Diffusion Imaging in the Rat Cervical Spinal Cord

The overall goal of this procedure is to obtain high quality diffusion weighted magnetic resonance imaging of the rat spinal cord for non-invasive characterization of tissue microstructure. This is accomplished by first preparing a rat in the MR Scanner with proper mobilization and respiratory monitoring. The second step is to perform a custom diffusion weighted MRI scan with respiratory gating.

Next, the images are corrected for susceptibility artifacts. The final step is to fit the MRI signal to a mathematical model. Ultimately, comparison of model parameters within regions of interest is used to show properties of tissue microstructure.

The main advantage of this technique over existing methods like ex vivo scanning, is that it can be translated to the clinic for diagnostic purposes. This method can help answer key questions in spinal cord injuries, such as identification of secondary injury mechanisms and prognosis for recovery. Demonstrating the procedure will be Natasha Wilkins and Matt Renquist technicians from my laboratory Before performing the steps in this protocol.

First, obtain approval from the appropriate institutional care and use committees for all procedures. Begin by anesthetizing the rat in an induction chamber using 5%isof fluorine in medical air. When the paw withdrawal and writing reflex are absent, reduce anesthesia to 2%Then transfer the animal to the scanner bed in a head first prone position.

Maintain 2%ISO fluorine through a nose cone throughout the procedure, and keep medical air at a flow rate at approximately one liter per minute. Also, apply a small amount of lubricating ointment to the rat’s eyes to avoid damaging the cornea while under anesthesia. Place a respiratory monitoring belt securely around the rat’s torso and connect it to a respiratory gating system.

Check the respiratory monitoring computer to ensure the respiratory cycle is clear and consistent. Adjust a belt if necessary since this step is imperative for image quality. Use a warm air heating system and monitor and maintain the animal’s body temperature through a rectal probe to ensure it is maintained at 37 degrees Celsius.

Maintain the respiratory rate between 30 to 45 breaths per minute by adjusting the level anesthesia between 1.2 and 2%Now, position the rat in the head holder with a bite bar and screw in ear bars and slide the head into a quadrature volume coil until the cervical spine is positioned in the center of the coil. Finally, advance the rat and supporting holders into the scanner bore if applicable, adjust attuning and matching capacitors of the coil to the proper frequency and the impedance according to the vendor instructions. Acquire a default three plane scout scan to ensure correct positioning.

This first scan activates the MRI system’s, automated procedures for detection of the resonance frequency shimming, calibration of the radio frequency power and adjustment of receiver gain. Check that the center of the cervical spine is aligned with both the center of the magnet and the center of the MRI coil. Then add an echo planer, diffusion weighted spin echo sequence to the imaging protocol.

Used a sequence default settings, but prescribed 12 slices with a 0.75 millimeter slice thickness, and orient these perpendicular to the main axis of the cervical cord. Use the base of the cerebellum as an internal reference to ensure consistent slice positioning between animals and across sessions. Turn on the saturation bands.

Then position four saturation bands with a thickness of 10 millimeters outside of the spinal cord to minimize the signal from these tissues and reduce their potential to induce artifacts. Also, be sure to turn on respiratory gating. Now, set up the sequence using the diffusion waiting settings as seen on screen here.

Then start the scan. The total acquisition time will be approximately 25 minutes throughout the scans. Monitored the respiratory gating software and adjust the delay period between the trigger and the signal to the MRI system so that acquisitions occur only in the quiescent portion of the respiratory cycle.

Note that a trigger delay between 100 and 400 milliseconds will be necessary depending on the animal’s respiration pattern. This will help to reduce artifacts that occur with respiratory motion. If available, repeat the sequence with the custom reverse blips set to on when imaging is completed.

Remove the animal from the holder and return it to its cage. Monitor the animal until it has regained consciousness. To maintain sternal recumbent.

Begin image processing by first performing susceptibility artifact correction. Then extract the B equals zero volumes from each scan into a single file using utilities provided with FSL or other MRI software packages. Sample code is seen here.

One file for each phase in code direction is required. Next, use the top-up command in FSL to create a corrected file with reduced image distortion artifacts. Apply this correction to the raw DWI images to be used for creation of parameter maps.

Load this corrected DWI file into FSL view and select file. Create mask from the menu. Use the pencil tools to draw a region of interest within one tissue type.

Save this file and repeat for any other desired regions of interest or ROIs. Use the ROI file to mask the DWI file, and then calculate the mean signal within the ROI for each image volume. Copy the first eight results into a numerical computing program such as MATLAB as a vector for transverse signal, and the second eight results as a vector for longitudinal signal, where eight is the number of B values used.

Also copy the B values into the program as a vector of eight B values. The B values for the transverse and longitudinal directions should be identical if possible. The effective B value rather than the nominal B value should be obtained from the scanner.

Use a curve fitting toolbox to fit the signal versus B value data to the desired model. By typing CF tools at the command prompt, select the B values as X data and the signal vectors as y data. Then select the fitting menu and enter an equation for fitting.

Note that when entering the equation, starting points and limits may need to be set for the variables that are more reasonable for the data. After applying the equations to the fit note the parameter values that will serve as quantitative markers. This image shows high quality diffusion weighted images obtained with diffusion applied transverse and longitudinal to the spinal cord main axis.

Different B values are shown for each direction that provide the best contrast between white and gray matter. Here, the left column shows a slice imaged with the DWI sequence. The middle column shows acquisition with reverse blips.

Note how features that appear stretched in the first image appear compressed in the middle column. The right column shows the diffusion weighted images corrected using top-up. The top row is the non diffusion weighted image.

The middle row shows diffusion weighting applied in the transverse direction, and the bottom row shows diffusion weighting in the longitudinal direction. Here we can see the normalized signal plotted as a function of diffusion weighting with the transverse and longitudinal diffusion encoding direction. High quality maps of diffusivity, kurtosis, and anisotropy are calculated from the signal at each voxel.

There is a clear difference in parameters between the white and gray matter as well as regional differences in the white matter regions. While attempting this procedure, it’s important to remember to carefully monitor the animal’s respiration and adjust getting delay in anesthesia level in order to reduce motion artifacts. This is especially important in injured rats which may have abnormal respiration rates or other physiological complications that may require adaptation of the procedures.