Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Summary

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer's disease.

Abstract

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 μm of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer's disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Introduction

The brain vasculature is a multi-cellular structure, which is anatomically and functionally coupled to neurons. A dynamic remodeling of vessels occurs throughout brain development and during the progression of pathologies of the central nervous system (CNS) ^1,2. It is widely accepted that cerebrovascular damage is a hallmark of several CNS diseases, including epilepsy, Alzheimer's disease (AD), traumatic brain injury and encephalitis ^3,4. Therefore, tracking cerebrovascular changes in vivo becomes significant when modeling CNS diseases, from onset and into chronic phases. As cerebrovascular modifications often occur concomitantly wit....

Protocol

Mice are allowed ad libitum access to food, water, and maintained on a 12-hr light-dark cycle. All procedures involving laboratory animals conformed to National and European laws and were approved by the French Ministry for Education and Scientific Research (CEEA-LR-00651-01). A total of 6 transgenic 5xFAD and 4 littermate wildtype (WT) control mice were used for this procedure.

1. Pre-operative Preparation

1. Intraperitoneally (IP) inject Methoxy-X04 (10 mg/kg) 48 hr before surgery to label Aβ deposits¹¹.
2. Prepare sterile artificial cerebrospinal fluid (aCSF) (120 mM NaCl, 26.2 mM NaHCO₃, 2.5 mM KCl, 1 mM N....

Results

This protocol describes a method for visualizing the cerebrovasculature and amyloid deposits overtime. Fluorescent dyes were injected to label amyloid depositions (methoxy-XO4) ¹¹ and to fill the cerebrovascular lumen (FITC-Dextran)¹. 3D image analysis software modules were used to create 3D images of a constant field of view captured at consecutive time points. Representative images obtained in the somatosensory cortex of 5XFAD mice⁵ (genetic model of AD).......

Discussion

The open-skull technique for in vivo two-photon microscopy offers the advantage of unlimited imaging sessions of large imaging fields^13,14. However, this technique also produces inflammation in the region of interest¹⁴, often incompatible or impacting neuro-vascular read-outs¹⁵. On the contrary, the thinned skull transcranial technique does not result in neuro-inflammation, enabling reliable imaging of the cerebrovascular structures and plaque accumulation^10,14. A seco.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
methoxy-X04	tocris	4920	use 10 mg/kg
FITC-Dextran 70 Kda	sigma	46945	use 100 mg/kg
gelfoam/Bloxang	Bausch and Lomb
micorsurgical blade	surgistar	6900	must be sharp and not dented
povidone-iodine	betadine		antisceptic solution
binocular stereomicroscope	olympus	SX10	optimal image contrast is crucial for this procedure
2-photon microscope	zeiss		Zeiss LSM 710mp
fine scissors-toughcut	Fine science tools	14058-09	this scissors are optimized for cutting skin and soft tissue