Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Hi, my name is Gurpreet. Our lab investigates mechanisms of lung inflammation and repair. Hi, my name is Jessica and I investigate ozone-induced lab rat alveoli by using fluorescent microscopic methods.

Hello everyone, my name is Manpreet. And I study lung inflammation with animal imaging methods. Lungs are continually faced with direct and indirect insults, arising from sterile, such as ozone, and microbial components, such as the bacterial lipopolysaccharide or LPS.

An overwhelming host response may result in compromised respiration and acute lung injury as characterized by lung neutrophil recruitment, due to unregulated host immune, coagulative, and tissue remodeling. We describe a comprehensive analysis of various compartments, namely the broncho-alveolar lavage fluid, that is BAL, the lung vascular perfusate, that is LVP, left lung cryo sections, peripheral blood and the sternal and femur bone marrow. For ozone exposures, gently place mice in the custom induction box and continuously expose the mice to 0.05 PPM of ozone for two hours.

For LPS exposures, anesthetize the mice under light intraperitoneal ketamine and xylazine mix. Now instill 50 microliters off the LPS solution into the mice external nares. Instill control mice with 50 microliters of sterile saline.

At specified time points, anesthetize the mice with the full dose of ketamine and xylazine mix. Next sanitize the mice and check for pedal reflexes, that is pinch either of the hind limb digits, and observe for retraction of the limb. No reflex means deep anesthesia.

Now make a cut below the sternum and expose the heart. Place a 25 gauge needle attached to heparinized syringe into the right ventricle and draw blood by cardiac puncture. Collect the blood in heparinized syringes and drain in microfuge tubes for further processing.

Carefully use tweezers or blunt forceps to clear off the tracheal region from overlying tissue. Now cut through the rib cage to expose the lungs. Pass a cotton ligature below the trachea, and leave it such for the time being.

Snip the trachea, spaced at around one third of its length from the lungs for tracheostomy. Insert a 28 gauge polyethylene cannula into the trachea. Firmly tie the cotton ligature to hold the cannula in place.

Make sure you don't collapse the cannula. Now gradually inject 0.5 ml of PBS into the cannula with the help of a one mL syringe. After injecting PBS aspirate out the syringe as long as it doesn't resist the suction.

Collect the aspirated fluid in a labeled microphage tube, and repeat this procedure two more times. Cut the descending thoracic aorta at the location between the thoracic and abdominal halves to avoid any backup during lung perfusion. Blot the cavity near the cut aorta end free of blood.

Next perfuse the lungs with 0.5 ML of room temperature heparinized saline, inject it through the right ventricle. Collect the vascular perfusate from the cavity at the cut end of the descending thoracic aorta. After ligating the right bronchus, let the left lung inflate with 0.5 ML of paraformaldehyde for five minutes.

Now sanitize the abdominal portion. Cut the abdominal part and deskin it from the pelvic bone. Collect the pelvic bone and isolate the left and right femur bones in a saline filled petri dish kept on ice.

Collect the ventral rib cage and sternum into a saline-filled Petri dish, which is also kept on ice. Perfuse the cut ends of the bones with four times with 0.5 ML of saline using well-fitted needles onto 1 ML syringes. Collect the fractions from each bone into labeled Falcon tubes, fitted with filters and placed on ice.

Centrifuge all the samples for 10 minutes at 3000 RPM. Collect the supernatants and flash freeze them. Reconstitute the cells in a minimum of 200 microliters of PBS.

Perform total leukocyte counts. Stain another liquid of BAL with a mix of calcein green and the red ethidium homodimer one stain. Analyze the supernatant for chemokines and total protein concentration.

Centrifuge the cells to prepare two cytospins on each slide and stain for biochemical and immune proteins. Perform modified H and E staining on lung cryosections for all the groups. Manually outline 150 to 200 cells in the merged image panel.

Copy the outlined regions of interest to all the channels. Measure the preset parameters for all the channels. Divide the fluorescent intensity of the stained molecule by that of DAPI or CD61.

These are termed as DAPI or CD 61 normalized fluorescent intensity ratios. Next plot the parameters to evaluate any changes after exposure. Although, combined exposures did not induce any changes in the total BAL, LVP or sternal bone marrow cell counts, the mice displayed systemic leukocytosis at 24 hours, which was followed by leukopenia at 72 hours after exposure.

These are displayed in the peripheral blood cell counts. The femur bone marrow cell counts show the late spike at 72 hours when compared to all the time points. Please note the absence of Siglec-F and CX3CRI staining in polymorphonuclear cells at 24 hours in the image of BAL cytospin.

BAL and LVP cytospins showed presence of large CD11b and GR1 positive mononuclear cells at zero hours. Polymorphonuclear cells presented as a predominant cell type in all the compartments after exposure. BAL total protein content was highest at 36 hours after combined exposure.

However, the LVP protein was unchanged after exposure. Levels of eotaxin-2 and interleukin-2 were highest at the four hours in LVP. These chemokines were reduced in the BAL fraction.

Interestingly, the neutrophil alarming IL-16 and many innate chemokines were altered in the LVP, but not the BAL fraction. At baseline more than 95%of the BAL cells were mono nuclear showing cortical actin, lamellipodia, stress and tubulin fibers, and they were strongly positive for reduced mitotracker staining. At four hours, we observed circular BAL cells, which had lost actin, but showed a spread out microtubule network as shown in red and lower reduced mitotracker staining.

Early after the combined exposure, BAL cells appeared double positive for calcein and ethidium homodimer-1, which indicates partially compromised cells. At 36 hours, the BAL cells were largely viable, that is green. We observed discrete ATP alpha and Ly6G staining in BAL alveolar macrophages, and neutrophils.

We observed a reduction in DAPI normalized ATP alpha and an increase in intracellular Ly6G protein content. The combined exposures induced transient expressions of Bal, natural killer cell and K1.1, neutrophil ATP, sub unit PETA, proliferation factor, Ki-67 and sustained expressions of neutrophil GR1, platelet CX3CRI and the anti-angiogenic angiostatin protein in BAL cells after exposure. Although bronchiolar and alveolar septal damage was expected, it was surprising to observe cellular aggregates in larger vessels at 36 hours after exposure.

Finally, we have summarizer results in this table. We hope that our murine model serves as a readily accessible prototype that can be reproduced in level two containment labs for studying the mechanisms of invasive cell death and infections. Thank you.