a murine skin wound infection model to study the immune response against a bacterial pathogen

A Murine Skin Wound Infection Model to Study the Immune Response against a Bacterial Pathogen

For Staph aureus inoculation, fill a 28-gauge insulin syringe with 50 microliters of the prepared bioluminescent bacterial inoculant, and use a finger to pull the dermis of the wounded animal to the side. Holding the syringe nearly parallel to the tissue, slowly insert the syringe into the tissue until a sudden decrease in resistance is felt, indicating piercing of the fascia.
With the needle placed in the center of the wound, slowly deliver the entire volume of the inoculant. Confirm that the inoculant forms a bubble at the center of the wound with minimal leakage or dispersion, and remove the syringe slowly from the animal. Then, return the animal to its cage with heat and monitoring until full recovery.

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Nanyang Technological University

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JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mouse Source and Housing
<ol>
	<li>Derive LysM-eGFP (enhanced green fluorescent protein (eGFP) inserted in the Lysozyme M (LysM) promoter region) mice on a C57BL/6J genetic background as described previously. Derive LysM-EGFP&#215;MyD88-/- mice by crossing LysM-EGFP mice with MyD88-/-&#160;(Myeloid differentiation primary response 88 protein) mice on a C57BL/6J background.</li>
	<li>House mice in a vivarium. For these studies, animals were housed at the University of California, Davis in groups prior to surgery and singly housed following surgery. Use mice between the ages of 10-16 weeks.</li>
</ol>
2. Bacterial Preparation and Quantification
<ol>
	<li>Remove the bioluminescent Staphylococcus aureus strain of interest from -80 &#176;C storage to thaw on ice. Streak on a 5% bovine blood agar plate. Incubate the streaked plate in a humidified incubator at 37 &#176;C overnight (16 h). 
	NOTE: In this protocol, the ALC2906 SH1000 strain was used. This strain contains the shuttle plasmid pSK236 with the penicillin-binding protein 2 promoters fused to the luxABCDE reporter cassette from Photohabdus luminescens.</li>
	<li>Prepare tryptic soy broth (TSB) by mixing 0.03 g of TSB powder per mL of pure water, and autoclave TSB to sterilize. When cooled, add any necessary antibiotics using a sterile technique. In this protocol, add 10 &#181;g/mL of chloramphenicol to the TSB to select for expression of the pSK236 shuttle plasmid, which contains the bioluminescence luxABCDE cassette.</li>
	<li>Pick 3-4 separate colonies from the S. aureus plate into TSB with 10 &#181;g/mL chloramphenicol to start an overnight culture. Incubate bacteria on an incubating shaker at 37 &#176;C overnight (16 h).</li>
	<li>Start a new bacterial culture from the overnight culture by diluting a sample 1:50 into TSB with 10 &#181;g/mL chloramphenicol. Culture in an incubating shaker at 200 rpm and 37 &#176;C.</li>
	<li>Two hours after splitting the S. aureus, monitor the optical density at 600 nm (OD600) on a spectrophotometer. Observe the OD600 vs. time to find mid-logarithmic phase growth. For the ALC2906 SH1000 strain, an OD600 of 0.5 is mid-logarithmic and corresponds to a concentration of 1 x 108 CFU/mL (CFU = colony-forming unit) (Figure 1).</li>
	<li>When OD600 is 0.5, wash bacteria 1:1 with ice-cold Dulbecco&#39;s phosphate-buffered saline (DPBS). Centrifuge the bacteria for 10 min at 3,000 x g and 4 &#176;C. Carefully decant the supernatant and add additional chilled DPBS and vortex thoroughly. Centrifuge once more for 10 min at 3,000 x g and 4 &#176;C.</li>
	<li>Carefully decant the supernatant. Resuspend the bacterial pellet at the desired concentration. For these studies, collect 3 mL of ALC2906 SH1000 and resuspend in 1.5 mL of phosphate-buffered saline (PBS), correlating to a bacteria concentration of about 2 x 108 CFU/mL. Keep bacteria on ice until use.</li>
	<li>To verify bacteria concentration, dilute 100 &#181;L of the bacterial sample 1:10,000 and 1:100,000 in PBS. Plate 20 &#181;L aliquots on an agar plate. Incubate at 37 &#176;C in a humidified incubator for 16 h. Count CFUs by gross examination and calculate a bacterial concentration the following day.</li>
</ol>
3. Excisional Skin Wounding and Inoculation with S. aureus
<ol>
	<li>Administer 100 &#181;L of 0.03 mg/mL buprenorphine hydrochloride (~0.2 mg/kg) to each mouse via intraperitoneal injection.</li>
	<li>Twenty minutes post-injection, place 2-4 mice in a chamber with 2-3 LPM oxygen with 2-4% isoflurane. Once mice are fully anesthetized, transfer the mice one at a time to a nose cone connected to 2-3 LPM oxygen with 2-4% isoflurane. Verify mice are fully anesthetized by firmly pinching each rear paw between a thumb and forefinger. Proceed to the next step if the animal does not respond to the pinch.</li>
	<li>Shave a 1-inch by 2-inch section on the back of the mouse with electric clippers and clear the area of fur clippings using a clean wipe or gauze. Avoid using depilatory lotion because it may cause excess inflammation.</li>
	<li>Clean the back of the mouse first with 10% povidone-iodine-soaked gauze and then with 70% ethanol-soaked gauze. Clean the area in a spiral pattern, moving outward from the center of the surgical area. Wait approximately 1 minute for the surgical area to dry prior to surgery.</li>
	<li>Hold the shaved back of the mouse loosely between two fingers and firmly press a sterile 6 mm punch biopsy at the center of the prepared surgical area. Do not pull the skin taut.
	<ol>
		<li>Twist the punch biopsy to create a circular outline on the skin that fully cuts through the skin in at least one section of the outline. Be careful not to cut into the underlying fascia or tissue.</li>
		<li>Use sterile scissors and forceps to cut through the epidermis and dermis following the circular pattern imprinted by the punch biopsy.</li>
	</ol>
	</li>
</ol>
4. S. aureus Inoculation
<ol>
	<li>Fill a 28 G insulin syringe with the desired bioluminescent bacterial inoculant. In this study, administer a concentration of 1 x 108 CFU/mL (50 &#181;L). Do not administer more than 100 &#181;L of volume.</li>
	<li>Inject 50 &#181;L of inoculant between the fascia and tissue in the center of the wound on the back of the mouse. Ensure that the inoculant forms a bubble at the center of the wound with minimal leakage or dispersion.
	<ol>
		<li>Pull the dermis to the side, hold the syringe nearly parallel to the tissue, and slowly push the syringe into the tissue until a sudden decrease in resistance is felt, which indicates piercing of the fascia. Carefully lead the syringe into the center of the wound and dispense the inoculant slowly. Remove the syringe slowly from the animal.</li>
	</ol>
	</li>
	<li>Inject the same volume of sterile PBS into the wounds of uninfected animals as described above.</li>
	<li>Return the animal to its cage. Place the cage under a heat lamp or on top of a heating pad, and monitor the animal until recovery from anesthesia.</li>
</ol>
5. In Vivo bioluminescence imaging (BLI) and fluorescence imaging (FLI)
<ol>
	<li>Initialize the whole animal imager through the instrument software. Anesthetize mice in a chamber receiving 2-3 LPM oxygen with 2-4% isoflurane. Deliver anesthesia to the nosecones inside the imager.</li>
	<li>Place the wounded and infected mouse into the imager. Position the mouse such that the wound is as flat as possible. Use the following sequence set-up to image the mice.
	<ol>
		<li>Select Luminescence and Photograph as the imaging mode. The exposure time is 1 min at small binning and F/stop 1 (luminescence) and F/stop 8 (photograph). The emission filter is Open. Click the Acquire button to record the image.</li>
		<li>Select Fluorescence and Photograph as the imaging mode. The exposure time is 1 s at small binning and F/stop 1 (Fluorescence) and F/stop 8 (photograph) with an excitation wavelength of 465/30 nm and an emission wavelength of 520/20 nm with a high lamp intensity. Click the Acquire button to record the image.</li>
	</ol>
	</li>
	<li>Return the animal to its cage and monitor it until it recovers from anesthesia.</li>
	<li>Image mice daily as described above.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>14 mL Polypropylene Round-Bottom Tube</td>
			<td>Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>6mm Disposable Biopsy Punch</td>
			<td>Integra Miltex</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioluminescent S. aureus</td>
			<td>Lloyd Miller, Johns Hopkins</td>
			<td>ALC 2906 SH1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Blood Agar, 5%, Hardy Diagnostics</td>
			<td>VWR</td>
			<td>10118-938</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenoprhine hydrochloride injectable</td>
			<td>Western Medical Supply</td>
			<td>7292</td>
			<td>0.3 mg/mL</td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>Jackson Labratory</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol (crystalline powder)</td>
			<td>Fisher BioReagents</td>
			<td>BP904-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (1X)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>Becton, Dickson and Company</td>
			<td>329461</td>
			<td>.35 mm (28 G) x 12.7 mm (1/2&#39;&#39;)</td>
		</tr>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image Software &#8211; IVIS Spectrum Series</td>
			<td>Perkin Elmer</td>
			<td>128113</td>
			<td></td>
		</tr>
		<tr>
			<td>LysM-eGFP Mice</td>
			<td>Thomas Graff Albert Einstein College of New York</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvolume Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>MyD88 KO Mice</td>
			<td>Jackson Labratory</td>
			<td>9088</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-woven sponges</td>
			<td>AMD- Ritmed Inc</td>
			<td>A2101-CH</td>
			<td>5 cm x 5 cm</td>
		</tr>
		<tr>
			<td>Povidone Iodine 10% Solution</td>
			<td>Aplicare</td>
			<td>697731</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Becton, Dickson and Company</td>
			<td>211825</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Anderson, L. S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates a technique to generate a murine model of skin wound infection. Upon infecting mice &#8212; engineered to express a fluorescent protein in neutrophils &#8212; with a genetically modified bioluminescent Staphylococcus aureus &#8212; a pathogenic bacteria. The bacterial burden at the infection site and the neutrophil recruitment dynamics can be studied using whole animal imaging.

<img alt="Figure 1" src="/files/ftp_upload/21695/21695_Figure1.jpg" /> 
Figure 1: Correlation between OD600 and CFU counts for ALC2906. 3-4 ALC2906 SH1000 colonies were picked from an agar plate and transferred into TSB with 10 &#181;g/mL chloramphenicol for overnight culture. The next day, the suspension was split 1:50 into TSB with 10 &#181;g/mL chloramphenicol and cultured. Optical density at 600 nm (OD600) was measured in regular intervals after 2 hours using a spectrophotometer. At each measurement, the bacteria were diluted 1:100,000 in ice-cold PBS and aliquoted onto an agar plate for overnight incubation. CFUs were counted the following day to calculate the initial concentration and correlated to OD600. N = 3 with 4 different OD600 measurements per experiment.

Source: Anderson, L. S., et al. A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection. J. Vis. Exp. (2019).
This video ...

Take an anesthetized mouse engineered to express a fluorescent protein in neutrophils. Shave the dorsum and create a circular cut. Excise the cut skin to reveal the fascia &#8212; a connective tissue layer underneath.
Take a syringe containing a suspension of Staphylococcus aureus &#8212; a pathogenic bacterium &#8212; genetically modified to produce bioluminescence. Inject the suspension between the fascia and the underlying tissue generating a localized wound infection.
The pattern recognition receptors on stromal cells in the fascia recognize the pathogen-associated molecular patterns on the bacteria. This binding induces the release of pro-inflammatory cytokines and chemoattractants &#8212; resulting in inflammation.
Chemoattractants recruit circulatory neutrophils at the infection site. Incoming neutrophils engulf the bacteria into a phagosome that fuses with cytoplasmic granules, causing antimicrobial peptide release.
NADPH oxidase &#8212; an enzyme complex &#8212; assembles on the phagosome membrane and produces reactive oxygen species or ROS. The antimicrobial peptides and ROS cause bacterial degradation.
As a counter-response, the bacteria secrete virulence factors that permeabilize the neutrophil membrane, causing cell lysis. The bacteria also release enzymes for the deposition of fibrin to avoid phagocytosis.
The immune response leads to an abscess &#8212; a fibrin capsule containing bacterial cells, neutrophils, and dead cells.
Record the neutrophil fluorescence and bacterial bioluminescence to visualize the bacteria-neutrophil interaction.

72 Views. Model zakażenia rany skóry myszy do badania odpowiedzi immunologicznej na patogen bakteryjny

A Murine Skin Wound Infection Model to Study the Immune Response against a Bacterial Pathogen

Transkrypcja

A Murine Skin Wound Infection Model to Study the Immune Response against a Bacterial Pathogen