Name: precise brain mapping to perform repetitive in vivo imaging of neuro-immune dynamics in mice
Uploaded: 2020-08-07T15:00:00
Description: Watch this Scientific Journal Video about Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice at JoVE.com

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).

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.
<ol>
	<li>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...

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...

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 understand neural cell behavior and interactions in the native environment of the intact brain in vivo. In this protocol, we describe a method to map in vivo regions of interest and precisely re-image those regions in future imaging sessions to track the complex interactions between the various CNS cell types over extended periods of time.
The development of in vivo imaging approaches has provided significant gains for the proper understanding of neural function11,12,13,14,15. Specifically, these approaches provide several advantages over traditional in vitro and ex vivo approaches. First, in vivo imaging systems have physiologically relevant cell and tissue components such as the vasculature with the full repertoire of cellular interactions to provide a complete understanding of neural network physiology. Second, recent findings suggest that when removed from their native environment, certain neural cells (such as microglia) lose important features of their identity and thus physiology16,17 which can be preserved in the in vivo setting. Third, in vivo imaging systems provide the opportunity for stable longitudinal investigations of weeks to months to study CNS cellular interactions. Finally, given the growing evidence for contributions from the peripheral immune system18,19 and the microbiome20,21 in CNS physiology, in vivo systems provide a platform to interrogate such contributions and effects on CNS cells. Thus, approaches that employ longitudinal in vivo imaging to study neuro-immune physiology and interactions in healthy, injured, and diseased states are a great complementary addition to traditional approaches.
In this protocol, we describe a reliable approach to image different cell types in the brain including microglia, neurons and NG2+ cells as examples. Two approaches to visualize neural cells in vivo have been developed: the thinned skull approach and the open skull with a cranial window approach. Although thinned skull approaches are in use and are preferred because they overcome some of the disadvantages of the open skull approach such as glial cell activation, higher-than-physiological spine dynamics and the use of anti-inflammatory agents22,23,24,25, thinned skull approaches also show a few critical drawbacks. First, the thinning procedure is a very delicate procedure that many researchers find difficult to perfect especially when re-thinning is necessary. This is the case because it is often difficult for experimenters to ascertain that they have thinned the skull to a ~20 &#181;m depth. Second, for adequate comparisons between mice, thinning would need to be identical and a variety of thinning success between imaging sessions or mice could complicate visualization of neural structures. Third, when employed for longitudinal imaging, animals with thinned skulls can only be used for a limited number of sessions when re-thinning of the skull is employed. Forth, since some of the bone tissue still remains, clarity in depth of imaging could be compromised from the thinned skull approach allowing for great visualization of more superficial but not as much with deeper regions. In the light of this, deeper brain structures such as the hippocampus, cannot be successfully imaged with the thinned skull approach. These considerations raise the need for alternative and complementary approaches that could overcome these concerns.
Alternative to the thinned skull approach, the open skull window implantation approach uses a procedure in which the skull is replaced with an optically clear glass coverslip. This allows for a near-unlimited number of imaging sessions. Moreover, given the replacement of the skull with the glass coverslip, this method allows for a clear viewing window of fluorescently tagged brain cells for extensive periods of times from hours to months and, therefore, can be employed to study cell activity and interactions that are relevant for physiology, aging and pathology.
Overall, we detail steps that can be followed to do implant chronic cranial windows through a stereotaxic craniotomy that enables in vivo imaging of brain regions of interest. We also describe how the grossly stable brain vasculature or the fluorescently labeled dendrites could be used to generate a coarse or a fine map, respectively of the brain regions of interest. This approach can then be used for repeated imaging over several sessions. The importance of this technique, therefore, lies in its ability to image the long-term changes or stasis in brain elements including the arrangement, morphology, and interactions of the different cellular types.

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precise brain mapping to perform repetitive in vivo imaging of neuro-immune dynamics in mice

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.

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...

Watch this Scientific Journal Video about Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice at JoVE.com

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

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.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

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