Small animal imaging enables researchers to examine below the surface and understand cellular processes in a relevant biological environment. While in vitro and ex vivo methods provide valuable information about a drug’s mechanism of action, tracking a compound’s biological effects in an animal after administration provides real time information for rapid decision making. Imaging enables researchers to monitor disease progression, biological phenomena and therapeutic efficacy over time in the same animal, something that ex vivo methods simply can not do making imaging a powerful method compared to traditional immunochemistry or histopathology.
While there are various small animal imaging methods available, this article will focus on optical molecular imaging as it is the most widely used small animal imaging modality due to its relative affordability and high throughput capabilities. By using imaging or contrast agents, researchers can rapidly survey targets associated with disease pathology in hundreds of animals per day.
Targeted imaging strategies include molecular probes that react in response to proteolytic enzyme activity. The two main types of active protease probes are substrate probes and activity based probes (ABP). A substrate probe fluoresces upon cleavage by its respective protease, allowing the fluorescent signal to migrate away from the target site. In contrast, activity based probes are small molecules that contain a substrate recognition sequence flanked by a fluorophore and a reactive functional group (often termed a warhead) that covalently binds to the active site of the target protease. Irreversible covalent binding of the ABP to the enzyme results in a fluorescent signal that is retained at the site of proteolytic activity. After imaging, tissue can be biochemically analyzed to evaluate ABP targets. Such biochemical monitoring of targets is not possible when substrate probes are used.
Figure 1: Fluorescent protease probes for non-invasive imaging. (A) Schematic diagram illustrating activation of substrate probes by a target protease. The probe is quenched by close proximity. Upon cleavage by a protease, smaller fragments containing the unquenched fluorophore are released and may not be retained at the site. (B) Schematic diagram illustrating activation of fluorescent activity based probes (ABP) labeling a target protease. The probe covalently binds in the active site of the target protease and is retained at the target location. The probe emits fluorescence even in the absence of protease. (C) Schematic diagram illustrating activation of a quenched ABP. The probe is optically silent by close proximity to a quenching group that is released upon covalent modification of the target protease.
Activty based probes enable researchers to study protease activity associated with disease pathology or therapeutic efficacy following drug administration. Both caspases and cathepsins are proteases of interest, particularly in the field of cancer. Cathepsins are highly upregulated in tumors making them ideal targets for following tumor progression and metastasis, while treatments causing cell death by apoptosis can be monitored with ABPs that recognize the caspase family of proteases.
The smart iABPTM
In an effort to minimize background signals of activity based probes, quenching mechanisms to render the probes optically silent until activated by their target enzyme are often employed. While auto-quenching and dye-dye self-quenching probes are two quenching strategies used, the fluorophore-quencher probe design in the smart iABPTM generates extremely low background fluorescence yielding a superior in vivo signal-to-background ratio. Since the smart iABPTM only fluoresces once activated, the wash step prior to imaging can be eliminated from many in vitro and ex vivo staining applications, making iABPTM ideal for high content screening.
Establishing the study design for non-invasive imaging
Typically, molecular probes are administered intravenously (IV) at peak protease activity. Animals are imaged at regular intervals for whole body fluorescence to capture the desired image once optimal clearance has occurred. When using a smart iABPTM probe whole body fluorescence is less of a concern since it is optically silent until bound by the target. Following live imaging, tissue can be excised and imaged ex vivo as well as homogenizing tissue for SDS-PAGE analysis, sectioning the tissue for fluorescence microscopy or digesting tissue for flow cytometery.
Figure 2: Optical imaging experiment in 4T1 tumor bearing mice using the smart iABPTM probe. (Top) Live animal imaging time course of a targeted smart iABP compared to control probe. (Middle) Tumor tissues were removed and imaged ex vivo. Following ex vivo imaging, tissues were homogenized and analyzed by SDS-page. Fluorescence intensity was compared between live animal, ex vivo and fluorescence gel imaging. (Bottom) Tissue sections were prepared and analyzed by fluorescence microscopy for iABP, CD68 and the overlay showing co-localiztion of the probe with CD68, a macrophage marker.*
Optimizing animal imaging studies
While molecular imaging probes offer researchers powerful tools for providing significant insight into disease progression and monitoring therapeutic efficacy of a drug, optimization is essential for successful imaging. In addition to choosing a high-quality probe as discussed above, the following points provide guidance for optimizing conditions.
Fluorophore selection
With optical imaging, the fluorophore chosen will have an impact on both the tissue autofluorescence as well as depth of penetration. Light does not travel in a straight line through biological tissue but rather is scattered and absorbed. Therefore, a fraction of the excitation and emission light is lost in optical imaging, however this phenomenon is reduced when using longer wavelenths such as near-infrared (NIR). As shown in the spectrum below, greater depth can be probed in the far red and NIR spectral region as the absorborption coefficient is at least one magnitude lower than the visible range. In addition to deeper photon tissue penetration in this spectrum, far red and NIR wavelengths have less light scatter, minimal tissue autofluoresence and higher optical contrast.
Figure 3: Spectral region for visible and near infrared wavelenths.
Low fluorescent rodent chow
Standard rodent chow contains high amounts of unrefined chlorophyll ingredients (e.g. Alfalfa), which can create autofluoresence in the most common imaging range of 650 to 700 nM. Therefore, a diet of low fluorescent feed starting at least one week prior to imaging is helpful in reducing background autofluoresence.
Figure 4: Animal fed standard rodent chow (left) vs mice fed a purified Low fluorescence diet (right). Image courtesy of Research Diets Inc.
Hairless mice or hair removal
Dark pigment in the hair or skin of rodents will absorb light. Hair removal is important to improving image quality. Always remove hair in the areas to be imaged.
Animal handling, orientation and imaging multiple animals
Animal handling can impact the quality of an imaging study. Anesthesia will slow the metabolism and decrease the body temperature, which may affect the kinetics of probe circulation and clearance. It is recommended to administer the probe at the same time point in relation to anesthesia throughout the imaging study.
Detection of the probe may vary based on the position of the animal, which can affect tissue depth. Place the animal so that the probe is closest to the detector. If the location is unknown, it is recommended to image both sides of the animal. Intense signals for example caused by accumulated probe in the kidneys or liver are also recommended to be covered since extremely bright sources will dictate the sensitivity settings of the imaging equipment. If imaging multiple animals at once, utilize guards to minimize reflectance of signal from neighboring animals
Best location for the tumor
Tumor location selection can play an important part of imaging success. Most dye/probe combination are excreted by either the liver or the kidney. So placing tumor implants outside of this region can significantly reduce the overall background noise from biological clearance. The one tradeoff to consider is tumor growth. Tumor implants placed out on limbs can have slower growth rates than those closer to the center of the body. Additionally, the deeper the tumor placement, the more limiting it will be for optical imaging.
Injection tips
Most injections are done via the tail vein. It is important to image the tail vein location to assess the injection success. If a high amount of probe is visible at or near the injection site, this can be a sign of an inadequate injection. Making a notation of this can be helpful in the post imaging analysis.
Imaging timepoint(s)
Providing optimal time for biodistribution and circulation of the probes are critical. Each dye/probe and animal model will differ. Therefore, a time course study is always recommended for initial optimization. In general, one advantage of using quenched ABPs is that the overall lower background will generally mean that the optimal time point from injection to image will be on the lower side. Of course, depending on the target being imaged such as with caspases, determining the optimal injection of the probe following drug administration is also crucial and will require optimization as each drug may have a different mechanism.
Controls
There are many variables when imaging in vivo, therefore it is highly recommended to have strong control groups (known positive control and placebo control). Additionally, including baseline imaging prior to dye/probe injection will be useful during data analysis.
Conclusion
Small animal imaging has greatly advanced researcher’s perspectives on cellular events involved in disease progress and/or therapeutic modulation of disease. Following these tips and suggestions as well as the evolution of advanced molecular probes has enabled scientists to look at changes in disease due to treatment over time.
*Data generated by Matt Bogyo’s lab at Stanford University, developers of the smart iABPTM commercialized by Vergent Bioscience. Permission obtained by J. Am Chem Soc.
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