Today we are presenting a method of detecting central nervous infection by an alpha virus within Aurine species using bioluminescent technology. This method utilizes TC 83, the live attenuated vaccine strain of Venezuelan equine encephalitis with an inserted firefly luciferase gene, which we can use to track the replication of the virus as its spread within the host. In a recently published vaccine paper, we demonstrate our capability to visualize the invasion of CNS and use it as a study endpoint for testing the efficacy of vaccines and antiviral treatments before clinical disease manifested in our controls.
Hello, my name is Allison Ard, and I'm an animal technologist in the high containment labs here at the University of Texas Medical Branch. The work that you'll see here in this video was completed by me, Michael Patterson, who's a graduate student here at UTMB and Katie Taylor, a senior graduate student in our lab. All of the work that you'll see was completed here in the Galveston National Laboratory with full IACUC approval following environmental health and safety regulations Utilizing in vivo imaging systems.
IVUS allows us to reduce the number of animals required within our studies due to our capability to generate images on a daily basis for each individual animal. An overview today will be showing the preparation of the animal, including forehead shaving BMDS, chipping intranasal infection with TC 80 D three interperitoneal injection of Lucifer for imaging the handling and transfer of the animal from the Biocon containment cabinet into the imaging box. From the imaging box, we transfer will be transferring the animal into the caliper's imaging unit, the actual imaging process, and two and three dimensional imaging and the transfer of the animals following imaging back into the biosafety cabinet.
Our animal lab has one Class two biosafety cabinet where all in vitro and in vivo procedures take place. Next to the biosafety cabinet is the Cali ivu spectrum with attached isof fluorine, vaporizer, and computer interface. The Isof fluorine vaporizer allows for precise delivery of inhalation anesthetic and is a quicker and safer means of anesthetizing mice compared to injectable anesthetics.
The XI C3 imaging box is a sealed unit complete with HEPA filtration, allowing us to Safely transport animals within the lab. In previous studies, we have determined That darker haired marine species have inhibited bioluminescent signal direct directly related to their fur. We recommend shaving of the forehead and snout to improve Signal detection throughout the study.
Throughout our studies, we utilize BMDS transponder chips, which allow us to collect temperature and animal identification information wirelessly. This method of collection allows for reduced animal handling and stress. The fur is matted with an ethanol wipe and the chip is inserted subcutaneously.
While the mouse is under anesthesia. We anesthetize the animals for Intranasal infection. Once unconscious, we hold each animal gently by the scruff with the snout pointing upwards.
20 microliters is pipetted into each nare. For a total of 40 microliters per mouse, the animals are allowed to breathe between drops so the inoculum does not collect in the back of the throat. For the IP luciferian injection, the animals are gently restrained by scruff and placing the tail under the pinky and ring fingers.
Luciferian is then injected into each animal's abdomen to ensure maximum diffusion of Lucifer within the bloodstream. We wait five to 10 minutes after injection before anesthetizing the mice for imaging. Once anesthetized, the animals can be moved into the XI C3 imaging containment box.
Their heads are placed gently on strips of black electrical tape, which reduces cranial displacement. Due to respiration movement will interfere with the imaging process for all imaging, including three dimensional imaging, full limb protraction, and a prone position within the XI C3 unit ensures optimal topographic mapping. When all animals are properly placed, seal the XI C3 box and wipe the outside of the unit with ethanol disinfect hands before removing the box from the biosafety cabinet.
When placing the XI C3 box within the ivus Imaging Unit, ensure the anesthetic in and outlines are correctly connected to the HEPA filters attached to the XI C3 box. Also, ensure the imaging grid completely encompasses the mice before closing the imaging unit And turning on the flow of anesthesia. Once you have opened the living image software, you will need to log in under your username.
You then initialize the ivus unit. When the IVUS unit is ready, the temperature indicator will turn green, which occurs around negative 90 degrees Celsius Ferrari work. We use an auto detection exposure time and leave all other settings at their default setting.
Once the image is acquired, it will appear on the primary screen. You have the option to select regions of interest, which will allow you to automatically detect specific areas of strong signal strength. It will also allow you to manually input regions of strength you wish to visualize, which the system does not automatically identify four three dimensional imaging.
Select a sequence setup option, select imaging wizard, and select bioluminescence. Once bioluminescence is selected, click DLIT and click next. Under DLIT ensure that the proper reporter is selected for US Firefly and click Next.
Ensure all your information is correct. Ours is imaging subject of a mouse with a field of view of a C size. We then click next and it produces a five image set of varying wavelengths from five 60 nanometers to six 40.
Within our first image, we also have the topographic image taken to allow for three dimensional imaging. We then click acquire sequence. Once all five images are acquired, we have the ability to close the imaging sequence set up and start a manipulation of our image.
To develop a three dimensional reconstruction, we will have to first ensure we have a strong topographic map. We set our subject to a fur mouse and a dorsal orientation and reconstruct it. We highlight the mouse itself, avoiding highlighting any of background area of the box.
We click next, and this section may take some time dependent upon your image and the computer system you're utilizing. Once finished, you'll have a topographic map of your mouse body. You then go onto DLIT 3D reconstruction.
Ensure your threshold settings and your tissue properties are correctly set. Ours or brain select start and then select reconstruct. This is another time point which may take some time based upon your computational system.
When finished, you'll see a signal that shows on your topographic map. You then need to click 3D tools, click registration, organ atlas, and for us female dorsal orientation. We can then change the opacity of the organs to better the we click linear fit to fit the topographic map to the digital organ set.
We can remove all organs except for the organs we wish to actually identify with the signal for US brain. Using this technology, we're also able to create a three dimensional video, which allows us to rotate, change opacity, change view, zoom in, remove organs, and finally zoom out. When imaging is completed, turn off the ISO fluorine vaporizer and detach the XI C3 containment box.
Return the sealed box to the biosafety cabinet and gently remove the mice from the electrical tape. For longer imaging durations. Within the ABS L two, the mice can be warmed through proper gloved handling.
We closely observe the animals To ensure complete recovery from anesthesia. We would like to thank Dr.Pessler and everybody In the laboratory who contributed to this project. We would also like to thank the Galveston National Laboratory, which provided the facilities and equipment as well as the Institute for Translational Sciences, which provided the Funding for this research.
