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1Experimental Pathology, University of Texas Medical Branch
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Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.
Keywords: Virology, Issue 70, Immunology, Medicine, Neuroscience, Molecular Biology, Pathology, IVIS, in vivo modeling, VEE, CNS, Neuroinvasion, Hume’s 3Rs, Encephalitis, bioluminescence, luciferase, virus
Poussard, A., Patterson, M., Taylor, K., Seregin, A., Smith, J., Smith, J., et al. In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus. J. Vis. Exp. (70), e4429, doi:10.3791/4429 (2012).
Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.
1. Animal Preparation
2. Animal Imaging
Disclaimer: Keep mice within their housing unit, the biosafety cabinet (BSC), or the XIC containment box at all times. The approved BSCs for this protocol are a Class II Type B1 or B2.
3. Return Mice and Data Analysis
With a genetically modified virus, TC83-Luciferase, we saw an increase in bioluminescent signal strength as the virus replication moves from the nasal region into the central CNS (Figure 1). Due to the high viral replication rate, we expect to see high levels of bioluminescent signal (Figure 2A) dependent upon the vector and the animal immune response to the vector. We expect this signal increase to continue to a peak, between days 5-7 post infection, in combination with viral replication peak within the CNS (Figure 2B). Following the signal and viral peak, we expect to see a decrease in signal both due to a decrease in viral replication and due to a loss of the of the luciferase gene. With these signal values we expect to be able to quantify viral load based upon the strength of the signal, providing an in vivo method to accurately calculate progressively increasing viral load in a single animal specific to TC83-Luciferase.
Utilizing this model as a means to analyze vaccines and antiviral treatments, we expect to be able to detect a difference between efficacious treatments based upon a reduction in bioluminescent signal. In our case, against TC83-Luciferase, we can utilize IP Ampligen (a Toll-like receptor 3 agonist) or vaccination three weeks before infection, to significantly reduce viral infection and bioluminescent signal throughout the study (Figure 3). This decrease can be directly correlated to a decrease in viral load within the CNS (data not shown). As a way to fully develop and visualize the technology, IVIS software LivingImage is capable of making 3-dimensional images of the bioluminescent signal (Figure 4) which allows us to easily identify locations of high viral replication based upon tissue depth and composition.
Figure 1. IVIS Imaging of TC83-Luciferase Three C57BL/6 mice were infected through intranasal challenge with TC83-Luciferase and imaged at 2, 4, 6, and 8 days post infection (DPI). Images were made following IP luciferin injection with an autoexposure length and open filter. Click here to view larger figure.
Figure 2. Both strains of mice had similar bioluminescent signal strength following IP injection of luciferin with a delay of 10 minutes post injection before imaging was completed (A). Viral load in the brain (pfu/gram tissue) shows a strong correlation throughout the infection to the biolumiescent signal (B).
Figure 3. Treatment Comparison Utilizing IVIS. Treatment comarison following TC83-Luciferase infection in C3H/HeN mice. Mice immunized with subcutaneous TC83 23 days prior to challenge with TC83-Luciferase presented with no bioluminescent signal (A). Mice receiving IP Ampligen at -4 and +48 hr post infection (B) showed reduced levels compared to mice receiving no treatment (C).
Figure 4. 3-Dimensional reconstruction imaging. Utilizing 3-Dimensional reconstruction we visualized the peak location of TC83-Luciferase infection utilizing LivingImage’s DLIT reconstruction tool. We are able to localize the depth and location of the primary signal within the CNS utilizing this technique.
While this protocol covers the imaging aspects for in vivo analysis, it is important to recognize the bioluminescent vector as a key factor for future studies. Our utilization of TC83, an attenuated vaccine strain of VEEV, as a vector for expression of luciferase ensures that large quantities of the enzyme are being produced due to the high replication rate of the virus in the CNS as previously described1-4. While the addition of a second subgenomic promoter and the luciferase gene results in further attenuation of the recombinant virus compared to wild type TC83, this attenuation does not appear to alter the clinical course of the disease within the first 6 days of infection5.We expect to see a loss of the luciferase gene through replication of the virus over time but again this is not seen until later in the development of disease. Development of other pathogenic vectors, viral and bacterial, confirms the potential and efficiency of IVIS and luciferase across multiple fields of pathogen research6-9.
The imaging process itself has a few critical steps that must be completed before a full study can be implemented. An initial analysis of the bioluminescent strength of the vector should be finished in a pilot study containing a minimal group of animals. The expected location in vivo of luciferase accumulation should be known beforehand due to the vector utilized but the expected signal strength should be determined before initiating a large study. With the new software an exposure length analysis is not as necessary due to the auto exposure detection feature now built into the software but a time delay based upon the injection of luciferin will still need to be determined. Even with all of these signal calculations, bioluminescent detection can still be limited due to vector depth and overall animal pigment and fur. We strongly recommend the shaving of animals before imaging begins to gain the strongest signal possible.
Even accounting for vector attenuation and enzyme production, we believe we have designed an accurate and more efficient model utilizing luciferase then could be generated with a standard fluorescent molecule system for in vivo analysis. Bioluminescence is not limited by having an initiating light source, which initially must pass through tissue resulting in a loss of signal. It also does not have the high background auto fluorescent problems seen with mice on their fur and even from their diet. The risk of toxicity from the luciferin substrate is easily controlled through proper filtration and dosage, making bioluminescent just as safe for the animal. Both fluorescent and bioluminescent systems can drastically reduce animal usage due to in vivo analysis which will be important for future research due the changing political attitude towards animal research and further adherence to Hume's 3Rs (replacement, reduction, refinement) towards animal use10.
For our application of viral pathogenesis and antiviral efficacy analysis, in vivo modeling is more efficient than current systems11,12 for multiple reasons. We are able to detect specific key steps for disease development, CNS invasion1 in the case of TC83, before any clinical disease develops. From initial invasion detection, we are able to repeatedly visualize in a single animal the spread and replication of the virus providing a more accurate means of studying disease progression5. The model we have designed is also safer for researchers due to the decreased requirement of sacrifice and organ collection, the capability to detect key disease progression points before clinical disease can lead to animal irritability, and, specific to our system, we can complete these studies in an attenuated viral vector in the ABSL2 instead of the ABSL3 utilizing a select agent.
Future disease studies will increasingly utilize bioluminescent IVIS modeling. Advancements in modern molecular biology techniques and cloning allow the insertion of a luciferase gene quickly and cheaply into both viral and bacterial vectors. The ability to develop transgenic mice expressing a promoter dependent luciferase gene also provides another system for bioluminescent study. Finally, newly developed bioluminescent probes such as Calipers RediJect Inflammation probe reacts with myeloperoxidase allowing for in vivo visualization of phagocyte inflammation reaction. These new technologies combined with the increasing efficiency of the cameras and software makes bioluminescent IVIS a viable system for future research in many different fields of study.
No conflicts of interest declared.
Institute for Translational Sciences UTMB-NIH grant 1UL1RR029876-01 and Alisha Prather for her assistance with video editing for this manuscript.
|Xenogen IVIS System (Spectrum)||Caliper Life Sciences|
|XGI-8-gas Anesthesia System||Caliper Life Sciences|
|XIC-3 Containment Box||Caliper Life Sciences|
|LivingImage 4.0 Software||Caliper Life Sciences|
|Telemetry/identification chips||Bio Medic Data Systems||IPTT-300||Animal ID and Temperature|
|BD Integra 1ml TB syringe with 26 g x 3/8” needle||Fisher Scientific||305279|
|Vet Bond tissue adhesive||Fisher Scientific||NC9259532|
|Vetropolycin Ophthalmic Ointment||Webster Veterinary Products||78444656|
|Dulbecco's Phosphate Buffered Saline 1X||Invitrogen||14190-144|
|BMDS Chip Reader||Bio Medic Data Systems||DAS-7007S|
|DAS-HOST Software||Bio Medic Data Systems||Used to download probe information|
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