Abstract
The mouse has been a popular organism for studying mechanisms of acute lung injury (ALI), given the small size, rapid life cycle, and genetic malleability of this animal. The major features of ALI in mice include disruption of the alveolar capillary barrier, histopathological changes in lung architecture, localized inflammation, and physiological dysfunction. In this chapter, we describe the basic techniques that we use in our laboratory to qualitatively and quantitatively assess ALI in mice, focusing particularly on permeability and inflammation. Detailed methodology for performing bronchoalveolar lavage and analyzing components of the lavage is outlined, along with guidelines for processing and evaluation of lung tissue. Mastery of these essential tools will allow any investigator to reliably and reproducibly measure the experimental outcome in multiple mouse models of acute lung injury.
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Notes
- 1.
This step can be omitted if blood is to be collected.
- 2.
If the left lobe is not to be lavaged (for example, if it is intended for an assay in which prior manipulation is contraindicated), it can be tied off at the bronchus using a separate suture. Carefully tease away the pleura attaching the left lobe to the cavity, lift up the lobe using the curved forceps, and slide the suture underneath.
- 3.
If the left lobe is tied off, we use a volume of 0.8 ml to lavage the right lung.
- 4.
A typical recovery is 75–90 % of the fluid volume.
- 5.
Injured mice often yield a BAL that is visibly bloody.
- 6.
If the left lobe is tied off, at this point it can be removed by cutting below the suture. We usually weigh the lobe and flash freeze it for downstream applications.
- 7.
Either via the retro-orbital sinus or tail vein.
- 8.
Keep BAL and plasma protected from light.
- 9.
Both BAL and plasma can be stored up to 48 h at −20 °C before the fluorescence measurement.
- 10.
In BAL from rodents at baseline, we and others have observed the FITC-dextran concentration to be <1 nM, using an administered dose of 10 µM. This level in the BAL fluid can increase by an order of magnitude with injury.
- 11.
The concentration of FITC-dextran can also be expressed as mg FITC-dextran per ml BAL fluid and compared to levels in uninjured mice.
- 12.
Each corner square is surrounded by triple lines. Cells within the triple lines are counted, as well as cells that touch the lines on two perpendicular sides (but not on the other two sides) (Fig. 4.2a).
- 13.
Ideally, a total of 100–200 cells should be counted for the most accurate determination.
- 14.
The RBC concentration range in BAL fluid from uninjured mice (using 1 ml saline for the lavage) is typically 104–105 cells per ml.
- 15.
Alternatively, BAL can be diluted for counting RBCs in the 4 corner squares. Any dilution amount needs to be factored into the calculation to obtain cells per ml.
- 16.
If desired, crystal violet can be used to visualize WBCs: Mix 20 µl BAL with 20 µl crystal violet and count WBCs in the 4 corner 1-mm2 squares of the hemacytometer. Add values and multiply by 2500 and by 2 to obtain WBCs per ml.
- 17.
The leukocyte count in BAL fluid (1 ml used for lavage) from animals at baseline usually ranges from 1 to 2 × 105 cells/ml.
- 18.
This step results in lysis of the RBCs.
- 19.
Alveolar macrophages are the dominant cell type in BAL from uninjured mice. Basophils are rarely observed in BAL fluid.
- 20.
Absorbance values will increase over time.
- 21.
The coefficient of variance (CV), which is the standard deviation divided by the average and multiplied by 100, ideally should be <10 % for replicates.
- 22.
Alternatively, microplate reader software can be used to calculate the concentration of unknowns based on the standards, and a four-parameter (quadratic) fit generally gives more reliable data than a linear fit.
- 23.
Total protein in BAL from uninjured mice normally ranges from 100 to 200 µg/ml, whereas in our experience the level increases at least an order of magnitude with acute injury.
- 24.
We generally use 4 % paraformaldehyde, although buffered formalin is an excellent alternative and other fixatives can be substituted as well. If prepared in the lab, paraformaldehyde should be fresh or used within a week (and stored at 4 °C or frozen). We also purchase 16 % paraformaldehyde from Electron Microscopy Sciences (catalog #15710) and dilute that to 4 % with PBS (store at 4 °C).
- 25.
As described in Section “Basic technique for in situ BAL of euthanized mice,” the left lung (or the right lung) can be tied off at the bronchus with a separate suture, if desired for other assays.
- 26.
For frozen sections, we generally fix the lungs for 2 h at room temperature and transfer to cold 18–30 % sucrose overnight. Lungs are then immersed in Optimal Cutting Temperature (OCT) compound added to an appropriately sized cryomold and frozen in a dry ice/ethanol bath.
- 27.
If desired, cut away the heart and thymus before embedding the lungs in paraffin or OCT.
- 28.
Our protocol was originally provided by Dr. Carol Feghali-Bostwick’s laboratory.
- 29.
The kit from Sigma uses a smaller amount of tissue and volume than called for in our protocol. In addition, after hydrolysis, a portion of the sample (5–25 %) is added to the 96-well plate and either dried under vacuum or in an oven at 60 °C. This step obviates the need to adjust the pH.
- 30.
Alternatively, omit addition of the H2O and use pH paper/strips to check the pH. Vortex the sample between drops of HCl and NaOH. Adjusting the pH is the most time-consuming step in the procedure, especially if a large number of samples is being analyzed.
- 31.
In uninjured mice, expect a range of 30–40 µg hydroxyproline per left lung. As a reference, this range typically increases to 60–80 at day 21 post bleomycin (a time point characterized by significant fibrosis during the resolution phase of injury).
References
Chen H, Wu S, Lu R, Zhang YG, Zheng Y, Sun J. Pulmonary permeability assessed by fluorescent-labeled dextran instilled intranasally into mice with lps-induced acute lung injury. PLoS ONE. 2014;9(7):e101925.
Dames C, Akyuz L, Reppe K, Tabeling C, Dietert K, Kershaw O, et al. Miniaturized bronchoscopy enables unilateral investigation, application, and sampling in mice. Am J Respir Cell Mol Biol. 2014;51(6):730–7.
Daubeuf F, Frossard N. Performing bronchoalveolar lavage in the mouse. Curr Protoc Mouse Biol. 2012;2(2):167–75.
Guery BP, Nelson S, Viget N, Fialdes P, Summer WR, Dobard E, et al. Fluorescein-labeled dextran concentration is increased in bal fluid after antu-induced edema in rats. J Appl Physiol (1985). 1998;85(3):842–8.
Horobin RW, Walter KJ. Understanding romanowsky staining. I: the romanowsky-giemsa effect in blood smears. Histochemistry. 1987;86(3):331–6.
Leishman WB. Note on a simple and rapid method of producing romanowsky staining in malarial and other blood films. Br Med J. 1901;2(2125):757–8.
Martin TR, Matute-Bello G. Experimental models and emerging hypotheses for acute lung injury. Crit Care Clin. 2011;27(3):735–52.
Matute-Bello G, Downey G, Moore BB, Groshong SD, Matthay MA, Slutsky AS, et al. An official american thoracic society workshop report: Features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol. 2011;44(5):725–38.
Matute-Bello G, Frevert CW, Martin TR. Animal models of acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2008;295(3):L379–99.
Mizgerd JP, Skerrett SJ. Animal models of human pneumonia. Am J Physiol Lung Cell Mol Physiol. 2008;294(3):L387–98.
Neuman RE, Logan MA. The determination of hydroxyproline. J Biol Chem. 1950;184(1):299–306.
Parker JC. Acute lung injury and pulmonary vascular permeability: use of transgenic models. Compr Physiol. 2011;1(2):835–82.
Parker JC, Townsley MI. Evaluation of lung injury in rats and mice. Am J Physiol Lung Cell Mol Physiol. 2004;286(2):L231–46.
Polikepahad S, Barranco WT, Porter P, Anderson B, Kheradmand F, Corry DB. A reversible, non-invasive method for airway resistance measurements and bronchoalveolar lavage fluid sampling in mice. J Vis Exp. 2010;38:1720.
Walters DM, Wills-Karp M, Mitzner W. Assessment of cellular profile and lung function with repeated bronchoalveolar lavage in individual mice. Physiol Genomics. 2000;2(1):29–36.
Ware LB. Modeling human lung disease in animals. Am J Physiol Lung Cell Mol Physiol. 2008;294(2):L149–50.
Wittekind D. On the nature of romanowsky dyes and the romanowsky-giemsa effect. Clin Lab Haematol. 1979;1(4):247–62.
Woronzoff-Dashkoff KK. The wright-giemsa stain. Secrets revealed. Clin Lab Med. 2002;22(1):15–23.
Wright JH. A rapid method for the differential staining of blood films and malarial parasites. J Med Res. 1902;7(1):138–44.
Acknowledgments
The authors thank previous members of Lynn Schnapp’s laboratory for their practical contributions to these protocols. We also thank Dr. Sarah Stephenson for reading the manuscript and Dr. Tetsuya Nishimoto for help with details of the hydroxyproline assay.
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Wilson, C.L., Felton, L.M., Chow, YH. (2017). Fundamental Methods for Analysis of Acute Lung Injury in Mice. In: Schnapp, L., Feghali-Bostwick, C. (eds) Acute Lung Injury and Repair. Respiratory Medicine. Humana Press, Cham. https://doi.org/10.1007/978-3-319-46527-2_4
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