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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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-cultured1,2,3,4,5 (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 CX3CR1GFP/+ mice26 to visualize microglia in vivo. Typically, juvenile to young adult 4 to 10-week-old mice that weigh 17-25 g are used.......

Representative Results

To visualize microglial dynamics in vivo, double transgenic CX3CR1GFP/+:Thy1YFP 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 imaging37,38. 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.......

Acknowledgements

We thank members of the Eyo lab for discussing the ideas presented in this manuscript. We thank Dr. Justin Rustenhoven from the Kipnis Lab at the University of Virginia for the gift of NG2DsRed mice33. This work is supported by funding from the National Institute of Neurological Disorders and Stroke of the National Institute of Health to U.B.E (K22 NS104392).

....

Materials

NameCompanyCatalog NumberComments
Coverglass (3mm)Warner Instruments64-0726
Cyanoacrylate glue (Krazy Glue)Amazonhttps://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 SystemDental Health Products, Inc910860-1
Dental DrillOsada: www.osadausa.eduEXL-M40
Drill BitFine Science Tools#19008-07
Eye OintmentHenry Schien1338333
iBond Total Etch (Primer glue)Chase Dental Supply (Heraeus Kulzer)66040094
Rhodamine BMillipore Sigma81-88-9 (R6626)
Tetris Evoflow glue (Final glue)Top Dent (Ivoclar Vivadent)#595956
Wahl Brav Mini+Amazonhttps://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

References

  1. Engle, S. J., Blaha, L., Kleiman, R. J. Best Practices for Translational Disease Modeling Using Human iPSC-Derived Neurons. Neuron. 100 (4), 783-797 (2018).
  2. Osaki, T., Shin, Y., Sivathanu, V., Campisi, M., Kamm, R. D.

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