In vivo Imaging Method to Distinguish Acute and Chronic Inflammation
August 16th, 2013
We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.
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.
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Overview
This study presents a non-invasive imaging technique to differentiate between acute and chronic inflammation in living mice. By utilizing chemiluminescent substrates, luminol and lucigenin, the method allows for the visualization of inflammation stages based on neutrophil and macrophage activity.
Key Study Components
Area of Science
- Neuroscience
- Immunology
- In vivo imaging
Background
- Inflammation is a critical response to injury and infection.
- Acute inflammation is characterized by rapid onset and resolution.
- Chronic inflammation can lead to various diseases.
- Current methods for assessing inflammation can be invasive.
Purpose of Study
- To develop a non-invasive method for visualizing inflammation.
- To distinguish between acute and chronic inflammatory responses.
- To utilize chemiluminescent substrates for real-time imaging.
Methods Used
- Subcutaneous injection of inflammatory triggers (e.g., PMA).
- Intraperitoneal injection of luminol for acute inflammation detection.
- Intraperitoneal injection of lucigenin for chronic inflammation detection.
- Bioluminescence imaging to visualize luminescence signals.
Main Results
- Luminol luminescence indicates areas of acute inflammation.
- Lucigenin luminescence reveals sites of chronic inflammation.
- Sequential imaging allows for quantitative analysis of inflammation.
- Different inflammatory responses can be monitored over time.
Conclusions
- The method effectively distinguishes between inflammatory stages.
- Non-invasive imaging can enhance understanding of inflammation.
- This technique may improve research on inflammatory diseases.
Frequently Asked Questions
Executive Industry Relevance
Non-invasive imaging of neutrophil and macrophage activity enables mechanistic de-risking in early inflammation research. By distinguishing acute and chronic phases, the method supports target validation and predictive confidence in therapeutic development. This approach reduces biological ambiguity in preclinical models of inflammatory diseases.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by visualizing neutrophil-mediated acute inflammation via luminol-dependent MPO activity.
- Scientific Value: Clarifies pathway involvement by tracking macrophage-mediated chronic inflammation through lucigenin-dependent Phox signaling.
- Operational Value: Enables biological de-risking through longitudinal, non-invasive monitoring of phagocyte dynamics in living models.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for compound screening by establishing baseline inflammatory phenotypes.
- Operational Value: Supports assay standardization through quantitative bioluminescence readouts in photons per second.
- Operational Value: Enhances screening readiness via reproducible, longitudinal imaging across multiple time points.
Translational & Preclinical Research
- Scientific Value: Aligns with disease-relevant systems by modeling neutrophil-to-macrophage transition in tissue repair.
- Scientific Value: Facilitates translational biomarker identification through differential substrate emission kinetics.
- Operational Value: Enables risk-adjusted advancement decisions by monitoring inflammation resolution over time.
Pipeline & Workflow Integration
The method integrates into discovery biology workflows by providing real-time, quantitative readouts of inflammatory cell activity prior to lead identification.
- Discovery Biology: Supports hypothesis testing by distinguishing MPO-driven neutrophil activity from Phox-driven macrophage activity.
- Screening: Delivers assay readiness through standardized ROI-based flux measurements and sequential imaging protocols.
- Analytics: Enables comparative condition analysis via peak total bioluminescence signal quantification.
- Translational Research: Connects discovery to preclinical validation by modeling inflammatory resolution and tissue repair phases.
- Enterprise Reuse: Functions as a reusable imaging platform for longitudinal study of inflammatory responses across therapeutic areas.
Operational & Enterprise Impact
- Scientific Value: Provides predictive confidence by reducing mechanistic ambiguity in inflammation pathway interpretation.
- Operational Value: Ensures standardization and reproducibility through defined substrate dosing and imaging parameters.
- Strategic Value: Improves go/no-go decisions by enabling early detection of chronic inflammation signals.
- Portfolio Impact: Supports risk-adjusted prioritization by visualizing therapeutic impact on inflammation phase transition.
Implementation Considerations
- Requires expertise in bioluminescence imaging and inflammatory model handling.
- Dependent on bioluminescence imager sensitivity and sequential acquisition capabilities.
- Necessitates cross-team standardization of ROI selection and flux quantification methods.
- Involves adaptation considerations for different mouse strains and inflammatory triggers.
- Includes practical limitations such as substrate specificity and signal attenuation over time.
