This document will outline the necessary information required for Collaborations and provide a go-to spot for documentation needed.

Typical Collaborations:

What is different in the brain of my mouse?

  • With all collaborations it is important to establish an agreement between both our lab and yours on the work that is to be done prior to preparation of the brains for scanning.

Simple collaborations are composed of two different groups: an experimental group and a control group.  Group sizes will depend on the different comparisons required, but typical group sizes range from 12-14 mice per group.  More complex studies will require similar numbers of mice in each group of interest.

Common Sequences Used:

A multi-channel 7.0 Tesla MRI scanner (Agilent Inc., Palo Alto, CA) is used to image the brains within skulls.  Sixteen custom-built solenoid coils will be used to image the brains in parallel (Bock et al., 2005).

  • Anatomical Imaging Scan - T2- weighted, 3-D fast spin-echo sequence
    • This scan is used for optimizing the gray/white matter contrast, which is required for registration and analysis.
    • Current Parameters:  Cylindrical acquisition of k-space, with a TR of 350 ms, and TEs of 12 ms per echo for 6 echoes, field-of-view of 20 x 20 x 25 mm3 and matrix size = 504 x 504 x 630 giving an image with 0.040 mm isotropic voxels.  Total imaging time is currently ~14 hours.
  • Diffusion Tensor Imaging - 3D Diffusion Weighted Fast Spin-Echo sequence
    • This scan is used for calculating differences in the tissue microstructure, particularly the white matter.
    • Current Parameters: Echo train length of 6 will be used, with a TR of 270 ms, first TE of 32 ms, and a TE of 10 ms for the remaining 5 echoes, one average, field-of-view 14 x 14 x 25 mm3 and a matrix size of 180 x 180 x 324 yielding an image with 0.078 mm isotropic voxels.  Five b=0 s/mm2 images and 30 high b-value images (b=2147 s/mm2) in 30 different directions were acquired, using the Jones30 scheme (Jones et al. Magnetic Resonance in Medicine, 1999).  Total imaging time is currently ~12 hours.
  • P7 Brain scan - Diffusion Tensor Imaging - Used for Gray/White Matter contrast in young brains.
    • This scan was needed to enhance the gray/white matter contrast at the younger ages prior to large scale myelination.

    • This diffusion sequence is a 3D diffusion-weighted FSE, with TR= 270 ms, echo train length = 6, first TE = 30 ms, TE = 10 ms for the remaining 5 echoes, one average, FOV = 25 mm × 14 mm × 14 mm, and a matrix size of 450 × 250 × 250, which yielded an image with 56 µm isotropic voxels.  One b=0 s/mm2 image is acquired and 6 high b-value (b = 2147 s/mm2) images were acquired at the following directions (1,1,0), (1,0,1), (0,1,1), (-1,1,0), (-1,0,1) and (0,1,-1) corresponding to (Gx,Gy,Gz).  Total imaging time was ~ 14 hours. 

    • After the scan is complete, the high b-value images are averages to create a diffusion weighted image that has acceptable contrast for the image registration pipeline.

Preparation of Brains for Scanning:

The following perfusion protocol has been refined over the years.  The current version will always be posted on this webpage

Quick Summary of the Protocol: Initially the mice are anesthetized with ketamine/xylazine and intracardially perfused with 30mL of 0.1M PBS containing 10U/mL heparin (Sigma) and 2mM ProHance (a Gadolinium contrast agent) followed by 30mL of 4% paraformaldehyde (PFA) containing 2mM ProHance (Spring et al., 2007). Perfusions were performed with a Pharmacia minipump at a rate of approximately 1mL/minute. After perfusion, mice are decapitated and the skin, lower jaw, ears, and the cartilaginous nose tip were removed. The brain and remaining skull structures are incubated in 4% PFA + 2mM ProHance overnight at 4oC then transferred to 0.1M PBS containing 2mM ProHance and 0.02% sodium azide for at least 7 days prior to MRI scanning.

*  NOTE - p7 Brains:  For the younger brains ~ p7 and under we recommend following the same protocol, with a minor modification.  Because of the size of the brain it is unnecessary to use a volume of 30mL in the PBS stage and again in the PFA stage.  In both cases we recommend using only 15 mL.  Additionally as the brain and the skull at that stage are extremely fragile, after the perfusion mice just need to be decapitated.  The skin and other structures DO NOT need to be removed as that can cause damage to the brain.

*  NOTE -  Brains younger than p7: Mice under this age are too small to undergo perfusion. In this case, immersion fixation can be used. Decapitate the head, and without removing any skin/jaw/etc, put the whole head into a vial filled with 4% PFA + 2mM ProHance. Keep the sample in solution for one day, and then transfer to 0.1M PBS containing 2mM ProHance and 0.02% sodium azide.


Attached File: Perfusion Protocol ver 11


NOTE:

Prohance - This is a contrast agent required for optimization of the gray whitter matter contrast and increased scanning efficiency.  It can be purchased from Bracco Diagnostics at http://www.braccoimaging.com/ch-en/node/185.  As some institutions may be unable to purchase Prohance (it is usually necessary to be affiliated with a hospital), we can also provide Prohance in limited quantities.

Shipping of Brains:

Brains will be perfused in the collaborators laboratory prior to shipping them to MICe for scanning and analysis. Brains should be stored in the storage solution listed in the above perfusion protocol and not shipped in PFA or Formalin.  The storage solution is Phosphate Buffer Solution (PBS) with 2mM Prohance and 0.02% Sodium Azide.  Brains can be shipped at room temperature or with a few ice packs.  It is better that the brains arrive at room temperature than frozen.

The best address for shipping is:

  • Mouse Imaging Centre (MICe)
    The Centre for Phenogenomics
    25 Orde Street
    Toronto Ontario Canada
    M5T 3H7

Important points to note:  It should be indicated on the FedEx form or whatever shipping method that the contents are:

  1. Mouse Brain Specimens
  2. For Research Purposes Only
  3. Non-Hazardous
  4. Non-Toxic

Scanning, Analysis and Timeline:

Scanning will commence as soon as possible.  There are currently two determinants on when scanning can begin.

  1. Scanning needs to wait until the contrast agent has fully saturated and normalized throughout the sample.  This seems to occur after one month, and yields consistent scanning independent of time since perfusion after that.
  2. Current scanner backlog.  We prioritize brains scanning based on time the brains arrived in Toronto.  So if a lot of brains have arrived from other labs just prior there may be additional wait time.
  3. Typical analyses will take ~ 2 weeks after scanning has finished, again with considerations due to current backlog.

Analysis - What we will provide:

  1. High resolution figures and tables outlining the volumetric differences in the brain of your mouse.
  2. Access to a password protected website to investigate your findings in detail and create figures and download your data.
  3. Data will be provided in two different ways:
    1. Voxelwise Differences - Highlighting differences across the brain in individual voxels.
    2. Regional Differences - Highlighting differences in 159 different regions throughout the brain. These regions were based on the following three papers: