Diffusion-Weighted Imaging of the Rat Hippocampus with Mild Traumatic Brain Injury

Abstract

Source: Braeckman, K., et. al. Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury. J. Vis. Exp. (2019)

This video demonstrates the use of diffusion-weighted imaging to assess microstructural changes in a rat hippocampus following mild traumatic brain injury (mTBI), highlighting axonal injury through precise MRI scanning and diffusion analysis.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Diffusion magnetic resonance imaging (MRI)
   NOTE: Diffusion-weighted imaging is performed before and 1 day following trauma induction.

1. Anesthetize the rat in a small induction chamber filled with a mixture of isoflurane (5%) and O₂. When the rat is non-responsive to a paw or tail pinch reduce the anesthesia to 2% with a flow rate of 500 mL/min. Transfer the animal to the scanner bed in the head-first prone position.
2. Position the rat in the head holder with the teeth bar and nose cone, delivering the anesthesia, and slide the head forward until the center of the brain is at the level of the center of the quadrature volume MRI coil. Apply lubricating ointment to the eyes in small amounts to prevent any damage to the cornea. Fixate the head with a small piece of tape to avoid movement during scanning.
3. Place a pressure pad under the thorax of the rat to monitor respiration and cover the rat with a circulating warm water heating blanket and bubble wrap to keep the rat warm. Before the scanning, check the respiratory monitor to ensure that the signal is clear without noise and that the respiratory cycle is consistent. Relocate the pressure pad if necessary.
   NOTE: The respiratory rate should be kept between 1 breath per 1,200–1,700 ms by adjusting the level of anesthesia between 1%–2%.
4. Slide the quadrature volume coil over the head. Adjust the tuning and matching capacitors of the coil to the proper frequency and impedance according to the instructions provided by the coil vendor. Advance the scanner bed into the scanner bore to start scanning.
5. Obtain a default three-plane scout scan (“tripilot”) to ensure correct positioning.
   1. Load the tripilot sequence into the Scan Control by clicking New Scan and selecting the tripilot sequence from the protocol list. Next, click the traffic light button to start the scan.
   2. When the scan is finished, load the scan in the image display and ensure that 1) the head is lying straight and 2) the brain is positioned in the center of the magnet and coil. Adjust the position of the head and/or the scanner bed, if necessary, and acquire a new tripilot scan.
6. Adjust the local magnetic field using an automated second-order shimming protocol: load the second-order shim protocol into the Scan Control as described in step 1.5.1. Next, click on the Acq tab | Current Adjustments | Method-specific adjustment for the Local Field Homogeneity in the Spectrometer Control Tool window to start automated shimming.
7. Load a new T2 Rapid imaging with Refocused Echoes (RARE) sequence into the Scan control as described in step 1.5.1.
   1. Acquire T2 weighted images using the default settings, except for the following parameters:
   2. Open the Edit Scan tab and adjust the repetition time (TR) and echo time (TE) to 5,500 ms and 37 ms, respectively. Also, modify the field of view and matrix size to allow for a higher in-plane resolution of 109 μm x 109 μm (default resolution = 156 μm x 156 μm). Make sure that the slice thickness is 600 μm, the number of slices is set to 45, and the RARE factor is set to 8.
   3. Open the Geometry editor and place the slice package in the correct position, including the bulbus of the brain and the cerebellum.
8. Load three new echo-planar diffusion-weighted spin-echo sequences (DtiEpi) from the B_DIFFUSION folder into the Scan Control protocol as described in step 1.5.1.
   NOTE: Using three different diffusion “shells”, the diffusion tensor imaging (DTI) model, diffusion kurtosis imaging (DKI) model, and white matter tract integrity (WMTI) model can all be estimated. It is recommended to use at least three different b-values, with the highest b-value having a maximum of 3000 s/mm² with at least 15 evenly spaced directions per imaging shell.
   1. Acquire diffusion-weighted images (DWIs) using default settings, apart from the following settings:
   2. Open the Edit Scan tab and adjust the geometrical parameters under the Geometry tab. Adjust the field of view and matrix size to 105 x 105 to ensure a resolution of 333 μm x 333 μm.
   3. Set the slice orientation to axial and the number of slices to 25, resulting in a slice thickness of 500 μm and interslice distance of 600 μm. Amend the readout direction to the right.
   4. Click the Contrast tab to adjust the echo time to 24 ms and repetition time to 6,250 ms.
   5. Set the bandwidth to 250,000 Hz and turn the fat suppression on. Adjust the number of averages to one.
   6. Click on the Research tab and change the number of averages (EPI segments) to 4.
   7. Click on the Diffusion tab within the research tab. Perform this step separately for each of the three diffusion shells.
      1. Adjust the number of diffusion directions to 32 for the first shell, 46 for the second shell, and 64 for the third shell.
      2. Adjust the gradient directions with custom gradient directions files.
      3. Change the number of B0 images to 5 for the first shell, 5 for the second shell, and 7 for the third shell.
      4. Adjust the b-value per direction to 800 s/mm² for the first shell, 1500 s/mm² for the second shell, and 2000 s/mm² for the third shell.
         NOTE: Adjusting the gradient directions with a custom gradient directions file can be done manually by setting Enter Diffusion Directions to yes or automatically by using the DTI_SET_DIRECTIONS macro.
   8. Open the Geometry editor and place the field of view between the bulbus and cerebellum, containing only the cerebrum to reduce artifact and scan time. Position six saturation bands of 5 mm outside the brain to reduce artifacts by clicking on Saturation and sliding the bands in the preferred position using the scroll bars.
      NOTE: The bulbus and cerebellum can be identified based on anatomical landmarks and the three images of the tri pilot scan.
9. Acquire the imported sequences by clicking on the traffic light symbol. Using the settings of the parameters described above, the acquisition time of the T2-RARE scan is 12 min, of the first DWI shell 15 min, of the second DWI shell 21 min and of the third shell 30 min. The total acquisition time is approximately 80 min (on a single receiver channel system).
10. At the completion of the scanning protocol, remove the animal from the scanner bed, and place the animal in a clean cage with a heating pad at 37 °C. Return the animal to the home cage when it regains consciousness.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Diffusion MRI
Preclinical MRI acquisition software	Bruker Biospin MRI GmbH	Z400_PV51_CENTOS55	ParaVision 5.1 MRI software
Preclinical MRI scanner	Bruker Biospin MRI GmbH	PharmaScan 70/16	7T MRI scanner
Quadrature volume coil	Bruker Biospin MRI GmbH	RF RES 300 1H 075/040 QSN TR	Model No: 1P T13161C3
Small animal physiological monitoring unit	Rapid Biomedical	EKGHR02-0571-043C01	Unit for respiratory monitoring
Water-based heating unit	Thermo Fisher Scientific	Haake S 5P	Model No: 1523051
Anaesthesia
Anaesthesia movable unit	Veterenary technics		BDO - Medipass, Ijmuiden
isoflurane: Isoflo	Zoetis	B506
Oxygen generator	Veterenary technics	7F-3	BDO - Medipass, Ijmuiden