This study investigates the use of Mycobacterium bovis BCG to induce granuloma formation in mice and the application of magnetic resonance imaging (MRI) to visualize the inflammatory response. The research focuses on the interaction between superparamagnetic nanoprobes and the granuloma to enhance imaging contrast.
Take Mycobacterium bovis BCG, an attenuated tuberculosis-causing bacteria, and intradermally inject it into a mouse.
An inflammatory response against the bacteria forms a granuloma comprising bacteria, epithelioid macrophages, conventional macrophages, and lymphocytes.
Perform magnetic resonance imaging or MRI. The magnetic field aligns randomly oriented protons in the tissue in its direction.
Apply a radio frequency pulse perpendicular to the magnetic field. The protons absorb energy from the pulse and tilt away from the field.
When the pulse stops, protons emit the absorbed energy, termed free-induction decay or FID, and realign with the field.
Measure the FID signal to obtain an image. The decay duration is tissue-specific, and areas with a longer decay appear lighter.
Take superparamagnetic nanoprobes conjugated to Mycobacterium-specific polyclonal antibodies and inject intravenously.
The antibodies bind to multiple epitopes on the bacteria, labeling the granuloma.
The nanoprobes shorten the decay of neighboring protons to make the granuloma appear darker, confirming mycobacterial infection.
To inoculate experimental animals with M. bovis BCG, first, reconstitute the lyophilized vaccine, or bacterial stock, in Sauton's medium, and dilute the reconstituted stock solution with saline. Next, load 100 microliters of the solution into one 1-milliliter syringe per animal, and inject the entire volume of bacteria, intradermally, into the left or right dorsal scapular skin of each mouse.
For in vivo magnetic resonance imaging of live nanoprobe injected animals, acquire baseline T2-weighted fast-spin echo images of each anesthetized experimental animal, before injecting two nanomolar of SPIO-Tb antibody probes, suspended in 200 microliters of saline into the tail vein of each mouse. Then, image the animals again, immediately after the injection, and every five minutes for the next 30 minutes.
At the end of the imaging session, quantitatively analyze the magnetic resonance images, using the signal intensity as a measurement of the defined regions of interest in comparable locations of an M. tuberculosis granuloma center, and the back muscle adjacent to a granulomatous area. Then, use the formula to calculate the relative signal enhancements, using the signal intensity measurement before, and, zero to three hours, after the injection of the contrast agents.
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