Fig. 4.1
Preparation of the mouse for BAL fluid collection and inflation of the lungs in situ with saline/PBS. a Twenty-gauge blunt needle (top) and beveled tubing adapter (bottom) for insertion into the trachea. b The arrow indicates the abdominal inferior vena cava. c The arrow points to the exposed trachea, flanked by the salivary glands and still covered by the musculature. d Dissection of the musculature reveals the cartilage rings of the trachea. e The thoracic cavity is shown completely open and the ribcage pinned on each side. This mouse was not injured, so the lung lobes appear healthy and pink. f A small nick (arrow) has been made in the trachea near the pharynx. The suture is loosely tied around the trachea at this step. g The adapter is inserted into the trachea through the nick and held in place by knotting the suture twice. h Inflation of the lung lobes is clearly evident after introduction of 1 ml PBS/EDTA using a 1-cc syringe attached to the adapter
1.5-ml polypropylene microcentrifuge tubes.
General Procedure:
- 1.
Euthanize the mouse either by exposure to 100 % isoflurane or intraperitoneal injection of a commercially available euthanasia solution.
- 2.
Place the mouse on a Styrofoam support in a flat supine position, with its head facing the investigator, and pin each foreleg.
- 3.
- 4.
Tape down the snout to straighten out the airway and spray the area from the chin to the diaphragm with 70 % ethanol.
- 5.
Starting at the chin of the mouse, cut the skin over the trachea and chest down to the diaphragm. Using forceps pull the salivary glands apart to expose the trachea and surrounding musculature (Fig. 4.1c). Carefully tease away the muscle tissue so that the cartilage rings of the trachea are evident (Fig. 4.1d).
- 6.
Cut along the center of the ribcage to just above the diaphragm, keeping the scissors flush against the sternum to avoid puncturing the underlying heart or lungs, then cut along both sides of the ribcage. Using forceps, carefully pull apart the ribcage at the top and pin down on each side (Fig. 4.1e).
- 7.
- 8.
- 9.
Attach a 1-cc syringe containing 1 ml3 of warm PBS/EDTA to the tubing adapter or blunt needle. Slowly push in the entire volume, making sure that all lobes inflate (Fig. 4.1h). Then carefully pull back on the plunger to withdraw as much fluid as possible.4 If the pressure is high and fluid stops entering the syringe, try carefully adjusting the position of the adapter/needle. Alternatively, the syringe may need to be removed, and a Pipetman fitted with a tip can be used to collect the remaining fluid from the adapter/needle.
- 10.
Incorporation of Fluorescent Dextran into the BAL Technique
To assess permeability changes that accompany ALI, investigators have traditionally relied on using albumin or high molecular weight dextrans as tracers. Some approaches involve intranasal or tracheal administration of the tracer to measure its passage into the plasma [1]. Alternatively, the tracer can be added to a lung perfusate ex vivo or injected in vivo to monitor its accumulation in extravascular compartments. When using albumin as the tracer, it can be radioactively or fluorescently labeled and introduced into the animal, or the endogenous protein can be measured in BAL by colorimetric assays, ELISA, or binding of Evans Blue dye. Our group prefers to use a fluorescent dextran, such as fluorescein isothiocyanate (FITC)-dextran, because it is extremely stable at physiological pH and temperature with little risk of loss of the fluorescent label [4], and analysis requires minimal processing. Using a fluorescent compound also avoids the issue of dealing with radioactive waste. FITC-dextran is intravenously injected into the animal several hours prior to the lavage. Both blood and BAL are then collected, with the aim of measuring the concentration of fluorescent dextran in the BAL, normalized to that in the plasma. In this assay, increased fluorescent dextran in the airspace would be a significant indicator of enhanced vascular and epithelial permeability.
Materials (in addition to those listed in Section “Basic technique for in situ BAL of euthanized mice”):
70 kDa FITC-dextran, dissolved in sterile PBS to 10 µM (or 0.7 mg/ml)
Sterile 1-cc syringe and 26-G needle
Greiner MiniCollect tube containing EDTA (catalog #450474)
Sterile 3-cc syringe and 25-G needle
0.1 M EDTA, pH 7–8
96-well assay plate, black with clear bottom
Microplate reader with fluorescence measurement capability.
General Procedure:
- 1.
Three hours prior to lavage, anesthetize the mouse by exposure to isoflurane and, using the 1-cc syringe and 26-G needle, intravenously7 inject 100 µl of 10 µM FITC-dextran.
- 2.
Euthanize the mouse by intraperitoneal injection of a commercially available euthanasia solution and expose the thoracic cavity as described in Section “Basic technique for in situ BAL of euthanized mice,” omitting the IVC cut.
- 3.
Draw up a ml or so of 0.1 M EDTA into the 3-cc syringe and 25-G needle and expel (this coats the interior of the needle and syringe with anticoagulant without adding substantial volume). With the bevel side of the needle facing up, puncture the right ventricle of the heart and slowly draw blood into the syringe. Withdraw the needle and syringe from the heart.
- 4.
Pull back on the plunger slightly, remove the needle and insert the syringe into the MiniCollect tube. Press the plunger to transfer blood from the syringe into the tube.
- 5.
Gently mix the contents and place the tube on ice.
- 6.
Harvest BAL fluid as described in Section “Basic technique for in situ BAL of euthanized mice.”
- 7.
Centrifuge the BAL fluid at 4700–5200 × g for 5 min. Remove the supernatant and place on ice.8 Proceed with cell pellet as desired for other analyses.
- 8.
Centrifuge the blood at 4700–5200 × g for 10 min at 4 °C. Remove the supernatant (plasma) and transfer to a fresh tube.9
- 9.
Prepare dilutions of plasma and BAL (typically in the range of 1:5 for BAL and 1:20 or 1:50 for plasma) and FITC-dextran for the standard curve (ranging from 0.05 to 50 nM).
- 10.
Pipet 100 µl of each dilution, in duplicate or triplicate, into each well of the assay plate.
- 11.
Measure fluorescence using the microplate reader at an excitation wavelength of 493 nm and emission wavelength of 517 nm.
- 12.
Average the fluorescence readings for the standards and plot them as a function of FITC-dextran concentration. Determine the y-intercept and slope using a best-fit line.
- 13.
Average the fluorescence values for the BAL and plasma. Calculate the FITC-dextran concentration using the y-intercept and slope derived from the standard curve in #12 [using equation ((Average OD595 − y-intercept) ÷ slope) × dilution factor].10
- 14.
Express values as a ratio of fluorescence in the BAL relative to the plasma.11
BAL Total Cell Counts (Leukocytes and Red Blood Cells) and Differential Staining
BAL fluid is usually leveraged to obtain a qualitative and quantitative picture of the degree of inflammation and enhanced permeability, both hallmarks of ALI. Total numbers of cells can be assessed by manually counting a small sample of the BAL (either neat or diluted), typically using a hemacytometer. In addition, the composition of the leukocytes is evaluated by morphology coupled with differential staining, a process that employs more than one chemical dye to help the investigator differentiate the cell types from one another. The staining technique was developed by Dmitri Romanowsky and further modified by others (Wright, Giemsa, Leishman) [5, 6, 17–19] to combine methanol-based fixation and staining in a single step, using the basic dye methylene blue to detect nucleic acid and the acidic dye eosin to detect protein. Commercial kits (variously known as Diff-Quik, Diff-Quick, or Kwik-Diff™) are readily available that split the fixation and staining into three separate steps. A sample of cells from the BAL is smeared or centrifuged onto glass slides and taken through the staining process, which is extremely rapid and easy to perform. As previously mentioned, in ALI the expectation is that the total number of leukocytes will be elevated, with increased permeability and transmigration from the tissue through the epithelium into the airspace. The presence and load of neutrophils, which are rarely seen in the airspace at baseline, are particularly indicative of early stages of injury.
Materials:
Lavage fluid from Section “Basic technique for in situ BAL of euthanized mice”
1X and 10X PBS
Bright-Line™ hemacytometer, Improved Neubauer (Hausser Scientific), with glass coverslip
Crystal violet, 0.01 % (in 1 % (v/v) glacial acetic acid)
Inverted phase microscope
0.5- and 1.5-ml polypropylene microcentrifuge tubes
Microcentrifuge
Shandon Cytoslide (Thermo Scientific, catalog #5991056)
EZ Single Cytofunnel (Thermo Scientific, catalog #5991040)
Shandon Cytospin 3 instrument (or equivalent)
Shandon Kwik-Diff™ Staining kit (Thermo Scientific, catalog #9990700)
Coplin jars or 50-ml conical polypropylene tubes for staining
Conventional microscope with bright field capability.
General Procedure:
- 1.
Clean the hemacytometer and cover glass with 70 % ethanol and dry each with a Kimwipe. Place the coverslip over the central chamber of the hemacytometer.
- 2.
Carefully pipet 10 µl of the BAL fluid under the coverslip and allow the cells to settle. At the microscope, use the 40× objective to count the number of red blood cells (RBCs) in 4 corner 1-mm2 squares of the hemacytometer (each of these corner squares is further divided into 16 smaller squares)12,13 One of the 4 corner squares is highlighted in yellow in Fig. 4.2a. Add these 4 values and multiply the sum by 2500 to obtain the number of RBCs per ml.14 For BAL that is overtly bloody, count the RBCs in 5 diagonal squares (highlighted in blue in Fig. 4.2a) out of the 25 that comprise the central area of the chamber. Add these 5 values and multiply the sum by 50,000 to obtain the number of RBCs per ml.15
Fig. 4.2
Counting cells with a hemacytometer. a The grid layout of an Improved Neubauer hemacytometer is illustrated. One of the 4 corner squares, composed of 16 smaller squares, is highlighted in yellow. By convention, cells (gray circles) within or touching the lines defining the perimeter of the yellow square are counted, excluding cells on the top and right side, as shown by the X’s. The internal squares highlighted in blue are used for counting red blood cells (RBCs) when they are numerous in the BAL fluid. b Photomicrograph at 400× depicting cells in 10 µl BAL fluid (harvested from an uninjured mouse) loaded into a hemacytometer. The arrows point to leukocytes and the arrowhead indicates an RBC (note the size difference)
- 3.
- 4.
Centrifuge the BAL fluid at 4700–5200 × g for 5 min.
- 5.
Remove the supernatant and save at −20 °C for later protein analysis (see Section “Total protein determination in BAL”).
- 6.
Resuspend the cell pellet in H2O,18 rapidly (within 30 s) followed by the appropriate volume of 10X PBS, to yield a concentration of ~1.5 × 105 cells/ml.
- 7.
Load a cytoslide into a cytofunnel and snap it closed. Pipet 200 µl, or ~30,000 cells, into the chamber of the cytofunnel. Centrifuge in a Cytospin 3 for 2 min at 1000 rpm (use a balance cytofunnel and slide if only one sample is being centrifuged). After removing the cytofunnel from the centrifuge, carefully open and remove the slide. Allow the slide to air dry.
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