Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

Summary

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

Abstract

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2⁺ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

Introduction

The central nervous system (CNS) is governed by a complex interplay of interactions between various resident cell types including neurons, glia and vessel-associated cells. Traditionally, neural cells were studied in isolated, co-cultured^1,2,3,4,5 (in vitro) or excised brain tissue (ex vivo)^6,7,8,9,10 contexts. However, there is need to further u....

Protocol

All steps are in accordance with the guidelines set and approved by the Institutional Animal Care and Use Committee of the University of Virginia.

1. Mouse preparation for cranial window implantation

NOTE: Various transgenic mouse lines with florescent tags are suitable for imaging.

1. Use CX3CR1^GFP/+ mice²⁶ to visualize microglia in vivo. Typically, juvenile to young adult 4 to 10-week-old mice that weigh 17-25 g are used.
   NOTE: Although, this approach is even apt for pre-weaned mice, the need to return the mice to their cage with their mothers for feeding, may ....

Results

To visualize microglial dynamics in vivo, double transgenic CX3CR1^GFP/+:Thy1^YFP mice were used. The Thy1-YFP H line is used as opposed to the Thy1-GFP M line to avoid florescence overlap of microglia (GFP) and neurons (YFP). Alternative approaches could use a reporter line in which microglia are labeled with e.g., tdTomato and then the Thy1-GFP M line can be used. A drawback of the H line is that YFP labels a lot of neurons and the label increases with increasing age (personal observation). The M li.......

Discussion

The advent of in vivo two-photon imaging has opened opportunities to explore the plethora of cellular interactions and dynamics that occur in the healthy brain. Initial studies focused on using the open skull craniotomy approach to image neuronal dendrites by both acute and chronic imaging^37,38. This can also be used to elucidate neuroimmune interactions in the brain. This protocol describes a method for the reliable imaging of fluorescently tagged cells (especia.......

Materials

Name	Company	Catalog Number	Comments
Coverglass (3mm)	Warner Instruments	64-0726
Cyanoacrylate glue (Krazy Glue)	Amazon	https://www.amazon.com/Krazy-Glue-Original-Purpose-Instant/dp/B07GSF31WZ/ref=sr_1_2?keywords=krazy+glue&qid=1583856837&s=pet-supplies&sr=8-2
Demi Ultra LED Curing Light System	Dental Health Products, Inc	910860-1
Dental Drill	Osada: www.osadausa.edu	EXL-M40
Drill Bit	Fine Science Tools	#19008-07
Eye Ointment	Henry Schien	1338333
iBond Total Etch (Primer glue)	Chase Dental Supply (Heraeus Kulzer)	66040094
Rhodamine B	Millipore Sigma	81-88-9 (R6626)
Tetris Evoflow glue (Final glue)	Top Dent (Ivoclar Vivadent)	#595956
Wahl Brav Mini+	Amazon	https://www.amazon.com/Wahl-Professional-Animal-BravMini-41590-0438/dp/B00IN24ILE/ref=asc_df_B00IN24ILE/?tag=hyprod-20&linkCode=df0&hvadid=167141013968&hvpos=&hvnetw=g&hvrand=12368793083893626704&hvpone=&hvptwo=&hvqmt=&hvdev=c&hvdvcmdl=&hvlocint=&hvlocphy=9008337&hvtargid=pla-332197544154&psc=1