A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Summary

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

Abstract

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12–15 and 5–6 µm per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

Introduction

Neuroscientists have relied on lesions for a long time in order to understand the relationship between function and location in the brain. For example, our understanding of the hippocampus as being indispensable for learning and memory and of the prefrontal cortex as being key for impulse control were both products of serendipitous lesions in humans^1,2. The use of animal models, however, has allowed neuroscientists to harness the power of lesions by going beyond serendipity, and the function of countless brain areas has been elucidated through systematic studies of structure-function relationships through lesi....

Protocol

All care and experimental manipulation of animals were reviewed and approved by the Harvard Institutional Animal Care and Use Committee. The perfusion described here is specific for rats, but the procedure is applicable to any animals with smaller or similarly sized brains.

1. Perfusion

1. Prepare 1x phosphate-buffered saline (PBS). For a rat (age: 0.5–1.5 years old, weight: 250–600 g), 800–1,000 mL should be sufficient. Use 400 mL to perfuse the animal and an additional 400 mL to dilute the fixative.
2. Prepare the fixative consisting of 2% (w/v) paraformaldehyde (PFA) and 2.5% (w/v) glutaraldehyde (GA) in....

Results

Traditionally, brains are sectioned and stained in order to quantify lesions and locate electrodes, but this method is error-prone, labor-intensive, and typically requires estimation of the results. By preparing whole brains for micro-CT imaging, the probability of damaging the samples is greatly reduced, features of interest may be analyzed in the context of the entire brain, and the method lends itself to parallel processing of many samples, considerably speeding up sample preparation.<.......

Discussion

The following are critical steps to the protocol: first, the use of a combination of PFA and GA to perfuse the animal and subsequently post-fix the brain was paramount to achieving consistent full osmium penetration of the tissue. Although we did not test this explicitly, a plausible explanation is that PFA fixation is reversible¹⁵, whereas GA fixation is not reversible^16,17. Because a two-week incubation in osmium tetroxide is required fo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Paraformaldehyde (PFA)	Electron Microscopy Sciences (EMS)	15710	2% (w/v/) in 1X PBS
Glutaraldehyde (GA)	EMS	16220	2.5% (w/v) GA in 1x PBS
OsO4	EMS	19190	Work in fume hood
Ethanol	Decon Labs	Koptec	140, 190, 200 proof
Acetone	EMS	10015	Glass-distilled
Durcupan ACM resin	Sigma-Aldrich	44610	A, B, C and D components, resin for embedding
Disposable molds	Ted Pella	27114	Suggested
milliQ water (ultrapure water)	Millipore Sigma	QGARD00R1 (or related purifier)	Suggested
Parafilm (paraffin film)	Millipore Sigma	P7793	Suggested paraffin film
Micro-CT scanner	Nikon Metrology Ltd., Tring, UK	X-Tek HMS ST 225	Used by authors
Software for visualizing and analyzing micro-CT scans:
	Volume Graphics	VG Studio Max	Used by authors
	FEI / Thermo Scientific	Avizo	Used by authors
	FEI / Thermo Scientific	Amira	Similar to Avizo
	Mark Sutton & Russell Garwood	Spiers	Free, http://spiers-software.org/
	Pixmeo Sarl	Osirix Lite	Free, https://www.osirix-viewer.com/
	Open Source	FIJI	Free, https://fiji.sc/
	Adobe	Photoshop	Good for analyzing one slice at a time