The overall goal of this reporter enzyme fluorescence imaging procedure is to facilitate the sensitive and specific detection of mycobacterium tuberculosis in pre-clinical animal models for pathogenesis, therapeutics, and vaccine research. This method can help answer key questions in the tuberculosis field including virulence mechanisms of bacterial species as well as creating therapeutics and understanding vaccines. So the main advantage of this technique is that it allows for the same group of animals to be imaged at multiple timepoints.
This greatly reduces the cost, allows us to study the host pathogen interactions, and also increases the statistical power because more number of animals can be maintained readily at the same time. To begin, grow up any M.tuberculosis bacteria in MOADCTW medium at 37 degrees Celsius to an optical density of 0.5 measured at 600 nanometers. All procedures involving the manipulation of infectious materials must be conducted within biosafety cabinets or other approved physical containment devices.
Once ready, make a series of one to 10 dilutions of the culture in the same medium. Then, plate the dilutions in triplicate onto selective 7H11 plates to make an accurate determination of the CFU. Incubate the plates for four weeks at 37 degrees Celsius or until the colonies can be accurately counted to determine the CFU.
Before setting up an inoculation, always make certain that the biosafety hood is flowing air at 50 liters per minute. To prepare an inoculum that can infect up to 90 mice, measure out the desired CFU in a 15-milliliter falcon tube. Between 10, 000 and 1 million CFU are typically used.
Then pellet the bacteria. Next wash the bacteria once with 10 milliliters of saline. Then suspend them in 15 millileters of saline.
Then in a biosafety cabinet, load the inoculum into a glass jar that can attach to a nebulizer. Next connect the nebulizer unit and adjust the vertical stainless steel tube so that the lower end is about a quarter of an inch below the meniscus of the suspension. Now transfer the assembly to the inoculation chamber in a leak-proof transport container.
To proceed, load animals of known weight into the powered-up inoculation chamber. The chamber can fit up to 90 mice. Then close all the latches on the door.
Next connect the nebulizer using a steel clamp. Then press the start button on the chamber. And set the airflow rate to between three and five liters per minute.
The compressed air will flow at about 22 to 26 pounds per square inch and importantly it should be possible to see the mist of inoculum in the chamber. After 15 minutes, a red light on the front of the control panel will appear and an audible signal will indicate the end of the run. Then press the reset button to reset the timers.
To release the vacuum, press the small red button on the door of the chamber. Then open the chamber door and return the mice to their home cages. Going forward, keep the animals in the containment room.
Next return the jar and the transport container to the biosafety cabinet. There, discard the remaining bacterial suspension into a designated waste container. Then place the used nebulizer jar inside a biohazard bag and seal the bag for transport to the autoclave.
To clean the chamber, spray the inside surfaces with buffered phenol and 70%ethanol. Allow these solutions to react on those surfaces for 10 minutes. Then wipe them off thoroughly.
Dispose of all the wipes in the biohazard trash. Then autoclave the trash, waste container, and used nebulizer jars. Transfer the animals to the imaging room.
There, anesthetize an animal according to the text protocol and inject it with contrast substrate by intraperitoneal injection. In the imaging systems software, initialize the system and proceed when the temperature bar turns green. After initializing the system, place the mouse in the imaging chamber.
Make sure its nostrils are within the nose cone so it receives anesthetic during imaging. Now return to the computer. In the acquisition control panel, select either fluorescence, transillumination, for whole-animal visualizaiton, or epi-illumination to view lung tissues.
For a single mouse, set the field of view to B and the lamp level to high. For the exposure, use automatic time, medium binning, and two or three for the F/Stop. Then set the excitation filter to 745 nanometers and emission filters from 780 to 840 nanometers.
Next go to sequence set-up and select nine to 12 transillumination points in the area of interest. Now press the acquire button to collect images. Image analysis is covered in the text protocol.
To analyze the extent of the infection, euthanize the mouse using sodium pentobarbital. Be sure to check for a pedal reflex to ensure the animal is euthanized. And then explant the lungs using a standard dissection.
Next homogenize the lung tissue in one milliliter of PBS. Then make a ten-fold serial dilution of the homogenyn using PBS. Plate the diluted homogeny in triplicate and incubate the plate at 37 degrees Celsius until colonies can be counted.
Then use the CFU score to measure the severity of the infection. Mice were treated as described. An REF substrate was used to view progression of M.tuberculosis.
Fluorescence was quantified using transillumination. And it increased from week two to week six in the infected mice. The slight drop in signal in the control mice could be attributed to the increase in body mass and volume over the six-week period.
3D FLIT reconstruction of fluorescence sources was made from multiple transillumination points using the same excitation and series of emissions filters. NTF efficiency maps were made to check the reconstruction quality. The image taken from the specific excitation filter was normalized to the transmission image made with the same emission filter and an open excitation filter.
This provides a signal produced by the substrate alone. There was nearly no error in the reconstruction. Examining the NTF efficiency of the data in both the horizontal and vertical view showed a tight correlation.
Thus the 3D reconstruction had few artifacts, improved signal localization, and improved sensitivity over other methods. This technique paved the way for researchers in the field of tuberculosis to explore therapeutic efficacies of drugs against mycobacterium tuberculosis. After watching this video, you should have a good understanding of how to infect mice via aerosol route with mycobacterium tuberculosis with follow-up optical imaging using the REF substrate.
