The overall goal of the following protocol is to examine the effects of microbial infection on chronic inflammation in vivo using a physiologically relevant animal model. This is achieved by orally infecting mice with porphyromonas gingivalis to induce local and systemic chronic inflammation. MRI can then be utilized to measure the progression of systemic inflammation in live animals, while local inflammatory bone loss is assessed by micro CT and atherosclerotic plaques can be visualized by on face lipid staining after sacrifice.
Ultimately, differences in alveolar bone volume and the accumulation of atherosclerotic plaques between P gingivalis infected mice and uninfected controls can be measured based on micro CT analysis and on face lipid staining respectively. The major advantage of our animal model is that it allows you to examine both host and pathogenic mechanisms involved in chronic inflammation and specifically at sites distant from infection. So you can look at systemic sites as well as local sites of infection Using transgenic mice and defined bacterial mutants.
This model can help identify key aspects of pathogenesis, such as host and microbial factors contributing to disease. Although this method can provide insight into the specific host and pathogen mechanisms involved in inflammatory bone loss and atherosclerosis, it can also be adapted for other mouse models of disease, including rheumatoid arthritis, diabetes, and cancer. This is the high field MRI core facility for Boston University, and my postdoc fellow Ning is gonna demonstrate aspects of mouse imaging Begin by streaking frozen stalks of peach gingivalis 3 81 onto anaerobic blood agar plates, and incubate the plates in an anaerobic chamber at 37 degrees Celsius.
After three to five days, use the plate grown organisms to inoculate five milliliter liquid cultures of brain heart infusion broth, or BHI. Following overnight growth, transfer the cultures to 45 milliliters of BHI and incubate the cultures anaerobically for an additional 18 to 24 hours. At mid log phase, harvest the bacteria by centrifugation for 10 minutes at 7, 000 GS and room temperature, and then thoroughly re suspend the pellet in five milliliters of PBS with a serological pipette after washing the pellet three times in 50 milliliters of PBS Resus, the pellet in PBS such that a one to 10 dilution of the culture has an optical density of 1.0 at 660 nanometers.
Then way out enough medium viscosity carboxy methyl cellulose to achieve a 2%weight to volume solution and slowly add the carbox ethyl cellulose to the bacterial suspension while vortexing to avoid clumping. To measure the progression of the inflammation in C two. First place an animal holder with the mouse's head in a supine position into a 30 millimeter vertical probe and into the vertical bore of an 11.7 TMRI scanner.
After wobbling and shimming processes, use a rare sequence to acquire scout images along three orthogonal orientations to create axial coronal and sagittal images. Then after using low resolution magnetic resonance angiography to confirm the acquired images are within the target region, perform a high resolution magnetic resonance angiography of the innominate artery with an ungated 3D gradient echo sequence using the following parameters. A slab thickness of 1.5 centimeters, a flip angle of 45 degrees, a repetition time of 20 milliseconds, an echo time of 2.2 milliseconds, a field of view of 1.5 by 1.5 by 1.5 centimeters, a matrix of 1 28 by 1 28 by 1 28, and a number of the average of four for a total scan time of approximately 22 minutes.
Then select an axial image of the denominate artery 0.3 to 0.5 millimeters below the subclavian bifurcation for ROI analysis To pin the aorta, keeping the tissue covered in PBS at all times. Begin by laying the aorta in the anatomic position with the bulbs of the aorta on the left. Then place temporary mnuchin pins consecutively at the following five locations, the top of the aorta below the third branch of the arch, midway of the descending aorta near the lower end of the descending aorta and above the branch of the femoral artery.
Next, use extra fine spring scissors to cut up the left side of the aorta starting at the left branch of the femoral artery all the way up to below the lowest branch of the ascending aorta. Then after cutting and pinning each branch to expose its inner surface, continue to pin the tissue. The entire inner surface is exposed and clearly visible from above and free of visual interference from the pins.
To stain the lipids and quantify the lesions, begin by covering the pinned aorta with freshly prepared Sudan four or oil red O solution. After 50 minutes, wash the tissue with 70%isopropanol for one to five minutes, and then gently rinse the aorta with double distilled water until the water coming off the tissue is no longer red. Next, cover the aorta with PBS and place a ruler next to the tissue to assist in the image calibration.
Then capture images of the tissue with the high resolution camera attached to a dissecting microscope, saving the images as TIFF files. Finally, in image analysis software manually trace each intimal surface to determine its area. Then to calculate the lesion area using automated color thresholding set a threshold for each image to define a color intensity that discriminates the lesions from the normal areas.
The percentage of the intimal surface covered by atherosclerotic lesions can then be calculated in this representative experiment with p gingivalis infected mice. Volumetric analysis revealed that infected mice exhibit significant bone loss compared with uninfected controls. Visual inspection of the reconstructed Hemi maxilla illustrates an increase in exposed surface area of the molar roots in infected mice as compared to controls an atherosclerosis prone.
A POE deficient mice. P gingivalis induces a chronic inflammation that drives alveolar bone loss and inflammatory plaque deposition within the aortic sinus and a nominate artery as early as 24 hours following the last infection, which can be monitored in live mice by serial in vivo, MRI at various time points post-infection as in these representative images. Further on face measurements of Sudan four stain aorta demonstrate that p gingivalis infection significantly increases lipid deposition and lesional areas on the intimal surface.
Additionally, immunohistochemical analysis of aortic sinus lesions reveals an increased microphage infiltration and elevated expression of the innate immune receptor toll-like receptor two IVs infected mice. While attempting this procedure, it's important to consider the experimental duration time points at which inflammation is evaluated should appropriately align with disease progression. After MR Imaging of others, other methods like in face standing or immune histo chemistry can be performed to answer additional questions like the global atherosclerotic burden or the cellular components of the lesion.
After watching this video, you should have a good understanding of an in vivo mouse model, which allows you to examine both host and pathogenic factors involved in chronic inflammation. Don't forget that working with pathogens can be extremely hazardous and that precautions such as proper use of PPE should always be taken while performing this procedure.
