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.
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.
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 with neuronal damage or plasticity, imaging of the neuro-vasculature represents a key entry point to decipher CNS disease pathophysiology.
This protocol describes a longitudinal two-photon based procedure to track the remodeling of the cerebrovasculature in a mouse model of AD, a progressive pathology marked by cerebrovascular defects on large and small caliber vessels due to amyloidogenic plaque deposition 5-7. This procedure allows for the visualization of amyloid deposits and tracking of their position and growth with respect to neurovascular remodeling throughout the course of the disease. Vital fluorescent dyes are injected before each imaging session for the visualization of the cerebrovasculature and amyloid plaques in transgenic AD mice8. Repeated imaging sessions of a ROI through a thinned skull transcranial window is non-invasive and the method of choice to assess neurovascular remodeling in the living mouse brain 2,5,9,10.
The procedure below outlines the surgical protocol, image acquisition and processing. The early progression of cerebral amyloid angiopathy (CAA) mostly at large leptomeningeal and penetrating arterioles is characterized.
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
2. Vasculature Labeling and Thinned Skull Window Preparation (40 min)
3. Two-photon Microscopy (45 min)
4. Recovery and Re-imaging
5. Post-acquisition Three-dimensional Reconstruction and Image Analysis
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) show that most Aβ deposits appear between 3 and 4 months of age, growing in the parenchyma (Figure 2) and around penetrating vessels (Figure 3). Plaque deposition occurs simultaneously with significant remodeling and occasional occlusion of the neighboring cerebrovasculature (Figure 2). Only rare instances of plaque size reduction were observed, indicating that this is a process of plaque accumulation. Figures 2 and 3 illustrate that the amyloidogenic load in the parenchyma and around vessels is associated with angiogenesis and vascular occlusion in the somatosensory cortex. The relationship between vascular amyloid plaques and vessel occlusion remains unclear, given that most vascular plaques accumulated on large penetrating arteries, whereas occlusion occurred mostly at small caliber vessels.
Figure 1. Experimental Setup. (A) Head setup; Stage is custom made. Head mount device consists of stacked razor blades built as previously described10. (B) View of the mouse locked in the head mount device. (C) View of the device and skin glued to the skull. Edges are centered on the targeted region. Please click here to view a larger version of this figure.
Figure 2. Comparison of Two Reconstructed Images of the Microvasculature and Amyloid Depositions on a 5xFAD Mouse at 4 and 5 Months of Age. Amyloid plaques are indicated with a white asterisk, new plaques appearing at 5 months are indicated by yellow asterisks. A rare instance of plaque reduction is indicated with an orange asterisk Newly formed microvessels are indicated with a yellow arrow while vascular occlusion is indicated with a red arrow. Please click here to view a larger version of this figure.
Figure 3. Large and Small Caliber Vessels in the Somatosensory Cortex at 3, 4 and 5 months of age. (A) Growth of amyloid vascular deposits on large caliber vessels (white asterisks). (B) Amyloid plaques can be also observed on small caliber vessels (yellow asterisks). Red arrows indicate occluded vessels. White asterisks indicated growing amyloid plaques. Please click here to view a larger version of this figure.
The open-skull technique for in vivo two-photon microscopy offers the advantage of unlimited imaging sessions of large imaging fields13,14. 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 accumulation10,14. A second advantage presented by this technique is that thinned-skull window success rate can reach up to 80% to 90% for an experienced manipulator. A major setback for success is the lack of appreciation of bone thickness leading to excessive thinning and ultimate fracture; experience and excellent optics of the binocular microscope will limit this problem. A number of limitations do affect this technique however. First, the imaging depth is limited compared to the open-skull procedures. Second, over time, scar tissue overlying the bone can diminish the quality of the transcranial imaging. Third, it is difficult to proceed to more than 4 imaging sessions given that the skull has to be thinned a little more at each session. Consequently, it is important to determine the number of imaging sessions at the beginning of an experiment, in order to optimize the thinning increment between sessions. Alternatively, a chronic transcranial window consisting of a thinned-skull preparation glued with a coverglass has been previously described to limit the regrowth of the bone tissue over time16. Though efficient, we found that this technique only delayed bone regrowth for several weeks eventually interfering with the quality, depth and subcellular resolution of images when re-imaging after longer periods of time (Margarita, Arango-Lievano and Freddy Jeanneteau, unpublished data). Despite its limitations, two-photon transcranial microscopy through repeated thinned skull preparations is a method of choice to track fluorescent markers in chronic disease models where neuro-vascular changes and inflammation occur.
An optimal thinning of the skull represents a major technical step, as pressure applied to the bone should be minimized to avoid bleeding and to ensure a flat skull window. Bleeding and an uneven skull window can facilitate scarring and the uneven regrowth of the bone. Excessive thinning during the first imaging session, overheating after excessive drilling, or laser damage can also compromise the clarity of the signal through the skull window. The latter problems can be controlled by regularly changing the aCSF medium during drilling, cooling the preparation, drilling intermittently, and using clean sharp tools when polishing the bone. Additional critical steps include the (i) fixation of the skull to the head mount plate to reduce the movement associated with respiration, (ii) retro-orbital injection and reliable visibility of all vessels, (iii) mapping the region of interest for future relocation, and (iv) the pre-, per- and post-operative care in between imaging sessions.
The methodology summarized here allows for studies to assess the efficacy and safety of CNS drugs targeting neuro-vascular structures, e.g., molecules reducing or preventing amyloid depositions in the brain parenchyma and on vessels. Additionally, longitudinal two-photon microscopy of the living brain permits tracking of amyloidogenesis and its impact on the neurovasculature at the early stages of AD, prior to onset of cognitive deterioration. Lack of clearance of large amyloid plaques can have deleterious effects on brain vasculature, exacerbating disease pathophysiology17. The advent of new fluorescent vital dyes to label Lewy bodies, synucleopathy, prion aggregates, huntingtin aggregates, support future attainability of neurovasculature remodeling studies during the progression of other neurodegenerative disorders 18. This technique is adapted to several mouse strains combining dyes (SR101, Methoxy-X04, dextrans, lectins) and genetic markers (thy1YFP, CX3CR1-GFP, NG2-dsRed) to investigate cellular interactions in vivo and in experimental models where the structure and function of the neurovasculature deviates from normal physiology.
The authors have nothing to disclose.
The authors would like to acknowledge the Ligue Francaise contre l'épilepsie (to M.A-L), Institut National de la Santé et de la Recherche Médicale Grant AVENIR R12087FS (to F.J), grant from the university of Montpellier (to F.J) and grant from Federation pour la Recherche sur le Cerveau (to N.M). We acknowledge the technical assistance of Chrystel Lafont at the IPAM in vivo imaging core platform facility of Montpellier. We also thank Mary Vernov (Weill Cornell Medical College) for proofreading the manuscript.
methoxy-X04 | tocris | 4920 | use 10 mg/Kg |
FITC-Dextran 70Kda | 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 |