Acute inflammation preclinical and in vivo imaging


Overview


Acute inflammation is an initial biological response of vascular tissues to tissue injury or infection. Several diseases are characterized by acute inflammation, including bacterial and fungal infections, inflammatory bowel disease, Crohn’s disease, kidney, lung and liver diseases, trauma, and various degenerative diseases including arthritis. Acute inflammation is characterized by vascular changes, including vasodilation, increased vascular permeability, edema, and increased blood flow.

The difficulty in studying inflammation in vitro increases the importance of robust and quantitative in vivo techniques to assess progressive inflammation and therapeutic intervention. In vivo imaging targets for acute inflammation include proteases and other mediators involved in the inflammation process; leukocytes, monocytes and macrophages that migrate to the injured tissue; macromolecules that accumulate in inflamed tissues due to increased vascular permeability; and the vasculature itself.

The ability to non-invasively and quantitatively study the underlying biology of inflammatory immune responses in real time is critical in the development and monitoring of anti-inflammatory therapies. Understanding the pathophysiology of inflammatory disease processes at the cellular and biochemical level is a major challenge in basic research and drug development. 


Products for imaging acute inflammation


Our pre-clinical imaging agents fall into three main categories: Activatable, meaning they are optically silent until cleaved by a protease of interest; Targeted, meaning they bind to a receptor or other cellular membrane expressed in the disease of interest; or Vascular, meaning they localize to vasculature and are used to assess changes in vascular permeability. All three types of agents can be used to assess acute inflammation in vivo.  We also offer the cell labeling agent (VivoTrack™, see table below). Agent properties, protocols and instrument settings, can be found by clicking on the agent links below. All of our agents are compatible with FMT and IVIS imaging systems, provided your system has the capabilities to image the probes at the proper wavelengths.

AgentAgent TypeAgent MechanismAvailable Wavelengths and optimal in vivo imaging time (post-injection)Route of metabolism/ background tissue(s)Validated imaging methods
Cat B FAST™Activatable, fluorescentDetects cathepsin B activity, a lysosomal protease released by monocytes and macrophages to target inflammatory products, helping to degrade the cell matrix, cell debris and pathogens. Cathepsin B activity has been positively correlated with the degree of inflammation and neutrophil recruitment.680: 6-24 hours

750: 6-24 hours
Salivary glands, liver, kidneysIn vivo, flow cyometry, in vitro cell microscopy, frozen tissue labeling
MMPSense™Activatable, fluorescentDetects the activity of matrix metallo- proteases 2, 3, 7, 9, 12 and 13 (MMP2, MMP3, MMP7, MMP9, MMP12, MMP13), proteases secreted in response to acute inflammation. MMPs mediate degradation of essentially all components of the extracellular matrix.645 FAST*: 24 h (6-24 h)

680: 24 h (24-36 h)

750 FAST*: 12 h (12-24 h)
Liver, kidneys (645 FAST, 750 FAST)

Liver only (680)
In vivo
Neutrophil Elastase FAST™Activatable, fluorescentMeasures the activity of neutrophil elastase, which is secreted by neutrophils and macrophages during an inflammatory response.680: 3-6 hBladder, liver, intestinesIn vivo, frozen tissue labeling
ProSense®Activatable, fluorescentDetects cathepsin B, L, S and plasmin, which are released by immune cells in response to inflammatory mediators680: 24 h (24-48 h)

750: 24 h

750 FAST*: 6-24 h
Liver (680)

Low liver (750)

Low liver, bladder (750 FAST)
In vivo, flow cytometry, in vitro cell microscopy
RediJect COX-2 probeTargeted, fluorescentEfficiently target cyclooxygenase-2 (COX-2), which is normally absent from cells, but is found at high levels in inflammatory lesions and in many premalignant and malignant tumors600 nm: 3 hKidney, liver
In vivo, in vitro, cell microscopy
RediJect Inflammation ProbeTargeted, chemi- luminescentDetects myelo- peroxidase activity, which is released by activated phagocytes in both chronic and acute inflammationEmits at 425nm, 10 min post-injection, 5 min BLI acquisition time, internal fluorescent control imaged at 745 nm excitation and 800 nm emission 1-5 sec
Kidney
In vivo
FolateRSense™Targeted, fluorescentBinds to folate receptors, which are overexpressed in activated macrophages680: 6 h (6-24 h)KidneyIn vivo, flow cytometry, in vitro cell microscopy, frozen tissue labeling
AngioSense®Vascular, fluorescentWill accumulate in areas of vascular leakage associated with tumorigenesis and inflammation; used to assess vascular leak680: 24 h

750: 24 h
Low liver, lungIn vivo
AngioSPARK®Vascular, fluorescentNanoparticle for imaging vascularity, long PK profile, 20 hour half-life in plasma680: 24 h

750: 24 h
Long term tissue accumulationIn vivo
Superhance®Vascular, fluorescentLow MW albumin-binding agent that clears from the blood and tissue rapidly; used to image acute edema680: 3 h (1-3 h)BladderIn vivo
VivoTrack™Labeling, fluorescentCell labeling agent; can be used to label macrophages in vitro and track them in vivo to monitor recruitment and localization in acute inflammation680: variable hours In vivo, flow cytometry, in vitro cell microscopy
  1. Jochum, M., Machleidt, W. & Fritz, H. Proteolysis-induced pathomechanisms in acute inflammation and related therapeutic approaches. Agents Actions Suppl.42, 51–69 (1993).
  2. Warner, R. L. et al. Matrix metalloproteinases in acute inflammation: induction of MMP-3 and MMP-9 in fibroblasts and epithelial cells following exposure to pro-inflammatory mediators in vitro. Exp. Mol. Pathol.76, 189–195 (2004).
  3. Korkmaz, B., Horwitz, M. S., Jenne, D. E. & Gauthier, F. Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacol. Rev.62, 726–759 (2010).

Choosing the right agent

When choosing an agent for acute inflammation studies, several factors should be taken into consideration:

  • Fluorescent vs. Chemiluminscent agents: Be sure to choose the proper agent for your instrumentation. Some imagers can measure both chemiluminescence and fluorescence, while others can only measure one or the other. 
  • Dye excitation/emission wavelength: Some fluorescent agents are available with excitation wavelengths that range from 645 nm to 800 nm. Be sure to pick a wavelength that is appropriate for both your instrument and application. Most microscopes filters are not suitable for wavelengths above 680 nm.  However deep tissue imaging typically has less background fluorescence and works better with longer wavelengths (750 nm- 800 nm). 
  • Agent clearance time is typically faster for activatable agents (particularly the FAST™ platform) which allows for re-injection after shorter time periods.  
  • Agent specificity: For targeted and activatable agents, it is important to remember that the protein or enzyme that the agent is targeting needs to be present and well expressed in your mouse model. 
  • In vivo distribution: Some agents might accumulate in organs at timepoints which could interfere with your study. Be sure to check the biodistribution profile of each agent of interest. 
  • Type of imaging required for your study: Vascular agents for example, while useful in assessing vascularity changes in the mouse, do not make good agents for in vitro cell imaging or tissue analysis. If these types of analyses are desired, targeted or activatable agents might be a better choice for you.

For more information about agents to study chronic inflammatory models, please visit the Arthritis, Atherosclerosis, Bacterial Infection, or Pulmonary Inflammation sections.


Mouse model of acute inflammation


Carrageenan paw edema
A model commonly used to study acute inflammation in mice is carrageenan-induced paw edema (CPE). Inflammation and swelling are induced by injecting carrageenan into the footpad of the mouse. Carrageenan elicits an inflammatory response by stimulating pro-inflammatory agents such as neutrophils which migrate to the site of infection, resulting in edema within the first 3-4 hours. At later timepoints (~24 hours), edema decreases as macrophages infiltrate the site of injection. For a protocol on how to prepare carrageenan for mouse injections, click here.
In the examples below, PerkinElmer’s NIR imaging agents can be injected either at 3 or 24 hours after the administration of carrageenan to determine the extent of edema and cellular inflammation non-invasively. Unlike traditional methods (i.e. caliper measurements of footpad thickness) which effectively represent the magnitude of the inflammatory response, these fluorescent agents provide quantification of specific biological processes—such as vascular leak associated with edema.

inflammationfig1.jpg

Figure 1: Carrageenan is injected into the right hindpaw of a mouse. Vascular imaging agents are administered 3-24 hours post carrageenan injection. Topographic imaging using PerkinElmer’s FMT system allows quantitative detection through tissues, while 2D epifluorescence primarily measures surface weighted fluorescence in the outer mm of the tissue.


In Figure 2 below, paw thickness measurements at both early (2 hours) and late (24 hours) time points are compared to in vivo quantification of the vascular imaging agent AngioSense™ and the activatable imaging agents Neutrophil Elastase 680 FAST™ and Cat B 680 FAST™. 

inflammationfig2.jpg

Figure 2: Comparison of Paw Thickness Measurements to Imaging Agents Carrageenan Paw Edema. Paw thickness, vascular leak (AngioSense), neutrophil elastase activity (Neutrophil Elastase 680 FAST), and cathepsin activity (CAT B 680 FAST) were assessed in mice that were injected with 1% carrageenan at 2 hours (early) and 24 hours (late) post-CG injection.


In Figure 3, AngioSense is used to assess vascular leak in the presence or absence of the anti-inflammatory steroid dexamethasone. 

inflammationfig3.jpg

Figure 3: Assessment of Dexamethasone Therapy. Mouse hindpaws were injected with PBS, 10 mg/kg dexamathosone + 1% carrageenan, or 1% carrageenan alone. A) Tomographic images of three conditions. The fluorescent signal is represented as a maximum intensity projection (identical gains set for each mouse) of the 3D regions of interest established around each paw with quantitative color bars to represent fluorescence concentration (nM). B) Quantification of paw fluorescence for each of the three conditions. As expected, the steroid dexamethasone significantly inhibited the vascular response at 3 hours.



Application notes and posters


  • Application Note: A method of NIR fluorescent cell-labeling for in vivo cell tracking


Citations


Please visit our Citations Library to view publications on acute inflammation and our preclinical imaging agents.