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Basic Science Aspects

SUBANESTHETIC DOSE OF ISOFLURANE PROTECTS AGAINST ZYMOSAN-INDUCED GENERALIZED INFLAMMATION AND ITS ASSOCIATED ACUTE LUNG INJURY IN MICE

Mu, Jinglan*; Xie, Keliang*†; Hou, Lichao*; Peng, Daorong; Shang, Lei§; Ji, Genlin*; Li, Juntang; Lu, Yan*; Xiong, Lize*

Author Information
doi: 10.1097/SHK.0b013e3181cffc3f

Abstract

INTRODUCTION

Multiple organ dysfunction syndrome (MODS) is one of the leading causes of death in the intensive care unit (ICU). Lung is frequently the first failing organ during the development of MODS. It is reported that isoflurane (ISO), at anesthetic concentration ranging from 1.2% to 2.5%, reduces inflammatory cytokines and protects against sepsis and organ injuries including lung injury (1-5). However, anesthetic dose of ISO is limited to be applied to critically ill patients who may not tolerate the hemodynamic effects of anesthesia including vasodilation, myocardial depression, and bradycardia (6).

Inhaling ISO at a concentration of less than 1% (0.7 minimum alveolar concentration [MAC]) has been used in ICU patients for facilitating mechanical ventilation and sedation (7-9). Sedative dose of ISO selectively suppresses consciousness while leaving many autonomic functions intact, interferes very little with hemodynamics, and avoids anesthesia-related side effects (7). However, it has not been reported whether low-concentration (<1%, <0.7 MAC) ISO has protective effects on MODS including lung injury.

Recent studies show that systemic inflammatory response syndrome (SIRS) as well as the disorder of oxidant/antioxidant has been considered as the important mechanism in the development of MODS, and the improvement in the activities of endogenic antioxidant enzymes definitely reduces oxidative injuries and protects against MODS (2, 10, 11). It is reported that zymosan (ZY) has been used as a tool to induce SIRS/MODS in many studies using animal models (12, 13). The present study was designed to investigate the effects of ISO at subanesthetic dose (0.7%, 0.5 MAC) on ZY-induced generalized inflammation and its associated lung injury in mice and the roles of antioxidant enzymes in the effects.

MATERIALS AND METHODS

Animals

Male ICR (imprinting control region) mice (specific pathogen-free) were provided by the Laboratory Animal Center of Fourth Military Medical University. Animals were housed in a standard housing condition (in the plastic boxes with free access to food and water, 12-h light/dark cycle at 20°C-22°C). All experimental protocols were approved by the Institutional Animal Care and Use Committee of Fourth Military Medical University and were performed in accordance with the National Institutes of Health guidelines.

Zymosan-induced generalized inflammation

Zymosan (Sigma Chemical Co, St Louis, Mo) solution was prepared in normal saline (NS) to a final concentration of 25 mg/mL and was sterilized at 100°C for 80 min. All suspensions were freshly made before being used. Generalized inflammation model was induced by an aseptic i.p. injection of ZY at a dose of 1 g/kg (12, 14, 15). Same volume of NS was injected through the same route as the control.

Isoflurane treatment

The animals were put in a sealed plexiglass chamber with inflow and outflow outlets. Isoflurane was delivered by air into the chamber through a tube at a rate of 4 L/min, and carbon dioxide was removed from the chamber gases with Baralyme. The concentration of ISO in the outflow hose of the chamber was continuously monitored with a gas analyzer (Brüel & Kjae, Naerum, Denmark). Isoflurane concentration was maintained at the predetermined level during the treatment. The pentobarbital (PT) treatment was performed in the animal cages. The temperature of the room and the chamber was maintained at 20°C to 22°C.

Experimental design

Effects of 0.7% ISO treatment on the survival rate in ZY-challenged mice

One hundred sixty animals were randomly divided into eight groups (n = 20 per group): NS, NS + PT, NS + 0.7% ISO, NS + 1.4% ISO, ZY, ZY + PT, ZY + 0.7% ISO, and ZY + 1.4% ISO groups.

Generalized inflammation was induced in all the animals of ZY, ZY + PT, ZY + 0.7% ISO, and ZY + 1.4% ISO groups. The animals from the other four groups were given the same volume of NS as the control.

Isoflurane treatment was performed in the NS + 0.7% ISO, NS + 1.4% ISO, ZY + 0.7% ISO, and ZY + 1.4% ISO groups, with the animals exposed to 0.7% or 1.4% ISO for 1 h starting at 1 and 6 h after the NS or ZY injection, respectively. As a control, the animals from NS + PT and ZY + PT groups were given an aseptic i.p. injection of PT (12.5 mg/kg) at 1 and 6 h after the NS or ZY injection, respectively. The survival rate was observed on days 1, 2, 3, and 7 after the NS or ZY administration (Fig. 1).

Fig. 1
Fig. 1:
The schematic diagram for experimental protocols. The animals were randomly divided into eight groups (n = 20 per group): NS, NS + PT, NS + 0.7% ISO, NS + 1.4% ISO, ZY, ZY + PT, ZY + 0.7% ISO, and ZY + 1.4% ISO groups. Isoflurane treatment was performed in the NS + 0.7% ISO, NS + 1.4% ISO, ZY + 0.7% ISO, and ZY + 1.4% ISO groups, with the animals exposed to 0.7% or 1.4% ISO for 1 h starting at 1 and 6 h after the NSor ZY injection, respectively. As a control, the animals from NS + PT and ZY + PT groups were given an aseptic i.p. injection of sodium PT (12.5 mg/kg) at 1and 6 h after the NS or ZY injection, respectively. The survival rate was observed on days 1, 2, 3, and 7 after the NS or ZY administration.

Effects of 0.7% ISO treatment on generalized inflammation-associated lung injury in mice

Based on our previous experiments, the levels of serum inflammatory cytokines including TNF-α and IL-10 are increased most, and abnormal changes of lung histopathology are obvious at 24 h after the ZY injection (14). To study the effects of 0.7% ISO treatment on ZY-induced lung injury, additional animals were used to observe lung myeloperoxidase (MPO) activity (n = 6 per group), lung wet/dry (W/D) weight ratio (n = 6 per group), protein concentration in bronchoalveolar lavage fluid (BALF; n = 6 per group), and lung histopathology (n = 6 per group) at 24 h after the NS or ZY injection. The grouping method and experimental protocols were the same as it was described above.

Effects of 0.7% ISO treatment on cytokines as well as oxidant and antioxidant system in ZY-challenged mice

Additional animals were used in this study. The grouping method and experimental protocols were the same as it was described above. According to our previous experiments (14), serum and lung tissue proinflammatory cytokine TNF-α (n = 6 per group), antioxidant enzyme superoxide dismutase (SOD) (n = 6 per group), catalase (CAT) (n = 6 per group), and oxidation product 8-iso-prostaglandin F2α (8-iso-PGF2α; Cayman Chemical, Ann Arbor, Mich) (n = 6 per group) were detected at 24 h after the NS or ZY injection.

Effects of CAT activity inhibitor on the protection of 0.7% ISO treatment in ZY-challenged mice

To investigate the effects of CAT activity antagonist 3-amino-1,2,4-triazole (AT; Sigma-Aldrich Co, St Louis, Mo) on the protection of ISO treatment against ZY-induced generalized inflammation, animals were randomly divided into four groups (n = 20 per group): ZY, ZY + 0.7% ISO, ZY + 0.7% ISO + 0.09 g/kg AT, and ZY + 0.09 g/kg AT groups.

Generalized inflammation was induced in all animals. Isoflurane treatment was performed in the ZY + 0.7% ISO and ZY + 0.7% ISO + 0.09 g/kg AT groups with animals exposed to 0.7% (0.5 MAC) ISO for 1 h starting at 1 and 6 h after the ZY injection, respectively.

In the ZY + 0.7% ISO + 0.09 g/kg AT and ZY + 0.09 g/kg AT groups, animals were intraperitoneally administered with 0.09 g/kg AT. In the animals of ZY + 0.7% ISO + 0.09 g/kg AT groups, an i.p. injection of AT was performed before 0.7% ISO inhalation, which was repeated before the second 0.7% ISO inhalation (16). In the ZY + 0.09 g/kg AT group, AT was given at the same time points as that in the ZY + 0.7% ISO + 0.09 g/kg group. The survival rate was observed on days 1, 2, 3, and 7 after the NS or ZY administration.

Detection of TNF-α

At the predetermined time points, animals were anesthetized with PT 50 mg/kg, and blood samples were collected by cardiac puncture and then clotted for 30 min at 25°C. The serum was separated by centrifugation at 3,000g for 15 min at 4°C, aliquoted, and stored at −80°C until assayed. After the blood had been sampled, lung was removed immediately. Lung tissue homogenates were prepared in chilled phosphate buffer (0.1 M, pH 7.4) and were centrifuged at 10,000g at 4°C for 10 min. The supernatants were collected, aliquoted, and stored at −80°C until the analysis.

The levels of serum and lung TNF-α were detected by using specific enzyme-linked immunosorbent assay kits, respectively (TNF-α; R&D Systems Inc, Minneapolis, Minn) with microplate reader (CA 94089; Molecular Devices, Sunnyvale, Calif). All standards and samples were run in duplicate.

Detection of 8-iso-PGF2α

Measurement of 8-iso-PGF2α, free radical-catalyzed products of arachidonic acid, seems to offer a reliable approach for quantitative measurement of oxidative stress status in vivo (17). The levels of serum and lung 8-iso-PGF2α were detected by using specific enzyme-linked immunosorbent assay kits (8-iso-PGF2α; Ann Arbor, Mich) with microplate reader (CA 94089; Molecular Devices). All standards and samples were run in duplicate.

Assay of enzymatic activity

The serum and lung homogenates obtained above were also used for enzymatic activity assay. The activities of SOD and CAT were measured by using commercial kits purchased from Cayman Chemical Company (Ann Arbor, Mich). According to the manufacturer's instructions, total SOD activity was assayed by detecting superoxide radicals generated by xanthine oxidase and hypoxanthine. The reaction was monitored at 450 nm, and 1 U of SOD activity was defined as the amount of enzyme, which was needed to exhibit 50% dismutation of superoxide radical. The CAT activity was assayed by measuring the reduction of hydrogen peroxide at 540 nm, and 1 U of it was defined as the amount of enzyme that would cause the formation of 1.0 nmol of formaldehyde per minute at 25°C. All spectrophotometric readings were performed by using a spectrophotometer (DU 640B; Beckman, Fullerton, Calif). All assays were conducted in triplicates. The lung protein concentration was determined by using a standard commercial kit (Bio-Rad Laboratories, Hercules, Calif).

Measurement of lung MPO activity

Homogenated lung supernatants were prepared for detecting the activity of MPO, an indicator of neutrophil infiltration into the lung tissue, which was measured as previously reported (18). Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 μmol of peroxide per minute at 37°C and was expressed in milliunit per gram weight of wet tissue. The change in absorbance was measured spectrophotometrically at 590 nm by spectrophotometer (DU 640B; Beckman). The activity of MPO was measured by using commercial kits purchased from Cayman Chemical Company.

Lung W/D ratio

To quantify the magnitude of pulmonary edema, we evaluated lung W/D weight ratio. The harvested wet left lung was weighed and then was placed in an oven for 24 h at 80°C and weighed when it was dried.

Bronchoalveolar lavage and total protein assay

Animals were subjected to bronchoalveolar lavage to collect BALF by the methods described previously (19). Animals were anesthetized, and the trachea and lung were exposed by thoracotomy. A cannula was inserted into the trachea and was ligated by using a thread. Phosphate-buffered saline (pH 7.4) was injected into the lung via a syringe fitted with the tracheal cannula. The phosphate-buffered saline was allowed to stay in the lung for 30 s and then was instilled with the help of syringe, which was repeated for three times with the same solution (0.3 mL per lavage). Lavage samples were centrifuged at 1,500g for 10 min at 4°C. A supernatant was stored at −20°C. Total protein concentration in BALF was analyzed by the method described by Lowry et al. (20). Bovine serum albumin was used as a standard.

Lung histological observations

Lungs were taken at 24 h after the ZY or NS injection for observing histopathological changes. The tissue samples were fixed with 10% formalin for 48 h at room temperature, embedded in paraffin, and sectioned at 4- to 6-μm thickness. After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin. Based on the scoring standard in Supplemental Table 1 (see Table, Supplemental Digital Content 1, https://links.lww.com/SHK/A42) (14), the lung histological slides were blindly read and scored by two experienced pathologists.

Statistical analysis

The histopathological scores are expressed as median (range), and the survival rates are expressed as percentage. The measurement data are expressed as the mean (SD). The intergroup differences of antioxidant enzymatic, biochemical parameter, and inflammatory cytokines were tested by one-way ANOVA followed by LSD test for multiple comparisons. Survival data were calculated by Fisher exact probability test. The intergroup differences of histopathological scores were tested by Kruskal-Wallis H method followed by Nemenyi test for multiple comparisons. The statistical analysis was performed with SPSS 16.0 software (SPSS Inc, Chicago, Ill). In all tests, P < 0.05 was considered statistically significant.

RESULTS

0.7% ISO improved the survival rate in ZY-challenged mice

In the PT-treated groups, animals came to sleep within 5 min after PT treatment and awaked at 1 h and 10 min after PT treatment without any stimuli. In the 1.4% ISO-treated groups, animals became unmoved within 5 min after starting 1.4% ISO inhalation and awaked at 10 min after 1 h of 1.4% ISO treatment without any stimuli. In the 0.7% ISO-treated groups, animals became unmoved within 10 min after starting 0.7% ISO inhalation and started moving immediately after 1 h of 0.7% ISO treatment without any stimuli.

As was shown in Figure 2, the survival rate after ZY injection was 20% on day 1 and decreased to 10% on days 3 to 7 (P < 0.05 vs. NS group). Treatment with PT had no significant effects on survival rates on days 2 to 7 compared with those in the ZY group (P > 0.05); 0.7% ISO treatment increased the survival rates to 45% on days 3 to 7, which were significantly higher than those in the ZY (P < 0.05). However, the survival rates in the 1.4% ISO group on day 1, day 2, and days 3 to 7 were significantly higher (100%, 85%, and 80%) compared with those in the ZY group (P < 0.05). In the NS, NS + PT, NS + 0.7% ISO, and NS + 1.4% ISO groups, all animals survived during the observation period (Fig. 2).

Fig. 2
Fig. 2:
Isoflurane treatment improved the survival rates in ZY-stimulated mice. The grouping methods and experimental protocols were the same as in Figure 1. The values are expressed as survival percentage (n= 20 for each group). *P < 0.05 vs. NS group; P < 0.05 vs. ZY group; P<0.05 vs. ZY + PT group; § P < 0.05 vs. ZY + 0.7% ISO group.

0.7% ISO attenuated lung injury in ZY-challenged mice

As was shown in Figures 3 and 4, ZY injection induced lung injury, which was assessed by lung MPO activity, lung W/D ratio, and protein concentration in BALF, as well as lung histopathology.

Fig. 3
Fig. 3:
Isoflurane treatment attenuated lung injury in ZY-stimulated mice. A, Lung MPO activity (n = 6 for each group). B, Lung W/D ratio (n = 6 for each group). C, Protein in BALF (n = 6 for each group). The grouping methods and experimental protocols were the same as in Figure 1. The values are expressed as mean (SD). *P < 0.05 vs. NS group; P < 0.05 vs. ZY group; P < 0.05 vs. ZY + PT group; § P < 0.05 vs. ZY + 0.7% ISO group.
Fig. 4
Fig. 4:
Isoflurane treatment prevented lung histopathological changes in ZY-stimulated mice. The lungs were stained with hematoxylin-eosin (original magnification ×20). A, NS group. B, ZY group. C, ZY + 0.7% ISO group. D, ZY + 1.4% ISO group. E, ZY + PT group.

Zymosan caused a significant increase in lung MPO activity, lung W/D ratio, and protein concentration in BALF (P < 0.05 vs. NS group; Fig. 3). These abnormal changes were partly reversed by 0.7% ISO treatment (P < 0.05 vs. ZY group; Fig. 3). Pentobarbital treatment decreased lung MPO activity and lung W/D ratio (P < 0.05 vs. ZY group; Fig. 3, A and B), but had no any significant effect on protein concentration of BALF (P > 0.05 vs. ZY group; Fig. 3C).

Isoflurane at 1.4% also significantly decreased lung MPO activity, lung W/D ratio, and protein concentration in BALF (P < 0.05 vs. ZY group; Fig. 3), which have statistical significance compared with those with 0.7% ISO treatment (P < 0.05; Fig. 3).

In terms of histopathology, lung injury, which was characterized by alveolar wall thickening, infiltration of neutrophils into the lung interstitium and alveolar space as well as alveolar hemorrhage was present in mice injected with ZY (Fig. 4). As was shown in Table 1, the histopathological score for lung in ZY group was 3.5, much higher than that in NS group (P < 0.05). Both 0.7% and 1.4% ISO treatment significantly decreased the histopathological score for lung compared with those in ZY group (P < 0.05; Table 1) and ZY + PT group (P < 0.05; Table 1). However, the histopathological score for lung in ZY + PT group had no statistically significant difference compared with that in ZY group (P > 0.05; Table 1). Isoflurane at 1.4% had better protective effect on ZY-induced lung injury than 0.7% ISO did. No statistically significant difference existed in lung MPO activity, lung W/D ratio, and protein concentration in BALF, as well as lung histopathological score (data were not shown) between the NS, NS + PT, and NS + ISO groups.

TABLE 1
TABLE 1:
Effect of ISO treatment on lung histopathological scores in ZY-stimulated mice

0.7% ISO prevented the abnormal changes of antioxidant enzymes, oxidation product, and proinflammatory cytokine in ZY-challenged mice

In Figures 5 and 6, we observed the abnormal changes in the activities of SOD and CAT in lung and serum, as well as the levels of 8-iso-PGF2α and TNF-α in lung and serum after ZY injection in mice (P < 0.05 vs. NS group). Treatment with ISO at 0.7% increased the activities of SOD and CAT in lung and serum and decreased the levels of 8-iso-PGF2α and TNF-α in lung and serum. The activities of SOD and CAT in lung and serum in the 1.4% ISO treatment group were increased, and the levels of 8-iso-PGF2α and TNF-α in lung and serum were decreased significantly, compared with those in the ZY and ZY + 0.7% ISO groups (P < 0.05). However, mice treated with PT did not exhibit improvement in the activities of SOD and CAT in lung and serum (P > 0.05 vs. ZY group). In terms of the activities of SOD and CAT in lung and serum as well as the levels of 8-iso-PGF2α and TNF-α in lung and serum, ISO at 1.4% had better protective effect than 0.7% ISO did, and no statistically significant differences were present between the NS, NS + PT, and NS + ISO groups.

Fig. 5
Fig. 5:
Isoflurane treatment improved the activities of lung antioxidant enzymes and decreased the levels of lung oxidative product and proinflammatory cytokine in ZY-stimulated mice. A, SOD activity. B, CAT activity. C, Levels of 8-iso-PGF2α. D, Levels of TNF-α. The grouping methods and experimental protocols were the same as in Figure 1. The values are expressed as mean (SD) (n = 6 for each group). *P < 0.05 vs. NS group; P < 0.05 vs. ZY group; P < 0.05 vs. ZY + PT group; § P < 0.05 vs. ZY + 0.7% ISO group.
Fig. 6
Fig. 6:
Isoflurane treatment improved the activities of serum antioxidant enzymes and decreased the levels of serum oxidant product and proinflammatory cytokine in ZY-stimulated mice. A, SOD activity. B, CAT activity. C, Levels of 8-iso-PGF2α. D, Levels of TNF-α. The grouping methods and experimental protocols were the same as in Figure 1. The values are expressed as mean (SD) (n = 6 for each group). *P < 0.05 vs. NS group; P < 0.05 vs. ZY group; P < 0.05 vs. ZY + PT group; § P < 0.05 vs. ZY + 0.7% ISO group.

Inhibition of CAT activity prevented the improvement of survival rate in ZY-challenged mice treated with 0.7% ISO

As was shown in Figure 7, the CAT activity inhibitor AT at a dose of 0.09 g/kg decreased the 7-day survival rate from 35% (ZY + 0.7% ISO group) to 5% (ZY + 0.7% ISO + 0.09 g/kg group; P < 0.05). 3-Amino-1,2,4-triazole alone did not significantly change the survival rate of mice in the ZY group (P > 0.05 vs. ZY group).

Fig. 7
Fig. 7:
Inhibition to CAT activity decreased the survival rate of ZY-challenged mice with 0.7% ISO treatment. ZY group-the animals were treated with ZY at a dose of 1 g/kg. ZY + 0.7% ISO group-the animals were treated with 0.7% ISO for 1 h starting at 1 and 6 h after the ZY injection. ZY + 0.7% ISO + 0.09 g/kg AT group-the animals were treated with 0.09 g/kg AT before 0.7% ISO inhalation after ZY injection. ZY + 0.09 g/kg AT group-the animals were treated with 0.09 g/kg AT after ZY injection at the same time points as in ZY + 0.7% ISO + 0.09 g/kg AT group. The values are expressed as survival percentage (n = 20 for each group). *P < 0.05 vs. ZY group; P<0.05 vs. ZY + 0.7% ISO group.

DISCUSSION

The present study demonstrated that 0.7% ISO inhalation for 1 h starting at 1 and 6 h after ZY injection, respectively, attenuated the lung injury and increased the 7-day survival rate of ZY-challenged mice. The protective effect was associated with regulating the activities of antioxidant enzymes and the levels of oxidation product and proinflammatory cytokine. We also showed that inhibition of CAT activity prevented the improvement of 7-day survival rate in ZY-challenged mice treated with 0.7% ISO.

Zymosan, a substance derived from the cell wall of the yeast Saccharomyces cerevisiae, leads to systemic inflammation by inducing a wide range of inflammatory mediators (12). It is reported that ZY directly activates macrophages. After ZY is phagocytosed, macrophages release lysosomal enzymes, reactive oxygen metabolites, arachidonic acid, and TNF-α. Because ZY is not degradable, phagocytosis by macrophages results in a prolonged inflammatory response (12). It has been proved that i.p. injection of a high dose of ZY can induce a SIRS/MODS model in rats or mice (12, 13). Our previous experiments also suggest that i.p. injection of ZY (1.0 g/kg body weight) successfully induces a murine SIRS/MODS model (14, 15). In the present study, the generalized inflammation caused by ZY (1.0 g/kg body weight, i.p. injection) resulted in a 7-day mortality rate of 80% and increased the levels of TNF-α in lung and serum at 24 h after the ZY injection.

Based on our preliminary experiments, serum levels of inflammatory cytokine TNF-α and IL-10 are increased most at 24 h after the ZY injection, and at the same time point, we also find severe lung injury including large-area hemorrhage, macrophage infiltration, and proliferation (14). In the present investigation, we observed the abnormal changes in lung TNF-α level, lung MPO activity, lung W/D ratio, protein concentration in BALF, and lung histology at 24 h after the ZY injection, indicating that ZY caused acute lung injury (ALI).

Isoflurane is one of the most widely used anesthetic agents during the operative period. Anesthesia dose of ISO has been proved to protect against sepsis/MODS by modulating inflammatory cytokines. It is reported that 1.5% ISO anesthesia for 6 h attenuates lung inflammation and injury in mouse model of MODS (5). In murine septic peritonitis induced by cecal ligation and puncture (CLP), 1.2% (0.86 MAC) ISO anesthesia for 3 h after CLP reduces inflammatory processes, acute renal and hepatic injuries, and death (2). Based on some researches performed by Di Paola et al. (13) and Cuzzocrea et al. (21, 22), in the present study, twice inhalation of ISO was done with the animals for 1 h starting at 1 and 6 h after the NS or ZY injection, respectively. We showed that 1.4% ISO treatment attenuated the ALI and thus increased the 7-day survival rate in ZY-challenged mice. We also found that i.v. anesthetic PT treatment had no similar effects on this model. These results demonstrated that anesthesia dose of ISO had a protective action against ZY-induced generalized inflammation and its associated ALI. More importantly, we provided evidences that inhaling 0.7% (0.5 MAC) ISO for 1 h starting at 1 and 6 h after ZY injection, respectively, attenuated lung injury and improved the 7-day survival rate in mice. Because the protective effects of 0.7% (0.5 MAC) ISO treatment are not better than 1.4% ISOs, further studies on the inhalation time and therapy protocol are needed for increasing the effects of 0.7% (0.5 MAC) ISO treatment.

Systemic inflammatory response syndrome is associated with dramatically elevated levels of inflammatory cytokines such as TNF-α, IL-1β, and interferon γ. TNF-α is a major cytokine involved in the inflammatory response (23). The higher concentrations of TNF-α are found in the BALF from patients with sustained acute respiratory distress syndrome (24). We also observed a marked increase of TNF-α in serum and lung after the ZY injection. Excessive inflammatory responses, including high concentrations of TNF-α, can lead to organ injury and death during MODS. We demonstrated here that 0.7% and 1.4% ISO had a potent protective action by reducing TNF-α level both in lung and serum after the ZY injection.

The oxidative stress and inflammation can interact in many conditions, which are suggested by many studies (25, 26). The depletion of antioxidant enzymes, such as SOD, CAT, and the oxidative damage of lipids, is involved in the pathogenesis of sepsis/MODS (27-29). In rodent sepsis/MODS model induced by CLP, the activities of SOD, CAT, and glutathione peroxidase in lung were significantly decreased during the early and late phages, indicating that sepsis/MODS also sets up an environment favorable for oxidative stress in lung (30). The detection of products of lipid peroxidation has been widely used to estimate the overall status of oxidative stress in lung. The level of 8-iso-PGF2α seems to offer a reliable approach for quantitative measurement of oxidative stress status in vivo (17). In the present study, we observed the decrease in SOD and CAT in lung and serum and the increase in oxidation product 8-iso-PGF2α in lung and serum after ZY injection. We further showed that 0.7% and 1.4% ISO treatment improved the activities of CAT and SOD in lung and serum and decreased the levels of 8-iso-PGF2α in lung and serum. However, PT treatment had no significant influence on the activities of SOD and CAT in lung and serum. It is reported that CAT protects against cardiomyocytes injuries induced by proinflammatory cytokines (31). Hyperbaric oxygen treatment has beneficial effects on renal dysfunction in sepsis because of the increase in antioxidative capacity especially CAT activities (32). So the inhibitor of CAT activity was used in the present study for observing its effects on the protective action of 0.7% ISO treatment in ZY-challenged mice. We found that CAT inhibitor AT decreased the 7-day survival rate of mice with 0.7% ISO treatment, indicating that CAT is very important in these protective effects. These results suggest that the improvement in the activities of endogenous antioxidant enzyme SOD and CAT may be attributed to the protection of ISO treatment.

In addition, we also observed that, with PT treatment, the levels of TNF-α and 8-iso-PGF2α in lung as well as 8-iso-PGF2α in serum were decreased at 24 h after ZY injection, and the 1-day survival rate was significantly increased to 60% when compared with those in ZY group. It has been proved that several i.v. anesthetics have anti-inflammatory effects. For example, thiopental and ketamine inhibit the endotoxin-induced TNF-α, IL-1, and IL-8 responses and increase IL-10 release in vitro (33). In another investigation, Lee et al. (2) reported that PT does not decrease proinflammatory cytokines compared with ISO treatment. Further studies are needed in the future.

Our results demonstrated that, when inhaled for 1 h starting at 1 and 6 h after ZY injection, 0.7% ISO had protective effects on ZY-induced generalized inflammation in mice. A major limitation of this study was that we did not measure the hemodynamic and ventilatory responses in ZY-stimulated mice under ISO or PT treatment. As is known, it is very difficult to measure these indices in mice. According to research work done by Nasu et al. (34), it is shown that 0.5 MAC ISO inhalation fails to reduce arterial blood pressure in rats, but 1.0 MAC decreases it significantly.

We conclude that the treatment with 0.7% (0.5 MAC) ISO for 1 h starting at 1 and 6 h after ZY injection, respectively, is beneficial for ZY-induced generalized inflammation and its associated lung injury, which may be associated with the improvement in the activities of endogenous antioxidant enzymes. Systemic inflammatory response syndrome/MODS, when accompanied by ALI or acute lung respiratory distress syndrome, continues to be one of the leading causes of death in the ICU, with a mortality that has remained at more than 40% (2). As such, the development of novel strategies for the treatment of ALI is critical for the improvement of outcome of critically ill patients. As is known, ISO at anesthesia dose has adverse effects for critically ill patients, who may not tolerate its hemodynamic effects including vasodilation, myocardial depression, and bradycardia (6). Isoflurane at less than 1% (0.7 MAC) for sedation, which interferes weakly with hemodynamics, would be more beneficial for critically ill patients in the ICU (7). Therefore, the present study may be helpful for developing new effective therapeutic strategies for critically ill patients.

ACKNOWLEDGMENTS

The authors thank Prof Qing Li of the Department of Pathology, Fourth Military Medical University, for assisting in histopathological analysis; and Prof Shanlu Liu of the Department of Microbiology and Immunology, McGill University, Montreal, Canada, for his insightful comments.

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Keywords:

Multiple organ dysfunction syndrome; systemic inflammation response syndrome; acute lung injury; isoflurane; antioxidant enzyme

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