The overall goal of this procedure is to visually distinguish acute and chronic inflammation in living mice. This is accomplished by subcutaneous injection of four, ball 12 me state, 13 acetate or PMA to trigger an intense acute inflammatory response. Next intraperitoneal injection of chemiluminescent substrate, luminol, or lucigen is performed to induce light signals at the site of inflammation.
Luminol luminescence indicates acute inflammation. While lucigen in luminescence reveals areas of late stage or chronic inflammation, the animal is then placed inside a bioluminescence imager, which visualizes the luminescence signals produced by the chemiluminescence substrates at the sites of inflammation. Sequential imaging steps are then performed in order to capture the maximal luminescence emission for quantitative analysis, and the imaging procedure is repeated at various time points to follow up different inflammatory responses.
Ultimately, the longitudinal imaging results show differential inflammatory responses based on the emission kinetics of the two luminescent substrates. Given the important role of inflammation in variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions. Demonstrating the procedure will be Mrs.
Tanya Topper and imaging technologies from a lab. Begin this protocol with preparation of the reagents and solutions as described in the text protocol anesthetize NCR nude mice and confirm general anesthesia by loss of movement and a constant respiratory rate. Transfer an animal to a nose cone supplied with isof fluoride mixed gas to keep the animal under anesthesia.
Use an isopropyl alcohol wipe to clean and disinfect the injection site on the left flank. Using sterile technique, inject 50 microliters of the PMA inoculation solution into the subcutaneous space. On the left flank.
Remove excess fluid from the injection site by using an isopropyl alcohol pad. Move the animal back to the housing cage and closely monitor its recovery from anesthesia. In order to assist in the recovery, use a heating pad to keep the animal warm following animal anesthetization.
As before, prepare to intraperitoneal, inject 10 milligrams per milliliter of luminol solution for acute inflammation imaging, or 2.5 milligrams per milliliter of lucigen in solution for chronic inflammation imaging. The final dosage is 100 milligrams per kilogram for luminol and 25 milligrams per kilogram. For lucin, a lower dose of lucin can be used to avoid possible toxicity.
For some mouse strains, transfer the animal into the imaging chamber of a bioluminescence imaging system. Perform sequential bioluminescence imaging at one minute intervals. Each imaging step is comprised of a one minute acquisition time.
F-stop equal to one binning equal to 16 and a zero second delay. Within the image acquisition panel, enable sequential multi-step imaging by clicking on the sequence setting button. Provide 15 one minute imaging steps in the acquisition profile to determine maximal luminescence output.
The 15 minute imaging session allows sufficient time for substrate absorption and systemic circulation. Following image acquisition, transfer the animal from the imaging chamber to the housing cage. In order to assist in the recovery, use a heating pad to keep the animal warm.
During post-acquisition analysis, use the imaging software package to calculate peak total bioluminescence signal through standardized regions of interest or ROIs. The images are presented as radiance with minimal and maximal threshold indicated. Quantitative data are presented as total flux in photons per second.
Per R-O-I-P-M-A is a potent protein kinase C agonist that activates fox for superoxide ion production and triggers intense acute inflammatory responses. Subcutaneous injection of PMA in NCR nude mice caused rapid skin irritation and acute inflammation at injection sites. These images represent longitudinal bioluminescence imaging using luminol and lucin as substrates at early stages of inflammation.
Significant luminol bioluminescence is observed indicating high levels of neutrophil myelo peroxidase or MPO activity at the sites of PMA injection. Conversely, no significant lucid in bioluminescence was observed during these early stages of inflammation starting on day two, scab formation and therefore reduced luminescence. Intensity was observed as wound healing became apparent.
A study increase in lugen, in bioluminescence was observed. These results illustrate that luminol bioluminescence is associated with the acute phase of inflammation, whereas lugen in bioluminescence is closely associated with tissue repair in the late phases of inflammation.Together. This result demonstrate the ability to use differential sub specificity to distinguish acute and chronic inflammatory stages.
Importantly, imaging mediated by the endogenous enzymatic activity of the immune cells, and there's no need for cock expression of reporters.
