TITLE: Assessment of implant accuracy using high-resolution postmortem MRI
AUTHORS: Lucy Liang, Elvira Pirondini, Jonathan C Ho

[DESCRIPTION]
Deep brain implant accuracy is important for successful experiments in non-human primates. In this protocol, we describe the steps to use postmortem imaging to assess the accuracy of an implant by visualizing implant location with a small thermal ablation and comparing its coordinates to image based pre-surgical planning.

This protocol is supplementary to the manuscript: 
Liang, L., Zimmermann Rollin, I., Alikaya, A., Ho, J.C., Santini, T., Bostan, A.C., Schwerdt, H.N., Stauffer, W.R., Ibrahim, T.S., Pirondini, E., Schaeffer, D.J., 2024. An open-source MRI compatible frame for multimodal presurgical mapping in macaque and capuchin monkeys. BioRxiv https://doi.org/10.1101/2024.02.17.580767

[BEFORE_START]
Follow this protocol after all terminal experiments have been completed, but before euthanizing the animal.

Note: Perfusion with 4% PFA may cause some degree of tissue shrinkage. We observed an average of 3.3% shrinkage with our brain tissue.

[STEPS]
SECTION: Thermal ablation &amp; tissue preparation
1. Before euthanizing the animal according to protocol, perform a thermal lesion through the deep brain electrode. 

We use a radiofrequency cannula (S-100 5 mm ActiveTip Straight cannula 22G, Abbott) and a radiofrequency electrode (RF-SE-10 Reusable Stainless Steel Electrode, Abbott), with a radiofrequency generator (NeuroTherm NT 1100).

Parameters: ~80℃, 1min
SECTION: Thermal ablation &amp; tissue preparation
3. Perfuse transcardially with 1x phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA).
SECTION: Thermal ablation &amp; tissue preparation
4. Dissect out the brain with care. Carefully remove the dura and any blood clots from the surface of the brain.
SECTION: Thermal ablation &amp; tissue preparation
2. Remove electrode (not MR compatible) from the brain and euthanize the animal.
SECTION: Thermal ablation &amp; tissue preparation
5. Place the brain in 4% PFA for an additional 24 hours, then, move to 1x PBS.
SECTION: Imaging and implant error calculation
6. Collect T2 or T2* weighted images of the brain in a 9.4 T/31 cm horizontal-bore Bruker AV3 HD animal
scanner with the following parameters:
T2: 125 μm isotropic,TR/TE = 1500/60ms, FOV = 52×80×56 mm
T2*: 80 μm isotropic, TR/TE = 100/16 ms, FOV = 55×70×45 mm
SECTION: Imaging and implant error calculation
7. Visualize the images in FSL, and align the anterior commissure to posterior commissure (ACPC) line with the x-axis of the mid sagittal plane with the FLIRT function.
SECTION: Imaging and implant error calculation
8. Identif y the tip of the electrode (at the center of the thermal lesion) within the internal capsule (or your target of interest), and record the coordinates.

SECTION: Imaging and implant error calculation
9. Calculate the Xi, Yi, and Zi distance (in pixels) from the posterior commissure (PC) point. Convert to metric distance using pixel resolution.

(coordinate subscript "i" indicates implant coordinates, "t" indicates target coordinates)
SECTION: Imaging and implant error calculation
10. Calculate target implant error in each axis by subtracting implant coordinates and target coordinates (e.g. Xe = Xi - Xt)
SECTION: Imaging and implant error calculation
11. Finally, calculate the overall target implant error (IE) using Euclidean distance IE = sqrt(Xe2+Ye2+Ze2)