Name: visualizing lung cellular adaptations during combined ozone and lps induced murine acute lung injury
Uploaded: 2021-03-21T14:00:00
Description: Watch this Scientific Journal Video about Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury at JoVE.com

The research conducted is funded by President&#39;s NSERC grant as well as start-up funds from the Sylvia Fedoruk Canadian Center for Nuclear Innovation. The Sylvia Fedoruk Canadian Center for Nuclear Innovation is funded by Innovation Saskatchewan. Fluorescence imaging was performed at the WCVM Imaging Centre, which is funded by NSERC. Jessica Brocos (MSc Student) and Manpreet Kaur (MSc Student) were funded by the start-up funds from the Sylvia Fedoruk Canadian Center for Nuclear Innovation.

The study design was approved by the University of Saskatchewan&#39;s Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines for humane animal use. Six-eight week old male C57BL/6J mice were procured. NOTE: Euthanize any animals which develop severe lethargy, respiratory distress or other signs of severe distress before scheduled end point.
NOTE: Prepare the following: 27-18 G needle-blunted (will depend on the mouse tracheal diameter), appropriately sized P...

<ol>
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The methods presented in the current study highlight the usefulness of multiple compartment analysis to study multiple cellular events during lung inflammation. We have summarized the findings in Table 2. We and many labs have extensively studied the murine response to intranasal LPS instillation, which is marked by rapid recruitment of lung neutrophils, which peaks between 6-24 h following which, resolution kicks in. And recently, we have shown that sub-clinical O3 (at 0.05 ppm for 2 h) alone...

Acute lung injury (ALI) is a crucial pathologic response of lungs to infectious or other harmful stimuli which is marked by simultaneous activation of coagulative, fibrinolytic and innate immune systems1. Neutrophils promptly sense microbial as well as intracellular damage patterns through the Toll-like receptor (TLR) family2,3,4. Neutrophils release preformed cytokines and cytotoxic granule contents, which can then cause collateral tissue damage. The ensuing alveolar damage is marred with secondary cell death leading to release of molecules such as adenosine triphosphate (ATP)5, thus setting in a vicious cycle of immune-dysregulation.
An unsolved problem in the understanding of ALI relates to the question of how the injury is initiated within the alveolar membrane. The electron transport complex V, F1F0 ATP synthase, is a mitochondrial protein known to be expressed ubiquitously, on cell (including endothelial, leukocyte, epithelial) plasma membrane during inflammation. The cell cytoskeleton which is comprised of actin and tubulin, harbors many cell shape and function modulating as well as mitochondrial proteins, respectively. We have recently shown that blockade of the ATP synthase by an endogenous molecule, angiostatin, silences neutrophil recruitment, activation and lipopolysaccharide (LPS) induced lung inflammation6. Thus, both biochemical (ATP synthase) and immune (TLR4) mechanisms might regulate the alveolar barrier during lung inflammation.
Exposure to ozone (O3), an environmental pollutant, impairs lung function, increases susceptibility to pulmonary infections, and short low-levels of O3 exposures increase the risk of mortality in those with underlying cardiorespiratory conditions7,8,9,10,11,12,13,14. Thus, exposure to physiologically relevant concentrations of O3 provides a meaningful model of ALI to study fundamental mechanisms of inflammation7,8. Our lab has recently established a murine model of low-dose O3 induced ALI15. After performing a dose and time-response to low O3 concentrations, we observed that exposure to 0.05 ppm O3 for 2 h, induces acute lung injury that is marked by lung ATP synthase complex V subunit &#946; (ATP&#946;) and angiostatin expression, similar to the LPS model. Intravital lung imaging revealed disorganization of alveolar actin microfilaments indicating lung damage, and ablation of alveolar septal reactive oxygen species (ROS) levels (indicating abrogation of baseline cell signaling) and mitochondrial membrane potential (indicating acute cell death) after 2 h exposure to 0.05 ppm O315 which correlated with a heterogeneous lung 18FDG retention16, neutrophil recruitment and cytokine release, most notably IL-16 and SDF-1&#945;. The take-home message from our recent studies is that O3 produces exponentially high toxicity when exposed at concentrations below the allowed limits of 0.063 ppm over 8 h (per day) for human exposure. Importantly, no clear understanding exists on whether these sub-clinical O3 exposures can modulate TLR4-mediated mechanisms such as by bacterial endotoxin17. Thus, we studied a dual-hit O3 and LPS exposure model and observed the immune and non-immune cellular adaptations.
We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid (i.e., BAL) which samples the alveolar spaces, the lung vascular perfusate (i.e., LVP) that samples the pulmonary vasculature and the alveolar septal interstitium in the event of a compromised endothelial barrier, left lung cryosections, to look into resident parenchymal and adherent leukocytes left in the lavaged lung tissue, peripheral blood which represents the circulating leukocytes and the sternal and femur bone marrow perfusates that sample the proximal and distal sites of hematopoietic cell mobilization during inflammation, respectively.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>33-plex Bioplex chemokine panel</td>
			<td>Biorad</td>
			<td>12002231</td>
			<td></td>
		</tr>
		<tr>
			<td>63X oil (NA 1.4-0.6) Microscope objectives</td>
			<td>Leica</td>
			<td>HCX PL APO CS (11506188)</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 350 conjugated goat anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11045</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488 conjugated goat anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11002</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488 conjugated phalloidin</td>
			<td>Invitrogen</td>
			<td>A12370</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 555 conjugated mouse anti-&#945; tubulin clone DM1A</td>
			<td>Millipore</td>
			<td>05-829X-555</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 568 conjugated goat anti-hamster IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21112</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 568 conjugated goat anti-rat IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11077</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 633 conjugated goat anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21070</td>
			<td></td>
		</tr>
		<tr>
			<td>Armenian hamster anti-CD61 (clone 2C9.G2) IgG1 kappa</td>
			<td>BD Pharmingen</td>
			<td>553343</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 J Mice</td>
			<td>Jackson Laboratories</td>
			<td>64</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>Leica TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>aliquot in 2 &#181;l stocks and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Inverted fluorescent wide field microscope</td>
			<td>Olympus</td>
			<td>Olympus IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine (Narketan)</td>
			<td>Vetoquinol</td>
			<td>100 mg/ml</td>
			<td>Dilute 10 times to make a 10 mg/ml stock</td>
		</tr>
		<tr>
			<td>Live (calcein)/Dead (Ethidium homodimer-1) cytotoxicity kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ATP5A1 IgG2b (clone 7H10BD4F9)</td>
			<td>Invitrogen</td>
			<td>459240</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ATP5&#946; IgG2b (clone 3D5AB1)</td>
			<td>Invitrogen</td>
			<td>A-21351</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-NK1.1 IgG2a kappa (clone PK136)</td>
			<td>Invitrogen</td>
			<td>16-5941-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660 nm protein assay</td>
			<td>Thermoscientific</td>
			<td>22660</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-angiostatin (mouse aa 98-116) IgG</td>
			<td>Abcam</td>
			<td>ab2904</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-CX3CR1 IgG (RRID 467880)</td>
			<td>Invitrogen</td>
			<td>14-6093-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-Ki-67 (clone SolA15) IgG2a kappa</td>
			<td>Invitrogen</td>
			<td>14-5698-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-Ly6G IgG2a kappa (clone 1A8)</td>
			<td>Invitrogen</td>
			<td>16-9668-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-Ly6G/Ly6C (Gr1) IgG2b kappa (clone RB6-8C5)</td>
			<td>Invitrogen</td>
			<td>53-5931-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD16/CD32 Fc block (clone 2.4G2)</td>
			<td>BD Pharmingen</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced mitotracker orange</td>
			<td>Invitrogen</td>
			<td>M7511</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>20 mg/ml</td>
			<td>Dilute 2 times to make a 10 mg/ml stock</td>
		</tr>
	</tbody>
</table>

visualizing lung cellular adaptations during combined ozone and lps induced murine acute lung injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined O3 and LPS exposure leads to systemic inflammation and bone marrow mobilization at 72 h: Cell counts in different compartments revealed significant changes in peripheral blood and the femur bone marrow total cell counts upon combined O3 and LPS exposures. Although combined O3 and LPS exposures did not induce any changes in the total BAL (Figure 1A) or LVP (Figure 1B</str...

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

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

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.

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