Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

The aim of this procedure is to quantify COPD, like chronic lung pathologies in the lungs of cigarette smoke exposed mice. This is accomplished by first exposing the mice to cigarette smoke or air for six months. Next, pulmonary function testing is performed on the mice using a rodent ventilator.

Then the lungs are inflated to a fixed pressure removed and fixed in 10%formalin. Finally, more informatory software is used to measure airspace size on gill stained lung sections and small airway extracellular matrix, protein layer thickness, or mason's trichome stained lung sections. Ultimately, the measurement of airspace enlargement in the lungs of cigarette smoke exposed mice is performed in a standardized, unbiased, and automated manner.

The main advantage of this technique over existing methods such as mean linear intercept measurement or the destructive index, is that this protocol is less time consuming to complete, less susceptible to observer bias and more effective at capturing the heterogeneous nature of the pathology in mice as it can analyze the disease in entire LOBs and or both lungs. After exposing mice to cigarette smoke and anesthetizing them according to the text protocol, shave the skin anterior to the trachea and use an iodine based solution followed by ethanol to disinfect the area using claved scissors. Make a midline incision through the skin and subcutaneous tissue anterior to the trachea and use forceps to separate the Sterno thyroid muscles.

To expose the trachea, pass a two inch length of silk suture posterior to the trachea, induce tracheal scissors to make a tracheostomy on the anterior aspect of the trachea. Then insert an 18 gauge cannula into the tracheostomy. Induce the suture to secure it in place via the tracheal cannula.

Connect the mouse to the Y tubing of the adapter of the mechanical ventilator and initiate mechanical ventilation using a tidal volume of 10 milliliters per kilogram and a respiratory rate of 150 breaths per minute. Next, inflate the lungs to a total lung capacity or TLC of three times per volume history. To measure the inspiratory capacity or ic and to reduce ectasis of the lungs, perform a single frequency forced oscillation, maneuver, and assess the dynamic resistance, RRS, elastin ERS and compliance CRS of the respiratory system.

Then carry out a broadband frequency, forced oscillation, maneuver, and measure central airway resistance, RN tissue resistance, G tissue, elastin H, and the ratio G over H.Finally, record quasi static compliance CST during the volume pressure flow maneuvers. Repeat each of these maneuvers five times until consistent readings are obtained. Inflating to TL C3 times between each repeated set.

Record the mean value for each parameter for each mouse. Remove the mouse from the ventilator and after euthanizing it according to the text protocol. Cut the diaphragm, open the thorax in the midline and remove the anterior ribs to expose the lungs.

Dissect the skin and subcutaneous tissue around the trachea and pass two two inch length silk sutures posterior to the trachea. Once pulmonary inflation has been quantified by performing a bronchoalveolar lavage and the air has been flushed outta the giving set for lung inflation, connect the intravenous giving set to the tracheal cannula. Open the valve and allow PBS to flow into the lungs by gravity until the lungs fully inflate.

Make a knot with the suture distal to the tracheal cannula. Use forceps to lift the trachea and cut it proximal to the knot. Then dissect the connective tissue posterior to the trachea and the lungs.

Carefully remove the lungs and place them in a tube containing 10%saline. Buffered formula in. Fix the lungs at room temperature overnight before using PBS to wash them twice.

Embed the lungs in paraffin and cut five micro run thick sections. Then use gill stain to stain the sections and image them as outlined in the text protocol. To prepare images for Scion image analysis, start Scion image and load the macros as indicated in the supplemental portion of the text protocol, select the open brightfield image one macro to select and open a TIFF file.

Use image edit tools to prepare the image. For analysis, select the paintbrush color by clicking on black or white at the bottom of the LUT window. Then click the brush tool to change the brush size.

Double click on the brush tool and enter an appropriate brush size. Use the paintbrush tool to force areas that are not airspace or alveolar walls to be treated as airspace or tissue by painting bronchi and vessels black so that they are analyzed as tissue. To measure the mean cord length of the airspace, select the macro cord length air two.

This step converts the gray scale image to a binary image with only black or white pixels threshold. The binary image by clicking near the center of the image and dragging the mouse up or down. To adjust the threshold value, click the mouse a second time to accept the value.

Adjust the threshold to make the alveolar walls the same thickness as in the original images. It is crucial not to under threshold and thereby create breaks in the alveolar walls that do not exist in the original image, which will produce artificially high cord length values. Observe a window which prompts a re thresholding of the binary image and continue or cancel the macro to threshold.

Again, answer why to the prompt and select the okay button. Observe the macro generated horizontal and vertical grid window with lines five pixels apart. The program measures the lengths of the horizontal vertical lines that overlap airspace.

Save the file in any folder using the default name. That includes a file format that is appended with C LA TXT for airspace cord length. This will allow Excel report macros to find the file.

To analyze the reports open X Excel report TWENTYX XLS. Choose the CL m multi macro to report alveolar cord length measurements for multiple folders corresponding to images from multiple mice from the file window. Select one folder at a time by highlighting the folder, then selecting, okay.

As each folder is selected, observe its worksheet that shows statistics for each CL txt file. Followed by statistics for cord length data combined from all files, open the file all CL to view data from all the worksheets. Finally, rename and save the spreadsheet.

The default file name is the name of the parent folder appended with c xls. A section from a well inflated lung is shown here. Macrophages in the alveolar spaces are painted white and vessels and bronchi are painted black.

Next, the image is converted to a binary image subjected to thresholding using the original image and then inverted such that all pixels in the alveolar space are black and or pixels in the areas of the lung that are not alveoli are white. Shown in these figures are the vertical and horizontal alveolar cord lengths that the software macro generates. This experiment illustrates that C 57 black six wild type mice exposed to smoke have a 23%increase in alveolar airspace size when compared to age and sex matched air exposed wild type mice.

Pulmonary function tests show modest left shifts in the pressure volume loops reflecting modest loss of elastic recoil of the lung, consistent with the mild emphysema that develops in C 37 black six wild type mice exposed to cigarette smoke. The six months shown here are images of massen tri chrome stained sections of lungs from C 57 black six wild type mice exposed to air or cigarette smoke for six months, illustrating increases in extracellular matrix protein deposition around small airways in the cigarette, smoke exposed animals While attempting this procedure. It's important to remember to be careful not to nick the lungs when removing them from the thoracic cage of the mice after it development.

This technique paved the way for searches in the field of pulmonary diseases to explore pathways that contribute to the pathogenesis of the chronic pro obstructive pulmonary diseases in mice, and assess the efficacy of novel therapies of our COPD in a preclinical model system.