Elsevier

Experimental Neurology

Volume 320, October 2019, 112972
Experimental Neurology

Review Article
Intravital imaging of immune cells and their interactions with other cell types in the spinal cord: Experiments with multicolored moving cells

https://doi.org/10.1016/j.expneurol.2019.112972Get rights and content

Abstract

Intravital imaging of the immune system is a powerful technique for studying biology of the immune response in the spinal cord using a variety of disease models ranging from traumatic injury to autoimmune disorders. Here, we will discuss specific technical aspects as well as many intriguing biological phenomena that have been revealed with the use of intravital imaging for investigation of the immune system in the spinal cord. We will discuss surgical techniques for exposing and stabilizing the spine that are critical for obtaining images, visualizing immune and CNS cells with genetically expressed fluorescent proteins, fluorescent labeling techniques and briefly discuss some of the challenges of image analysis.

Introduction

Intravital imaging of immune cell movement in the spinal cord has presented a number of specific challenges but has resulted in a greater understanding of immune cell interactions within the nervous system. Watching cellular movement in the spinal cord has provided compelling evidence for the role of the immune system in a variety of disorders. Immune mechanisms play a large role in evolving spinal cord pathology, whether it be from traumatic injury or disease processes such as multiple sclerosis or amyotrophic lateral sclerosis. A full, intact immune response requires: (1) signaling at the primary site; (2) signaling at secondary lymphoid organs such as the spleen, lymph nodes and bone marrow and; (3) immune cell movement throughout the body to the site of inflammation via the vascular system. Models that use explanted tissue or slices are intriguing but limit the full dynamics and complexity of the immune response. Tissue within an explant can be maintained at close to physiologic conditions, but the amount of trauma the tissue has sustained as well as the absence of intact circulatory and lymphatic systems make their use for immune applications limited. To this end, intravital imaging utilizing intact animals with fluorescently labeled cell populations to document the movements and cell-to-cell interactions of immune cells within the nervous system has provided spatiotemporal data that cannot be obtained by any other method. (Fig. 1)

Here, we will discuss some technical hurdles specific to investigation of the immune system in the spinal cord and some of the unique situations that may arise when planning intravital imaging experiments involving the immune system. These include methods of surgically exposing the spinal column to allow light penetration and then maintaining this exposure for the duration of imaging. This also requires stabilizing the tissue with one of a variety of fixation devices to minimize movement from breathing and cardiac function. The need to expose and stabilize tissue has yielded an assortment of methods including acute surgical procedures, chronically implanted glass windows and imaging chambers. A variety of techniques have been successfully utilized to image axonal movement after traumatic injury and blood vessel biology in several models but have proven particularly challenging in studying immune system dynamics due to the inflammatory reaction to implanted devices and to surgical procedures themselves. Beyond surgical techniques, complicated multi-color labeling schemes need to be carefully developed for each experiment, most often utilizing combinations of fluorescent labeling strategies to identify specific cellular subgroups with multiple distinguishable fluorophores to look at cellular interactions and potentially functional molecular markers.

Labeling strategies to identify and differentiate multiple structures simultaneously typically involve combinations of fluorescent labeling techniques including transgenic animals with fluorescent proteins expressed under cell type specific promoters, exogenous fluorescent dyes, cell transfer from either a genetically labeled animal or labeled cell culture as well as functional reporters such as calcium dyes. These methods can also be used alongside traditional techniques such as neuronal tract tracing and fluorescently labeled antibody staining. Often multiple strategies in concert are required to answer complex and interesting questions about cellular interactions. Hopefully, this technical discussion will provide a basic level of understanding of the complex experimental design needed to obtain useful information about the immune system in the spinal cord using live two-photon imaging and can guide initial discussions about experimental design.

Section snippets

Surgical techniques for exposure of the spinal cord and stabilization for imaging

Achieving adequate exposure and stability of the spinal column to allow for imaging with minimal movement artifact is critical to the success of intravital imaging of the spinal cord. This requires a single or multi-level dorsal laminectomy for more widespread imaging. However, if limiting imaging to the intervertebral spaces is acceptable, imaging is possible by removing part of the ligamentous tissue between the bones and imaging through this space (Kim et al., 2010). The spinal cord can then

Fluorescent labeling strategies specific for imaging of the immune system

Fluorescent labeling of multiple cellular groups has been essential for research involving immune cell function by allowing for identification, tracking and observation of cell-to-cell interactions that are critical to the function of the immune system. Some of the commonly imaged cell groups involve neurons, astrocytes, microglia, oligodendrocytes, macrophages, neutrophils and T-cells using combinations of genetic strategies, cell dyes and cell transplantation and lineage tracing methods. (

Conclusions

Intravital imaging is beginning to yeild exciting discoveries about immune cell responses and their interactions with the many other cell types in the spinal cord. The immune system presents specific challenges for intravital imaging beyond that with other models, requiring additional thought and planning when deciding on methods for exposure of the spinal tissue for imaging. Experimental design with multi-color labeling of immune cells, as well as endogenous components of the immune system,

Acknowledgements

There was not any government or NIH funding used, so no grant numbers are available.

Declaration of Competing Interests

The authors have no competing financial interests to disclose.

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