF-α, IL-1β, and IL-6
In separate rats (n = 6 each group) at 6 h, the left lung was removed and snap frozen in liquid nitrogen, then stored at −80°C for subsequent analysis. To determine the NO concentrations in collected samples, we chose to measure the sums of stable NO metabolites, and nitrite and nitrate concentrations using chemiluminescence, as described previously.22 TNF-α, IL-1β, and IL-6 concentrations were measured using a commercially available enzyme-linked immunoassay kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).
Real-Time Quantitative Polymerase Chain Reaction
Real-time quantitative polymerase chain reaction (PCR) estimation of mRNA levels was performed as described previously.23 Primers were commercially obtained (Shanghai Sangon Biologic Engineering & Technology and Service Co., Shanghai, China). The following primer sequences were used: for iNOS (forward: 5′-GGTGGAAGCAGTAACAAAGGA-3′; reverse: 5′-GACCTGATGTTGCCGTTGTTG-3′; expected size 231 bp) and for β-actin (forward: 5′-GCTCGTCGTCGACAACGGCTC-3′; reverse: 5′-CAAACATGATCTGGGTCATCTTCTC-3′; expected size 353 bp). Amplification and detection were performed using an ABI PRISM 7700 detection system (Applied Biosystems, Foster City, CA) as follows: 1 cycle at 95°C for 10 s, 40 cycles at 95°C for 5 s, and 1 cycle at 60°C for 30 s. Real-time PCR was performed in triplicate reactions with 20 ng complementary DNA in a final volume of 10 μL containing 1xSYBR Green Master Mix and 200 nM of both primers. Agarose gel electrophoresis, purification, and DNA sequencing confirmed the identity of the PCR products. The amount of gene transcript was measured using the comparative (2−ΔΔCT) method described by Applied Biosystems in their instructions. Reference gene β-actin, which was not altered by isoflurane or any other treatment applied in this study (data not shown), was used for normalization of the expression data.
Immunoblotting assays were performed as described previously.23 Briefly, 100 μg of protein was loaded onto a 10% sodium dodecyl sulfate/polyacrylamide gel, and after electrophoresis, it was blotted onto nitrocellulose membranes. The primary rabbit anti-rat iNOS (Abcam, Cambridge, MA) and β-actin monoclonal antibody (Sigma) were used at 1:500 or 1:4000 dilution. The anti-rabbit immunoglobulin G secondary antibody (KPL, Gaithersburg, MD) was used at 1:2000 dilution, and the signal was analyzed by enhanced chemiluminescence (Chemicon, Billerica, MA).
All data were tested for normality using the Shapiro-Wilk normality test and were determined to have a normal distribution. Homogeneity of variance was tested using Bartlett’s test. When Bartlett’s test indicated that the group comparisons had equal variance, 1-way analysis of variance (ANOVA) was used. Hemodynamic changes over time from baseline within each group were determined by repeated-measures ANOVA. Differences between the groups at each timepoint were evaluated by 1-way ANOVA and a post hoc Tukey test. The data are expressed as means ± sd. Nonparametric statistical analysis (Kruskal-Wallis 1-way ANOVA on ranks) was used for statistical evaluation of the histopathologic scores because of unequal variance. The survival rate was estimated by the Kaplan-Meier method and compared by log-rank test. Differences in values were considered significant at P < 0.05.
Alterations in Arterial Blood Pressure and Heart Rate
Isoflurane caused a decrease in MAP at 1-2 h. The administration of LPS caused significant systemic hypotension and tachycardia throughout the 6-h observation (Fig. 1). Isoflurane preconditioning alleviated the decrease in MAP but had little effect on the tachycardia in LPS rats. Isoflurane alone did not affect heart rate.
Effects of Isoflurane Preconditioning on ALI
Photomicrographs showed that IP administration of LPS caused infiltration of inflammatory cells into the lung interstitium and alveolar spaces, alveolar wall thickening, and intraalveolar exudation at 6 h after LPS administration (Fig. 2A). However, isoflurane preconditioning attenuated these histological changes. Semiquantitative assessment using a lung injury score demonstrated that the degree of lung injury in the ISO-LPS group was lower than that in the vehicle-LPS group at 6 h after LPS administration (Fig. 2B). The lung wet/dry weight ratio was significantly increased at 6 h after LPS administration (Fig. 3A). In terms of capillary permeability, Evans blue extravasation showed that LPS induced a significant increase in Evans blue leakage into the lung (Fig. 3B). When lungs were pretreated with isoflurane, both capillary leakage and lung edema were reduced significantly at 6 h after LPS administration (Fig. 3). Isoflurane alone did not affect these variables in sham rats.
Blood Gases and Acid-Base Status
Arterial blood gas analysis demonstrated that LPS caused a significant decrease in pH, Pao2, SaO2, and base excess, indicating that LPS caused impairment of pulmonary functions. Preconditioning with isoflurane in LPS rats improved pulmonary function (Table 1). Isoflurane alone did not affect the arterial blood gases and acid-base status.
Isoflurane Preconditioning Protects Against Endotoxin-Induced Death
The survival rate after LPS with vehicle administration was 40% on Day 2 and decreased to 20% on Days 4-10 (Fig. 4). Preconditioning with isoflurane, however, improved the survival rate to 70% on Day 2 and to 55% on Days 4-10, which was significantly higher than that for the LPS group treated with vehicle (P < 0.05; Fig. 4).
Lung Inflammatory Response to Endotoxin Is Attenuated in Rats With Isoflurane Preconditioning
As compared with the sham-vehicle group, 6 h of endotoxin induced a large increase of pulmonary NO metabolites, TNF-α, IL-1β, and IL-6 levels. Isoflurane preconditioning reduced the induction of nitrate/nitrite, TNF-α, IL-1β, and IL-6 by LPS. The values were significantly lower than those caused by LPS but still higher than the values in the sham-vehicle group (Fig. 5).
Isoflurane Preconditioning Inhibits LPS-Induced iNOS Expression in Lungs
Several experimental studies have indicated that NO release through the iNOS isoform is responsible for the pathogenesis of ALI or ARDS induced by endotoxemia.24–26 To study whether isoflurane pretreatment has any effect on iNOS gene expression in the lungs, iNOS mRNA and protein levels were determined 6 h after LPS administration. As demonstrated in Figure 6, iNOS mRNA and protein levels increased dramatically 6 h after LPS stimulation. Pretreatment with isoflurane, however, resulted in a significant decrease in the expression levels of the iNOS gene. This result indicates that isoflurane pretreatment inhibits the activation of the iNOS gene induced by LPS.
In this study, isoflurane preconditioning attenuated the decrease in MAP associated with LPS from 2 to 6 h but had no effect on MAP in sham-ISO rats, suggesting that isoflurane inhibits the effects of LPS rather than acting through mechanisms that directly increase MAP, and also indicating that isoflurane preconditioning is protective of the vasculature during LPS-induced inflammation, as described previously.6 Nevertheless, it is also possible that the increase in MAP reflects an increase in cardiac output secondary to myocardial protection, an effect that occurs with isoflurane preconditioning.27 Isoflurane pretreatment prevented the acidosis at 6 h associated with LPS. This was secondary to a trend toward a decrease in both the base deficit and arterial carbon dioxide partial pressure. Isoflurane pretreatment also increased Pao2 and SaO2 in the LPS rats, suggesting improved lung function. ALI was histologically confirmed in rats subjected to LPS administration. LPS also increased the lung water content and protein leakage, indices of ALI. Isoflurane preconditioning significantly attenuated these abnormalities, suggesting that isoflurane preconditioning ameliorates LPS-induced ALI in rats.
Although the powerful antiinflammatory effect of isoflurane is increasingly recognized, there is no research investigating whether isoflurane pretreatment alters mortality from severe sepsis. Isoflurane anesthesia has been shown to improve survival in a cecal and ligation puncture mouse model.28 Our results showed that all rats survived LPS toxicity for at least 6 h, but 60% of animals that received LPS died within 48 h, and isoflurane pretreatment significantly reduced the mortality rate to 30%. The surviving rats showed less severe lung injury than those evaluated at 6 h, suggesting a partial recovery from lung injury if animals survived. Therefore, isoflurane preconditioning may accelerate recovery from the ALI induced by LPS and may in turn enhance the animal’s chance of survival.
It is now widely accepted that ALI in the setting of sepsis is the result of the actions of an integrated network of soluble inflammatory mediators and a variety of inflammatory cells.29 In fact, in this study, pulmonary TNF-α, IL-1β, and IL-6 levels were markedly increased in rats administered LPS, and isoflurane pretreatment attenuated the accumulation of these cytokines. These results are supported by our earlier in vitro findings that isoflurane preconditioning reduced LPS-stimulated secretion of TNF-α in murine macrophages.30 TNF-α, a proinflammatory cytokine, participates in several important processes involved in the inflammatory response.31 On the other hand, IL-6 plays a pivotal role in neutrophil migration and serves as both a marker and a mediator for the severity of sepsis.32 There is evidence that the cytokines TNF-α and IL-1β are involved in a reduction of alveolar ion and the associated fluid transport and hence pulmonary edema clearance.33 TNF-α has been particularly strongly implicated in the pathogenesis of pulmonary edema, and its effect is mediated by NO.34 TNF-α-induced NO leads thereby to a reduction of the activity of the alveolar epithelial sodium channel and the basolateral sodium potassium adenosine triphosphatase, which are essential for alveolar sodium and fluid transport.33 Additionally, isoflurane preconditioning inhibits LPS-induced pulmonary NO accumulation and iNOS gene expression. Several reports have indicated that induction of the expression of iNOS and subsequently the overproduction of NO are involved in the pathogenesis of ALI or ARDS in animals or humans with endotoxemia.24,35 It has been demonstrated that iNOS activity during an inflammatory response is predominantly regulated at the transcriptional level, although there may be posttranscriptional and posttranslational regulation as well.36 In this regard, our results suggest that attenuation of the activation of an iNOS-NO pathway may be the mechanism for isoflurane preconditioning to induce the protection.
Isoflurane has been shown to induce a preconditioning effect against LPS-induced inflammation and injury in lungs and vasculature in rats and mice.6,7 Our findings that isoflurane pretreatment attenuates pulmonary proinflammatory cytokine release and iNOS-NO pathway activation may provide an explanation for the protection of isoflurane. Moreover, the fact that isoflurane pretreatment increased the 10-day survival rate after LPS administration confirms the notion that the protection conferred by isoflurane was global and not restricted to a particular organ. Thus, isoflurane pretreatment may be an effective treatment for severe sepsis-induced ALI.
Increased vascular permeability and PMN recruitment are 2 key factors in lung injury, and there is evidence that regulation of both is different and one might occur without the other.37 For example, vascular protein leakage is dependent on iNOS from alveolar macrophages; however, PMN recruitment is not.38 Although we did not specifically identify the nature of inflammatory cells in our histological examination, it has been shown that pretreatment with isoflurane reduced PMN recruitment and protected from lung damage when administered within certain time windows.7 Both attenuation of vascular permeability and PMN recruitment may thus be involved in the mechanisms of the protection of isoflurane.
In conclusion, isoflurane preconditioning, at a clinically relevant concentration, caused an improvement of endotoxin-induced hypotension and reduced lung water content and protein leakage. Isoflurane preconditioning also decreased pulmonary levels of nitrate/nitrite, TNF-α, IL-1β, and IL-6, and the lung pathology changes in severe sepsis-induced ALI. Furthermore, isoflurane preconditioning improved survival and pulmonary function of rats after endotoxin administration. Thus, isoflurane preconditioning may provide a therapeutic avenue to be explored for the treatment of severe sepsis-induced ALI. Early protection seems to be mediated partly through inhibition of iNOS-NO pathway activation.
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