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Variation of lipopolysaccharide-induced acute lung injury in eight strains of mice
Respiratory Physiology & Neurobiology 171 (2010) 157–164
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Variation of lipopolysaccharide-induced acute lung injury in eight strains of mice Ann-Sophie Alm b , Ka Li a , Hong Chen a , Diane Wang b , Roland Andersson b , Xiangdong Wang a,∗ a b
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China Department of Clinical Sciences, BMC F10, Lund University Hospital, Sweden
a r t i c l e
i n f o
Article history: Accepted 18 February 2010 Keywords: Susceptibility Mouse Acute lung injury Edema Inflammation Hyperinflation
a b s t r a c t Clinical and experimental evidence suggests that genetic variations may play an important role in the development of acute lung injury (ALI). Lipopolysaccharide (LPS)-induced ALI models has been widely applied for pathophysiological and pharmacological research. In order to understand the variation of acute pulmonary reactions between mouse strains and find the optimal strain for target-oriented study, the present study investigated the alterations of acute lung hyperinflation, inflammation and injury in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice after the intra-tracheal challenge with LPS. We found that LPS-induced ALI varied between measured variables, durations and strains. General score of LPS-induced acute lung hyperinflation, inflammation and edema followed the order CD-1, A/J, Balb/c, DBA/2J, C57BL/6J, DBA/1J, NMRI, C3H/HeN mice at 4 h, and CD-1, C57BL/6J, Balb/c, C3H/HeN, NMRI, A/J, DBA/2J, DBA/1 mice at 24 h. Thus, these data provide useful information to select sensitive or resistant strain mouse for understanding genetic variation of pathogenesis and screening of target-oriented drugs. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Acute lung injury (ALI) is defined as a phenomenon of acute diffuse lung inflammation followed by pulmonary functional and structural alterations responsible for more than 60% of mortality in the intensive care unit (Roupie et al., 1999). ALI and acute respiratory distress syndrome (ARDS) can be the primary insult leading to the development of extrapulmonary multiple organ dysfunction. ALI/ARDS has been considered as the first occurring form of organ dysfunction in the formation of multiple organ dysfunction syndrome (Bai and Wang, 2006). Acute inflammation and vascular leak are defined as critical features of ALI/ARDS, including the hyperproduction of inflammatory mediators (e.g. cytokines, chemokines, radicals and proteases). Local exposure of lipopolysaccharide (LPS) could induce ALI characterized by increased levels of neutrophils, protein content, cytokines and chemokines in the bronchoalveolar lavage fluid, associated with the severity of disease (Jansson et al., 2004; Nonas et al., 2006). The incidence and outcome of ALI were associated with the nature of the precipitating disease and individual susceptibility (Nonas et al., 2006). The role of the genetic background in susceptibility to lung inflammation was confirmed and evidenced by
∗ Corresponding author. Tel.: +86 21 6404 1990 2445; fax: +86 21 5496 1729. E-mail address: [email protected] (X. Wang). 1569-9048/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2010.02.009
identified candidate genes (Backus-Hazzard et al., 2004). Genetic susceptibility was found to play an important role in the induction and development of lung injury both in humans (Lagan et al., 2008) and animals (Moraes et al., 2006). For example, C57BL/6J (B6) mice were more inflammation-susceptible, while C3H/HeJ (C3) mice more inflammation-resistant, a model to investigate the susceptibility of acute and chronic inflammation, infection and carcinogenesis (Backus-Hazzard et al., 2004; Gong, 2006; Page et al., 2007). The present study aimed at investigating the variation of acute pulmonary reactions between animals with different genetic backgrounds and characterizing the optimal strain of mice for ALI research by measuring alterations of acute hyperinflation, inflammation and injury following LPS administration in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J, and C3H/HeN mice.
2. Materials and methods 2.1. Animals Female C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J, and C3H/HeN mice (6–8 weeks of age, Harlan, The Netherlands) were maintained in polyethylene cages with stainless steel lids at 20 ◦ C with a 12-h light/dark cycle and covered with a filter cap. Animals were fed food and water ad lib. The study was approved by the University Ethical Committee for Animal Experiments. Mice were anesthetized with efrane and placed in a supine position head
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up on a board tilted at 30◦ for intra-tracheal instillation of LPS or phosphate buffered saline (PBS). The mice remained in the position until the regain of consciousness and then had the access to food and water. All animals were pathogen free. Lung inflammation and hyperinflation were measured both 4 and 24 h after the intra-tracheal instillation of LPS (B.E. coli 026:B6, DIFCO Laboratories, USA). LPS was dissolved in PBS at a concentration of 2.5 mg/ml and a volume of 1 ml/kg body weight using a modified metal cannula. Animals received the same volume of PBS and manipulations as controls. Animals were terminated and lung inflammation and hyperinflation were measured (n = 10 mice/time point/group). Animal body weight was weighed both before the experiment and at termination. 2.2. ELGV measurement Excised lung gas volume (ELGV) was measured by Archimedes’ principle and based on the stable amount of air trapped within the excised lungs at a transpulmonary pressure of 0 cm H2 O. ELGV was characterized to measure lung hyperinflation in previous studies (Jansson et al., 2005, 2006). Briefly, animals for ELGV measurements were intraperitoneally injected with 0.1–0.2 ml of pentobarbitone sodium (50 mg/kg). After the chest was opened and the heart removed, the trachea was exposed and ligated with a 3–0 suture. The lungs were harvested and carefully trimmed of nonpulmonary tissue. A density determination kit (P3000, Mettler-Toledo GmbH, Sweden) and optional density determination software for the balance was used according to the principle that every solid body immersed in fluid looses weight, and is expressed as g/cm3 . After the system was set to zero in order to exclude the liquid density, the bracket weights, tissue weights outside the beaker and tissue buoyancy within the liquid were balanced. ELGV was determined by the difference between bracket weight and lung buoyancy in the liquid. Indication of lung tissue edema was calculated by the difference between lung tissue weight outside the beaker and bracket weight. The lung tissue density was determined by the ratio of the lung
weight (difference between lung tissue weight outside the beaker and holder weight) and the air volume within the lung (ELGV). 2.3. Bronchoalveolar lavage (BAL) The lungs from all animals were lavaged intra-tracheally with two injections of 0.5 ml PBS after the measurement of ELGV and about 0.2 ml per injection was collected into the individual tube. The BAL fluid was collected into plastic tubes on ice and centrifuged at 1000 rpm and 4 ◦ C for 10 min. The supernatant was stored at −80 ◦ C until the further analysis. The cell pellet was resuspended in PBS for counting total leukocyte number using a 15-parameter, semiautomated hematology analyzer (Sysmex F820, TOA Medical Electronics Co. Kobe, Japan). Cell differentiation was counted after the cytospin and staining with May Grynwald Giemsa. 2.4. Statistical analysis Data are presented as means ± standard error of the mean. Animal body weight change (%) was calculated by a formula of (body weights of mice before termination − body weights of mice before experiment)/body weights of mice before experiment × 100. Increased percentage (%) of pulmonary reactions to LPS was calculated by a formula of (measured values of animals with LPS − measured values of animals with PBS)/measured values of animals with PBS × 100. Based on the increased percentage of each variable measured in the present study, the alterative degrees were scored from the strongest as 8 to the lowest as 1. General alterative scores (GAS) were calculated by the sum of alterative degrees of ELGV, lung weight and total number of leukocytes and neutrophils in BAL fluid. Potential significant differences between groups and time points evaluated, was calculated by using Student’s t-test with two-tailed distribution and two sample equal variances. A p value of less than 0.05 was considered as statistically significant.
Fig. 1. Alterations (%) of mouse body weight 4 h (A) and 24 h (B) after intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). Animal body weight change (%) was calculated by a formula of (body weights of mice before termination − body weights of mice before experiment)/body weights of mice before experiment × 100. *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
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Table 1 Susceptibility of eight strains of mice indicated by increased percentage of responsesa to LPS 4 h after intra-tracheal challenge with LPS (2.5 mg/ml/kg).
Body weight ELGV Lung weight Lung density Total leukocytes Neutrophils Macrophages
C57BL/6J
Balb/cJ
DBA/1J
CD-1
NMRI
DBA/2J
A/J
C3H/HeN
33 (5) 45 (8) 14 (7) −19 14 (2) 10 (1) −36
−36 14 (4) 16 (8) 0 2 (1) 164 (8) −58
240 (7) 4 (2) 5 (2) 0 52 (4) 59 (6) −46
380 (8) 35 (7) 12 (5) −18 246 (7) 48 (5) 61 (7)
5 (4) 10 (3) 7 (4) 25 (6) 34 (3) 30 (3) −52
122 (6) 30 (6) 13 (6) 63 (8) 123 (5) 37 (4) 22 (6)
−11 25 (5) 7 (4) 53 (7) 386 (8) 99 (7) 88 (8)
−6 2 (1) 3 (1) 6 (5) 243 (6) 29 (2) −35
Based on the increased percentage of each variables measured in the present study, the alterative degrees were scored from the strongest as 8 to the lowest as 1 shown in the parenthesis. a Increased percentage (%) of pulmonary reactions to LPS was calculated by the following formula (measured values of animals with LPS − measured values of animals with PBS)/measured values of animals with PBS × 100. Table 2 Susceptibility of eight strains of mice indicated by increased percentage of responsesa to LPS 24 h after an intra-tracheal challenge with LPS (2.5 mg/ml/kg).
Body weight ELGV Lung weight Lung density Total leukocytes Neutrophils Macrophages
C57BL/6J
Balb/cJ
DBA/1J
CD-1
NMRI
DBA/2J
A/J
C3H/HeN
2 (3) 61 (8) 31 (5) −5 16 (6) 130 (4) 56 (7)
5 (5) 60 (7) 36 (7) 0 10 (3) 63 (2) −24
2 (2) 7 (1) 21 (1) 0 9 (2) 74 (3) −39
19 (7) 49 (6) 34 (6) −9 20 (7) 9432 (8) 86 (8)
7 (6) 30 (4) 43 (8) 15 (8) 7 (1) 1031 (5) −9
0.5 (1) 39 (5) 28 (4) −10 11 (4) 63 (1) −9
28 (8) 15 (3) 27 (3) 8 (6) 12 (5) 162 (6) 12 (6)
4 (4) 11 (2) 26 (2) 13 (7) 28 (8) 330 (7) −35
Based on the increased percentage of each variables measured in the present study, the alterative degrees were scored from the strongest as 8 to the lowest (shown in the parentheses). a Increased percentage (%) of pulmonary reactions to LPS was calculated by the following formula (measured values of animals with LPS − measured values of animals with PBS)/measured values of animals with PBS × 100.
3. Results Body weights of DBA/1J mice administered LPS significantly decreased, while body weights of CD1 mice significantly increased at 4 h, both compared with animals given PBS (Fig. 1A; p < 0.01). The body weight significantly increased in seven strains of mice 24 h after LPS challenge, but not in DBA/2J mice, as compared to those after PBS (Fig. 1B; p < 0.05 or less). The order of the three strains
with most weight loss was CD-1, DBA/1J and DBA/2J at 4 h (Table 1) and A/J, CD-1 and NMRA at 24 h (Table 2). ELGV in C57BL/6J and CD-1 mice were significantly increased at 4 h (Fig. 2A), and in C57BL/6J, Balb/cJ, CD-1 and NMRI mice at 24 h (Fig. 2B) after LPS challenge, as compared to the corresponding animals with PBS (p < 0.05 or less). Both DBA/2J and A/J mice with LPS had significantly lower values of ELGV than those with PBS at 4 h (Fig. 2A; p < 0.01). The leading order of the three strains in changed
Fig. 2. Values of lung hyperinflation measured by excised lung gas volume (ELGV) 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
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Fig. 3. Values of lung weight measured by excised lung gas volume (ELGV) 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
ELGV was C56BL/6J, CD-1 and DBA/2J at 4 h (Table 1) and C57BL/6J, Balb/c and CD-1 at 24 h (Table 2). The lung weight significantly increased in C57BL/6J, Balb/c, CD-1 and DBA/2J mice at 4 h (Fig. 3A) and in all strains at 24 h (Fig. 3B) after LPS challenge as compared with those after PBS (p < 0.05 or less). The leading order of the three strains in increased lung weight was Balb/c, C57BL/6J and DBA/2J at 4 h (Table 1) and NMRI, Balb/c and CD-1 at 24 h (Table 2). Values of lung density significantly increased in DBA/2J, A/J and NMRI mice, while the decreased in C57BL/6J mice 4 h after LPS challenge
(Fig. 4A, p < 0.05 or 0.01, respectively), but no changes were seen at 24 h (Fig. 4B). The total number of leukocytes in BAL fluid significantly increased in CD-1, DBA/2J and C3H/HeN mice at 4 h (Fig. 5A) and in all strains 24 h (Fig. 5B) after LPS challenge as compared to those with PBS (p < 0.05 or less). The leading order of the three strains in leukocyte influx in BAL fluid was A/J, CD-1 and C3H/HeN at 4 h (Table 1) and C3H/HeN, CD-1 C57BL/6J at 24 h (Table 2). The number of neutrophils in all strains of mice with LPS was significantly
Fig. 4. Values of lung density 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). * and ** stand for p value less than 0.05 and 0.01, respectively, as compared with the corresponding group challenged with PBS.
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Fig. 5. Total number of leukocytes in bronchoalveolar lavage (BAL) fluid 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
higher as compared to those with PBS both at 4 h (Fig. 6A) and 24 h (Fig. 6B). The leading order of the three strains in neutrophil influx in BAL fluid was Balb/c, A/J and DBA/1J at 4 h (Table 1) and CD1, NMRI and C3H/HeN at 24 h (Table 2). The number of both total leukocytes and neutrophils at 24 h was significantly higher as compared to that at 4 h after LPS challenge. The number of macrophages in BAL fluid was significantly lower in C57BL/6J, Balb/c and NMRI mice, while higher in CD-1 mice 4 h (Fig. 7A) and 24 h (Fig. 7B) after LPS challenge.
General alterative scores (GAS) of each variable are listed in Tables 1 and 2. From the sum of alterative scores of ELGV, weight, total leukocytes and neutrophils in BAL fluid, the order of the strains in lung severity was CD-1 (GAS = 24), A/J (GAS = 24), Balb/c (GAS = 21), DBA/2J (GAS = 21), C57BL/6J (GAS = 18), DBA/1J (GAS = 14), NMRI (GAS = 13), and C3H/HeN (GAS = 10) mice at 4 h, and CD-1 (GAS = 27), C57BL/6J (GAS = 23), Balb/c (GAS = 19), C3H/HeN (GAS = 19), NMRI (GAS = 18), A/J (GAS = 17), DBA/2J (GAS = 14) and DBA/1 (GAS = 7) mice at 24 h.
Fig. 6. Number of neutrophils in bronchoalveolar lavage (BAL) fluid 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
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Fig. 7. Number of macrophages in bronchoalveolar lavage (BAL) fluid 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). * and ** stand for p value less than 0.05 and 0.01, respectively, as compared with the corresponding group challenged with PBS.
4. Discussion Clinical and experimental studies have demonstrated that the development of acute lung inflammation, dysfunction and injury varies among human populations and mouse strains (De Maio et al., 2005; Bauer et al., 2004). This indicates that a genetic background responsible for the susceptibility may be a critical factor in the pathogenesis of ALI. The pulmonary inflammation, the characteristic of ALI, is driven by the infiltration of leukocytes and neutrophils to the lungs. In previous studies, we have shown that acute lung leukocyte/neutrophil infiltration, tissue edema and hyperinflation occurred in a LPS dose-dependent and time-associated pattern (Jansson et al., 2005, 2006). Differences of LPS-induced lung hyperinflation from inflammation indicate that the formation of lung inflammation may run in parallel with the development of acute lung injury rather than with lung hyperinflation. Studies on genes controlling the susceptibility for the inhaled irritants in seven strains of mice (A/J, AKR, C3H/HeN, C57BL/6J, CBA, DBA/2, FVB/N and specific F1 crosses) demonstrated that the A/J mouse strain was sensitive, the C3H/He intermediate, and the C57BL/6 resistant to nickel sulfate-induced acute lung injury (Chan et al., 2005). In order to investigate strain-dependent effects on the genetic susceptibility for ALI, Balb/c, DBA/2J, A/J and C57BL/6J mice were intra-tracheally challenged with RSO160401 oil and a strain-dependent acute response was found (Bauer et al., 2004). The present study compared the variation of LPS-induced ALI in eight strains, commonly used in preclinical research, since local administration of LPS has been used to induce both acute lung injury to mimic ARDS and chronic lung inflammation to mimic chronic obstructive respiratory diseases in humans (Vanhooren et al., 2007; Wesselkamper et al., 2000). These data will provide solid evidences for researchers to select correct mouse strains for the studies, although genetic differences between strains are not fully understood. Systemic responses to LPS, e.g. hypothalamus–pituitary– adrenal axis responses, cytokine production and hormone release, may be associated with the location, frequency and duration of LPS administration. Our results showed that body weight significantly
reduced in DBA/1J mice 4 h after LPS challenge, similar to previous findings in rats or mice after LPS challenge (Gonzalez et al., 1996; Kharitonov and Sjöbring, 2007). This effect might result from the acute desensitization of hypothalamus–pituitary–adrenal axis responses and hypophagia, increased plasma levels of adrenocorticotropic hormone, corticosterone, pituitary gland hormones, tumor necrosis factor-␣, corticotropin-releasing factor, acoustic startle reflex, prepulse inhibition and locomotor activity (Gonzalez et al., 1996; Kharitonov and Sjöbring, 2007). However, we also found a significant increase in body weight in CD-1 mice 4 and 24 h after the intra-tracheal administration of LPS, although the exact mechanism remains unclear. The polar responses to ALI between mouse strains were studied and compared. For example, sensitive C57BL/6J and resistant BALB/c mice to bleomycin were monitored by lactate dehydrogenase release and nuclear polymerase activity (Borges et al., 2007). The sensitive C57BL/6J and resistant C3H/HeJ mice to O2 toxicity were identified by an increased expression of mRNAs encoding metallothionein and the tissue inhibitor of metalloproteinases (Juszczak et al., 2008). The sensitive C57BL/6J and resistant 129X1/SvJ mice were marked by genetic complexity (Piedboeuf et al., 1994). The sensitive C3H/HeN and resistant C3H/HeJ mice to LPS were measured by neutrophil infiltration in the BAL fluid (Hoyt and Lazo, 1992). The sensitive A/J and resistant C57BL/6J mice to ozone were identified by genetic determinants (Chan et al., 2005; Piedboeuf et al., 1994). The sensitive A/J and resistant C57BL/6J mice to nickel sulfate, polytetrafluoroethylene or ozone were detected by pulmonary pathology and survival (Chan et al., 2005). The present study further investigated pulmonary susceptibility for LPS challenge between eight mice strains by comparing lung alterations of each variable and the sum of four variables. Our data indicate that the susceptibility to ALI is related to the measurement of disease-associated biomarkers and duration. Susceptibility to lung hyperinflation followed the order C57BL/6J, CD-1, DBA/2J, A/J, Balb/c, NMRI, DBA/1J and C3H/HeN at 4 h and C57BL/6J, Balb/c, CD-1, DBA/2J, NMRI, A/J, C3H/HeN and DBA/1J at 24 h, while to leukocyte infiltration in BAL fluid A/J, CD-1, C3H/HeN,
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DBA/2J, DBA/1J, NMRI, C57BL/6J and Balb/c at 4 h and C3H/HeN, CD1, C57BL/6J, A/J, DBA/2J, Balb/c, DBA/1J and NMRI at 24 h after LPS challenge, respectively. Since our general alterative score includes both numbers of leukocytes and neutrophils, it emphasizes the inflammatory cell infiltration. This is also evidenced by previous findings that C57BL/6J mice appeared sensitive and A/J mice resistant in the measurement of BAL fluid proteins and A/J sensitive and C3H/HeN resistant in BAL fluid neutrophils, while A/J sensitive and C57BL/6J resistant in the measurement of ALI (Chan et al., 2005). Those indicate a poor correlation between neutrophils and protein content within a strain. Variation of inflammatory responses among mouse strains was also evidenced by different production of cytokines like interleukin (IL)-1, IL-6, IL-10, IL-12, IL13, tumor necrosis factor-␣, chemokines (e.g. keratinocyte-derived chemokines, macrophage inflammatory protein-2, macrophage chemotactic protein (MCP)-1 and MCP-5), vascular endothelial growth factors and leptin in BALfluid (data unshown). Lung hyperinflation has been suggested to be one of the pathophysiological characteristics in ALI and asthma due to bronchial constriction or chronic lung diseases due to alveolar enlargement (Page et al., 2007; Jansson et al., 2006; Prows et al., 1997). Our present study reveals that LPS-induced lung hyperinflation may result from neuron-associated bronchial constriction at 4 h and from airway narrowness caused by airway tissue edema and inflammation at 24 h. This hypothesis coincides with recent findings that sensory lung receptors were activated for initiating lung reflexes during acute lung injury or inflammation (Lin et al., 2007; Yu et al., 2007). Although there is a variation in susceptibility to lung hyperinflation among strains and durations, C57BL/6J and CD1 were more sensitive and DBA/1J and C3H/HeN more resistant. Pulmonary edema has been considered as one of the earliest pathological changes in ALI development. Pulmonary edema resulted from damaged endothelial barrier integrity, increased permeability and impaired lymph flow to accelerate the process of lung dysfunction in ARDS (Prows et al., 2007). LPS seems to increase acute pulmonary edema in a dose- and time-dependent pattern (Jansson et al., 2005). The present study noticed that Balb/c and C57BL/6J (4 h) or NMRI (24 h) mice were more sensitive and C3H/HeN and DBA/1J mice more resistant to pulmonary edema. We also found that the susceptibility of mice to leukocyte/neutrophil infiltration changed by the time. C56BL/6J mice showed more sensitive at 24 h, while CD-1 mice at both 4 and 24 h. Further studies are needed to understand genetic mechanisms by which these differences exist. It would be more important to explore genetic mechanisms by comparing genetic differences of these animals and identify candidate genes responsible for such susceptibility, when the genes of these mice are mapped. In conclusion, the present study demonstrates the susceptibility of lung inflammation and acute lung injury to LPS in eight strains of mice. We found that the susceptibility to intratracheal LPS varied between measured variables, durations and strains. The ALI susceptibility to LPS followed the order CD-1, A/J, Balb/c, DBA/2J, C57BL/6J, DBA/1J, NMRI and C3H/HeN mice at 4 h, while CD-1, C57BL/1J, Balb/c, C3H/HeN, NMRI, A/J, DBA/2J and DBA/1 mice at 24 h. Thus, these data provide vital information to select sensitive or resistant strain mouse for understanding genetic variation of pathogenesis and screening of target-oriented drugs.
Conflict of interest All authors disclose no financial and personal relationships with other people or organizations that could inappropriately influence (bias) the work, including potential conflicts of interest from employment, consultancies, stock ownership, honoraria, paid
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Variation of lipopolysaccharide-induced acute lung injury in eight strains of mice Ann-Sophie Alm b , Ka Li a , Hong Chen a , Diane Wang b , Roland Andersson b , Xiangdong Wang a,∗ a b
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China Department of Clinical Sciences, BMC F10, Lund University Hospital, Sweden
a r t i c l e
i n f o
Article history: Accepted 18 February 2010 Keywords: Susceptibility Mouse Acute lung injury Edema Inflammation Hyperinflation
a b s t r a c t Clinical and experimental evidence suggests that genetic variations may play an important role in the development of acute lung injury (ALI). Lipopolysaccharide (LPS)-induced ALI models has been widely applied for pathophysiological and pharmacological research. In order to understand the variation of acute pulmonary reactions between mouse strains and find the optimal strain for target-oriented study, the present study investigated the alterations of acute lung hyperinflation, inflammation and injury in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice after the intra-tracheal challenge with LPS. We found that LPS-induced ALI varied between measured variables, durations and strains. General score of LPS-induced acute lung hyperinflation, inflammation and edema followed the order CD-1, A/J, Balb/c, DBA/2J, C57BL/6J, DBA/1J, NMRI, C3H/HeN mice at 4 h, and CD-1, C57BL/6J, Balb/c, C3H/HeN, NMRI, A/J, DBA/2J, DBA/1 mice at 24 h. Thus, these data provide useful information to select sensitive or resistant strain mouse for understanding genetic variation of pathogenesis and screening of target-oriented drugs. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Acute lung injury (ALI) is defined as a phenomenon of acute diffuse lung inflammation followed by pulmonary functional and structural alterations responsible for more than 60% of mortality in the intensive care unit (Roupie et al., 1999). ALI and acute respiratory distress syndrome (ARDS) can be the primary insult leading to the development of extrapulmonary multiple organ dysfunction. ALI/ARDS has been considered as the first occurring form of organ dysfunction in the formation of multiple organ dysfunction syndrome (Bai and Wang, 2006). Acute inflammation and vascular leak are defined as critical features of ALI/ARDS, including the hyperproduction of inflammatory mediators (e.g. cytokines, chemokines, radicals and proteases). Local exposure of lipopolysaccharide (LPS) could induce ALI characterized by increased levels of neutrophils, protein content, cytokines and chemokines in the bronchoalveolar lavage fluid, associated with the severity of disease (Jansson et al., 2004; Nonas et al., 2006). The incidence and outcome of ALI were associated with the nature of the precipitating disease and individual susceptibility (Nonas et al., 2006). The role of the genetic background in susceptibility to lung inflammation was confirmed and evidenced by
∗ Corresponding author. Tel.: +86 21 6404 1990 2445; fax: +86 21 5496 1729. E-mail address: [email protected] (X. Wang). 1569-9048/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2010.02.009
identified candidate genes (Backus-Hazzard et al., 2004). Genetic susceptibility was found to play an important role in the induction and development of lung injury both in humans (Lagan et al., 2008) and animals (Moraes et al., 2006). For example, C57BL/6J (B6) mice were more inflammation-susceptible, while C3H/HeJ (C3) mice more inflammation-resistant, a model to investigate the susceptibility of acute and chronic inflammation, infection and carcinogenesis (Backus-Hazzard et al., 2004; Gong, 2006; Page et al., 2007). The present study aimed at investigating the variation of acute pulmonary reactions between animals with different genetic backgrounds and characterizing the optimal strain of mice for ALI research by measuring alterations of acute hyperinflation, inflammation and injury following LPS administration in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J, and C3H/HeN mice.
2. Materials and methods 2.1. Animals Female C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J, and C3H/HeN mice (6–8 weeks of age, Harlan, The Netherlands) were maintained in polyethylene cages with stainless steel lids at 20 ◦ C with a 12-h light/dark cycle and covered with a filter cap. Animals were fed food and water ad lib. The study was approved by the University Ethical Committee for Animal Experiments. Mice were anesthetized with efrane and placed in a supine position head
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up on a board tilted at 30◦ for intra-tracheal instillation of LPS or phosphate buffered saline (PBS). The mice remained in the position until the regain of consciousness and then had the access to food and water. All animals were pathogen free. Lung inflammation and hyperinflation were measured both 4 and 24 h after the intra-tracheal instillation of LPS (B.E. coli 026:B6, DIFCO Laboratories, USA). LPS was dissolved in PBS at a concentration of 2.5 mg/ml and a volume of 1 ml/kg body weight using a modified metal cannula. Animals received the same volume of PBS and manipulations as controls. Animals were terminated and lung inflammation and hyperinflation were measured (n = 10 mice/time point/group). Animal body weight was weighed both before the experiment and at termination. 2.2. ELGV measurement Excised lung gas volume (ELGV) was measured by Archimedes’ principle and based on the stable amount of air trapped within the excised lungs at a transpulmonary pressure of 0 cm H2 O. ELGV was characterized to measure lung hyperinflation in previous studies (Jansson et al., 2005, 2006). Briefly, animals for ELGV measurements were intraperitoneally injected with 0.1–0.2 ml of pentobarbitone sodium (50 mg/kg). After the chest was opened and the heart removed, the trachea was exposed and ligated with a 3–0 suture. The lungs were harvested and carefully trimmed of nonpulmonary tissue. A density determination kit (P3000, Mettler-Toledo GmbH, Sweden) and optional density determination software for the balance was used according to the principle that every solid body immersed in fluid looses weight, and is expressed as g/cm3 . After the system was set to zero in order to exclude the liquid density, the bracket weights, tissue weights outside the beaker and tissue buoyancy within the liquid were balanced. ELGV was determined by the difference between bracket weight and lung buoyancy in the liquid. Indication of lung tissue edema was calculated by the difference between lung tissue weight outside the beaker and bracket weight. The lung tissue density was determined by the ratio of the lung
weight (difference between lung tissue weight outside the beaker and holder weight) and the air volume within the lung (ELGV). 2.3. Bronchoalveolar lavage (BAL) The lungs from all animals were lavaged intra-tracheally with two injections of 0.5 ml PBS after the measurement of ELGV and about 0.2 ml per injection was collected into the individual tube. The BAL fluid was collected into plastic tubes on ice and centrifuged at 1000 rpm and 4 ◦ C for 10 min. The supernatant was stored at −80 ◦ C until the further analysis. The cell pellet was resuspended in PBS for counting total leukocyte number using a 15-parameter, semiautomated hematology analyzer (Sysmex F820, TOA Medical Electronics Co. Kobe, Japan). Cell differentiation was counted after the cytospin and staining with May Grynwald Giemsa. 2.4. Statistical analysis Data are presented as means ± standard error of the mean. Animal body weight change (%) was calculated by a formula of (body weights of mice before termination − body weights of mice before experiment)/body weights of mice before experiment × 100. Increased percentage (%) of pulmonary reactions to LPS was calculated by a formula of (measured values of animals with LPS − measured values of animals with PBS)/measured values of animals with PBS × 100. Based on the increased percentage of each variable measured in the present study, the alterative degrees were scored from the strongest as 8 to the lowest as 1. General alterative scores (GAS) were calculated by the sum of alterative degrees of ELGV, lung weight and total number of leukocytes and neutrophils in BAL fluid. Potential significant differences between groups and time points evaluated, was calculated by using Student’s t-test with two-tailed distribution and two sample equal variances. A p value of less than 0.05 was considered as statistically significant.
Fig. 1. Alterations (%) of mouse body weight 4 h (A) and 24 h (B) after intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). Animal body weight change (%) was calculated by a formula of (body weights of mice before termination − body weights of mice before experiment)/body weights of mice before experiment × 100. *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
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Table 1 Susceptibility of eight strains of mice indicated by increased percentage of responsesa to LPS 4 h after intra-tracheal challenge with LPS (2.5 mg/ml/kg).
Body weight ELGV Lung weight Lung density Total leukocytes Neutrophils Macrophages
C57BL/6J
Balb/cJ
DBA/1J
CD-1
NMRI
DBA/2J
A/J
C3H/HeN
33 (5) 45 (8) 14 (7) −19 14 (2) 10 (1) −36
−36 14 (4) 16 (8) 0 2 (1) 164 (8) −58
240 (7) 4 (2) 5 (2) 0 52 (4) 59 (6) −46
380 (8) 35 (7) 12 (5) −18 246 (7) 48 (5) 61 (7)
5 (4) 10 (3) 7 (4) 25 (6) 34 (3) 30 (3) −52
122 (6) 30 (6) 13 (6) 63 (8) 123 (5) 37 (4) 22 (6)
−11 25 (5) 7 (4) 53 (7) 386 (8) 99 (7) 88 (8)
−6 2 (1) 3 (1) 6 (5) 243 (6) 29 (2) −35
Based on the increased percentage of each variables measured in the present study, the alterative degrees were scored from the strongest as 8 to the lowest as 1 shown in the parenthesis. a Increased percentage (%) of pulmonary reactions to LPS was calculated by the following formula (measured values of animals with LPS − measured values of animals with PBS)/measured values of animals with PBS × 100. Table 2 Susceptibility of eight strains of mice indicated by increased percentage of responsesa to LPS 24 h after an intra-tracheal challenge with LPS (2.5 mg/ml/kg).
Body weight ELGV Lung weight Lung density Total leukocytes Neutrophils Macrophages
C57BL/6J
Balb/cJ
DBA/1J
CD-1
NMRI
DBA/2J
A/J
C3H/HeN
2 (3) 61 (8) 31 (5) −5 16 (6) 130 (4) 56 (7)
5 (5) 60 (7) 36 (7) 0 10 (3) 63 (2) −24
2 (2) 7 (1) 21 (1) 0 9 (2) 74 (3) −39
19 (7) 49 (6) 34 (6) −9 20 (7) 9432 (8) 86 (8)
7 (6) 30 (4) 43 (8) 15 (8) 7 (1) 1031 (5) −9
0.5 (1) 39 (5) 28 (4) −10 11 (4) 63 (1) −9
28 (8) 15 (3) 27 (3) 8 (6) 12 (5) 162 (6) 12 (6)
4 (4) 11 (2) 26 (2) 13 (7) 28 (8) 330 (7) −35
Based on the increased percentage of each variables measured in the present study, the alterative degrees were scored from the strongest as 8 to the lowest (shown in the parentheses). a Increased percentage (%) of pulmonary reactions to LPS was calculated by the following formula (measured values of animals with LPS − measured values of animals with PBS)/measured values of animals with PBS × 100.
3. Results Body weights of DBA/1J mice administered LPS significantly decreased, while body weights of CD1 mice significantly increased at 4 h, both compared with animals given PBS (Fig. 1A; p < 0.01). The body weight significantly increased in seven strains of mice 24 h after LPS challenge, but not in DBA/2J mice, as compared to those after PBS (Fig. 1B; p < 0.05 or less). The order of the three strains
with most weight loss was CD-1, DBA/1J and DBA/2J at 4 h (Table 1) and A/J, CD-1 and NMRA at 24 h (Table 2). ELGV in C57BL/6J and CD-1 mice were significantly increased at 4 h (Fig. 2A), and in C57BL/6J, Balb/cJ, CD-1 and NMRI mice at 24 h (Fig. 2B) after LPS challenge, as compared to the corresponding animals with PBS (p < 0.05 or less). Both DBA/2J and A/J mice with LPS had significantly lower values of ELGV than those with PBS at 4 h (Fig. 2A; p < 0.01). The leading order of the three strains in changed
Fig. 2. Values of lung hyperinflation measured by excised lung gas volume (ELGV) 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
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Fig. 3. Values of lung weight measured by excised lung gas volume (ELGV) 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
ELGV was C56BL/6J, CD-1 and DBA/2J at 4 h (Table 1) and C57BL/6J, Balb/c and CD-1 at 24 h (Table 2). The lung weight significantly increased in C57BL/6J, Balb/c, CD-1 and DBA/2J mice at 4 h (Fig. 3A) and in all strains at 24 h (Fig. 3B) after LPS challenge as compared with those after PBS (p < 0.05 or less). The leading order of the three strains in increased lung weight was Balb/c, C57BL/6J and DBA/2J at 4 h (Table 1) and NMRI, Balb/c and CD-1 at 24 h (Table 2). Values of lung density significantly increased in DBA/2J, A/J and NMRI mice, while the decreased in C57BL/6J mice 4 h after LPS challenge
(Fig. 4A, p < 0.05 or 0.01, respectively), but no changes were seen at 24 h (Fig. 4B). The total number of leukocytes in BAL fluid significantly increased in CD-1, DBA/2J and C3H/HeN mice at 4 h (Fig. 5A) and in all strains 24 h (Fig. 5B) after LPS challenge as compared to those with PBS (p < 0.05 or less). The leading order of the three strains in leukocyte influx in BAL fluid was A/J, CD-1 and C3H/HeN at 4 h (Table 1) and C3H/HeN, CD-1 C57BL/6J at 24 h (Table 2). The number of neutrophils in all strains of mice with LPS was significantly
Fig. 4. Values of lung density 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). * and ** stand for p value less than 0.05 and 0.01, respectively, as compared with the corresponding group challenged with PBS.
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Fig. 5. Total number of leukocytes in bronchoalveolar lavage (BAL) fluid 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
higher as compared to those with PBS both at 4 h (Fig. 6A) and 24 h (Fig. 6B). The leading order of the three strains in neutrophil influx in BAL fluid was Balb/c, A/J and DBA/1J at 4 h (Table 1) and CD1, NMRI and C3H/HeN at 24 h (Table 2). The number of both total leukocytes and neutrophils at 24 h was significantly higher as compared to that at 4 h after LPS challenge. The number of macrophages in BAL fluid was significantly lower in C57BL/6J, Balb/c and NMRI mice, while higher in CD-1 mice 4 h (Fig. 7A) and 24 h (Fig. 7B) after LPS challenge.
General alterative scores (GAS) of each variable are listed in Tables 1 and 2. From the sum of alterative scores of ELGV, weight, total leukocytes and neutrophils in BAL fluid, the order of the strains in lung severity was CD-1 (GAS = 24), A/J (GAS = 24), Balb/c (GAS = 21), DBA/2J (GAS = 21), C57BL/6J (GAS = 18), DBA/1J (GAS = 14), NMRI (GAS = 13), and C3H/HeN (GAS = 10) mice at 4 h, and CD-1 (GAS = 27), C57BL/6J (GAS = 23), Balb/c (GAS = 19), C3H/HeN (GAS = 19), NMRI (GAS = 18), A/J (GAS = 17), DBA/2J (GAS = 14) and DBA/1 (GAS = 7) mice at 24 h.
Fig. 6. Number of neutrophils in bronchoalveolar lavage (BAL) fluid 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). *, ** and *** stand for p value less than 0.05, 0.01 and 0.001, respectively, as compared with the corresponding group challenged with PBS.
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Fig. 7. Number of macrophages in bronchoalveolar lavage (BAL) fluid 4 h (A) and 24 h (B) after an intra-tracheal administration of LPS at the dose of 2.5 mg/ml/kg in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J and C3H/HeN mice (n = 10/time point/group). * and ** stand for p value less than 0.05 and 0.01, respectively, as compared with the corresponding group challenged with PBS.
4. Discussion Clinical and experimental studies have demonstrated that the development of acute lung inflammation, dysfunction and injury varies among human populations and mouse strains (De Maio et al., 2005; Bauer et al., 2004). This indicates that a genetic background responsible for the susceptibility may be a critical factor in the pathogenesis of ALI. The pulmonary inflammation, the characteristic of ALI, is driven by the infiltration of leukocytes and neutrophils to the lungs. In previous studies, we have shown that acute lung leukocyte/neutrophil infiltration, tissue edema and hyperinflation occurred in a LPS dose-dependent and time-associated pattern (Jansson et al., 2005, 2006). Differences of LPS-induced lung hyperinflation from inflammation indicate that the formation of lung inflammation may run in parallel with the development of acute lung injury rather than with lung hyperinflation. Studies on genes controlling the susceptibility for the inhaled irritants in seven strains of mice (A/J, AKR, C3H/HeN, C57BL/6J, CBA, DBA/2, FVB/N and specific F1 crosses) demonstrated that the A/J mouse strain was sensitive, the C3H/He intermediate, and the C57BL/6 resistant to nickel sulfate-induced acute lung injury (Chan et al., 2005). In order to investigate strain-dependent effects on the genetic susceptibility for ALI, Balb/c, DBA/2J, A/J and C57BL/6J mice were intra-tracheally challenged with RSO160401 oil and a strain-dependent acute response was found (Bauer et al., 2004). The present study compared the variation of LPS-induced ALI in eight strains, commonly used in preclinical research, since local administration of LPS has been used to induce both acute lung injury to mimic ARDS and chronic lung inflammation to mimic chronic obstructive respiratory diseases in humans (Vanhooren et al., 2007; Wesselkamper et al., 2000). These data will provide solid evidences for researchers to select correct mouse strains for the studies, although genetic differences between strains are not fully understood. Systemic responses to LPS, e.g. hypothalamus–pituitary– adrenal axis responses, cytokine production and hormone release, may be associated with the location, frequency and duration of LPS administration. Our results showed that body weight significantly
reduced in DBA/1J mice 4 h after LPS challenge, similar to previous findings in rats or mice after LPS challenge (Gonzalez et al., 1996; Kharitonov and Sjöbring, 2007). This effect might result from the acute desensitization of hypothalamus–pituitary–adrenal axis responses and hypophagia, increased plasma levels of adrenocorticotropic hormone, corticosterone, pituitary gland hormones, tumor necrosis factor-␣, corticotropin-releasing factor, acoustic startle reflex, prepulse inhibition and locomotor activity (Gonzalez et al., 1996; Kharitonov and Sjöbring, 2007). However, we also found a significant increase in body weight in CD-1 mice 4 and 24 h after the intra-tracheal administration of LPS, although the exact mechanism remains unclear. The polar responses to ALI between mouse strains were studied and compared. For example, sensitive C57BL/6J and resistant BALB/c mice to bleomycin were monitored by lactate dehydrogenase release and nuclear polymerase activity (Borges et al., 2007). The sensitive C57BL/6J and resistant C3H/HeJ mice to O2 toxicity were identified by an increased expression of mRNAs encoding metallothionein and the tissue inhibitor of metalloproteinases (Juszczak et al., 2008). The sensitive C57BL/6J and resistant 129X1/SvJ mice were marked by genetic complexity (Piedboeuf et al., 1994). The sensitive C3H/HeN and resistant C3H/HeJ mice to LPS were measured by neutrophil infiltration in the BAL fluid (Hoyt and Lazo, 1992). The sensitive A/J and resistant C57BL/6J mice to ozone were identified by genetic determinants (Chan et al., 2005; Piedboeuf et al., 1994). The sensitive A/J and resistant C57BL/6J mice to nickel sulfate, polytetrafluoroethylene or ozone were detected by pulmonary pathology and survival (Chan et al., 2005). The present study further investigated pulmonary susceptibility for LPS challenge between eight mice strains by comparing lung alterations of each variable and the sum of four variables. Our data indicate that the susceptibility to ALI is related to the measurement of disease-associated biomarkers and duration. Susceptibility to lung hyperinflation followed the order C57BL/6J, CD-1, DBA/2J, A/J, Balb/c, NMRI, DBA/1J and C3H/HeN at 4 h and C57BL/6J, Balb/c, CD-1, DBA/2J, NMRI, A/J, C3H/HeN and DBA/1J at 24 h, while to leukocyte infiltration in BAL fluid A/J, CD-1, C3H/HeN,
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DBA/2J, DBA/1J, NMRI, C57BL/6J and Balb/c at 4 h and C3H/HeN, CD1, C57BL/6J, A/J, DBA/2J, Balb/c, DBA/1J and NMRI at 24 h after LPS challenge, respectively. Since our general alterative score includes both numbers of leukocytes and neutrophils, it emphasizes the inflammatory cell infiltration. This is also evidenced by previous findings that C57BL/6J mice appeared sensitive and A/J mice resistant in the measurement of BAL fluid proteins and A/J sensitive and C3H/HeN resistant in BAL fluid neutrophils, while A/J sensitive and C57BL/6J resistant in the measurement of ALI (Chan et al., 2005). Those indicate a poor correlation between neutrophils and protein content within a strain. Variation of inflammatory responses among mouse strains was also evidenced by different production of cytokines like interleukin (IL)-1, IL-6, IL-10, IL-12, IL13, tumor necrosis factor-␣, chemokines (e.g. keratinocyte-derived chemokines, macrophage inflammatory protein-2, macrophage chemotactic protein (MCP)-1 and MCP-5), vascular endothelial growth factors and leptin in BALfluid (data unshown). Lung hyperinflation has been suggested to be one of the pathophysiological characteristics in ALI and asthma due to bronchial constriction or chronic lung diseases due to alveolar enlargement (Page et al., 2007; Jansson et al., 2006; Prows et al., 1997). Our present study reveals that LPS-induced lung hyperinflation may result from neuron-associated bronchial constriction at 4 h and from airway narrowness caused by airway tissue edema and inflammation at 24 h. This hypothesis coincides with recent findings that sensory lung receptors were activated for initiating lung reflexes during acute lung injury or inflammation (Lin et al., 2007; Yu et al., 2007). Although there is a variation in susceptibility to lung hyperinflation among strains and durations, C57BL/6J and CD1 were more sensitive and DBA/1J and C3H/HeN more resistant. Pulmonary edema has been considered as one of the earliest pathological changes in ALI development. Pulmonary edema resulted from damaged endothelial barrier integrity, increased permeability and impaired lymph flow to accelerate the process of lung dysfunction in ARDS (Prows et al., 2007). LPS seems to increase acute pulmonary edema in a dose- and time-dependent pattern (Jansson et al., 2005). The present study noticed that Balb/c and C57BL/6J (4 h) or NMRI (24 h) mice were more sensitive and C3H/HeN and DBA/1J mice more resistant to pulmonary edema. We also found that the susceptibility of mice to leukocyte/neutrophil infiltration changed by the time. C56BL/6J mice showed more sensitive at 24 h, while CD-1 mice at both 4 and 24 h. Further studies are needed to understand genetic mechanisms by which these differences exist. It would be more important to explore genetic mechanisms by comparing genetic differences of these animals and identify candidate genes responsible for such susceptibility, when the genes of these mice are mapped. In conclusion, the present study demonstrates the susceptibility of lung inflammation and acute lung injury to LPS in eight strains of mice. We found that the susceptibility to intratracheal LPS varied between measured variables, durations and strains. The ALI susceptibility to LPS followed the order CD-1, A/J, Balb/c, DBA/2J, C57BL/6J, DBA/1J, NMRI and C3H/HeN mice at 4 h, while CD-1, C57BL/1J, Balb/c, C3H/HeN, NMRI, A/J, DBA/2J and DBA/1 mice at 24 h. Thus, these data provide vital information to select sensitive or resistant strain mouse for understanding genetic variation of pathogenesis and screening of target-oriented drugs.
Conflict of interest All authors disclose no financial and personal relationships with other people or organizations that could inappropriately influence (bias) the work, including potential conflicts of interest from employment, consultancies, stock ownership, honoraria, paid
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