Review ArticleIntravital imaging of immune cells and their interactions with other cell types in the spinal cord: Experiments with multicolored moving cells
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.
References (141)
- et al.
Focal transient CNS vessel leak provides a tissue niche for sequential immune cell accumulation during the asymptomatic phase of EAE induction
Exp. Neurol.
(2015) - et al.
In vivo two-photon imaging of motoneurons and adjacent glia in the ventral spinal cord
J. Neurosci. Methods
(2018) - et al.
Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy
J. Neurosci. Methods
(2008) - et al.
Imaging large-scale neural activity with cellular resolution in awake, mobile mice
Neuron
(2007) - et al.
Infection and spread of alphaherpesviruses in the nervous system
Adv. Virus Res.
(1998) - et al.
High-resolution intravital imaging reveals that blood-derived macrophages but not resident microglia facilitate secondary axonal dieback in traumatic spinal cord injury
Exp. Neurol.
(2014) - et al.
Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages
Blood
(2000) - et al.
Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP
Neuron
(2000) - et al.
Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice
Mol. Cell. Neurosci.
(2005) - et al.
Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites
J. Immunol. Methods
(2010)
A surviving intact branch stabilizes remaining axon architecture after injury as revealed by in vivo imaging in the mouse spinal cord
Neuron
Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance
Neuron
The transcription factor NFAT exhibits signal memory during serial T cell interactions with antigen-presenting cells
Immunity
Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool
Nat. Neurosci.
Transfer of a foreign gene into the brain using adenovirus vectors
Nat. Genet.
Imaging windows for long-term intravital imaging: general overview and technical insights
Intravital
An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein
Proc. Natl. Acad. Sci. U. S. A.
Extravascular CX3CR1+ cells extend intravascular dendritic processes into intact central nervous system vessel lumen
Microsc. Microanal.
Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions
Nature
Volatile anesthetics-induced neuroinflammatory and anti-inflammatory responses
Med. Gas. Res.
Methylprednisolone or naloxone treatment after acute spinal cord injury: 1-year follow-up data. Results of the second National Acute Spinal Cord Injury Study
J. Neurosurg.
A new approach for ratiometric in vivo calcium imaging of microglia
Sci. Rep.
Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene
J. Leukoc. Biol.
Overcoming macrophage-mediated axonal dieback following CNS injury
J. Neurosci.
Two-photon tissue imaging: seeing the immune system in a fresh light
Nat. Rev. Immunol.
Diversity of innate immune cell subsets across spatial and temporal scales in an EAE mouse model
Sci. Rep.
CellProfiler: image analysis software for identifying and quantifying cell phenotypes
Genome Biol.
Nontoxic, double-deletion-mutant rabies viral vectors for retrograde targeting of projection neurons
Nat. Neurosci.
Ultrasensitive fluorescent proteins for imaging neuronal activity
Nature
An in vivo duo-color method for imaging vascular dynamics following contusive spinal cord injury
J. Vis. Exp.
Fractalkine receptor (CX3CR1) deficiency sensitizes mice to the behavioral changes induced by lipopolysaccharide
J. Neuroinflammation
Anti-inflammatory properties of anesthetic agents
Crit. Care
In vivo imaging of the mouse spinal cord using two-photon microscopy
J. Vis. Exp.
ATP mediates rapid microglial response to local brain injury in vivo
Nat. Neurosci.
Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation
Nat. Commun.
Icy: an open bioimage informatics platform for extended reproducible research
Nat. Methods
Whole-brain vasculature reconstruction at the single capillary level
Sci. Rep.
NO mediates microglial response to acute spinal cord injury under ATP control in vivo
Glia
Influence of methylene blue on microglia-induced inflammation and motor neuron degeneration in the SOD1(G93A) model for ALS
PLoS One
Multiphoton excitation spectra in biological samples
J. Biomed. Opt.
Deficient CX3CR1 signaling promotes recovery after mouse spinal cord injury by limiting the recruitment and activation of Ly6Clo/iNOS+ macrophages
J. Neurosci.
Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord
Proc. Natl. Acad. Sci. U. S. A.
Regulation of myelin structure and conduction velocity by perinodal astrocytes
Proc. Natl. Acad. Sci. U. S. A.
Intravital analysis of vascular permeability in mice using two-photon microscopy
Sci. Rep.
Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures
Proc. Natl. Acad. Sci. U. S. A.
Chronic in vivo imaging in the mouse spinal cord using an implanted chamber
Nat. Methods
Characterization of blood flow in the mouse dorsal spinal venous system before and after dorsal spinal vein occlusion
J. Cereb. Blood Flow Metab.
Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows
J. Physiol.
Implanting glass spinal cord windows in adult mice with experimental autoimmune encephalomyelitis
J. Vis. Exp.
Long- and short-term intravital imaging reveals differential spatiotemporal recruitment and function of myelomonocytic cells after spinal cord injury
J. Physiol.
Cited by (3)
Repeat intravital imaging of the murine spinal cord reveals degenerative and reparative responses of spinal axons in real-time following a contusive SCI
2020, Experimental NeurologyCitation Excerpt :Our results agree with previous studies that observed optic nerve axonal swellings ex vivo, and found that some transected optic nerve distal segment axonal swellings remained static while others shrank or fused with neighboring spheroids (Beirowski et al., 2010). Intravital imaging of YFP+ axons following a dorsal column crush injury has shown that axonal retraction occurs by multiple axotomies along the proximal axonal segment of stalled axons (Evans et al., 2014; Evans et al., 2019). As axonal endbulb formation was a relatively rare event following a mild contusive SCI we did not determine the fate of transected axons in this model or the involvement of immune cells in this process.
Imaging the dynamic interactions between immune cells and the neurovascular interface in the spinal cord
2019, Experimental NeurologyCitation Excerpt :For example, there are different ways to expose the spinal cord for imaging either through the intervertebral space (Kim et al., 2010), or through one or serial laminectomies, and with or without implanting permanent spinal cord windows (Farrar et al., 2012; Fenrich et al., 2013; Haghayegh Jahromi et al., 2017; Sekiguchi et al., 2016). This special issue contains articles that describe in more detail all currently available spinal cord imaging protocols and mouse lines that have made longitudinal imaging of spinal cord biology possible (Cheng et al., 2019; Evans et al., 2019). In addition, others in this issue discuss different aspects of spinal cord injury (Denecke et al., 2019; Schaffran et al., 2019; Zheng et al., 2019) and findings regarding the execution phase of neuroinflammatory disease (Schumacher et al., 2019), as they were derived from spinal cord imaging studies in living animals.
Next Generation Imaging Techniques to Define Immune Topographies in Solid Tumors
2021, Frontiers in Immunology