Search

The central nervous system (CNS) has historically been viewed as an immunologically privileged site, but recent studies have uncovered a vast landscape of immune cells that reside primarily along its borders. While microglia are largely responsible for surveying the parenchyma, CNS barrier sites are inhabited by a plethora of different innate and adaptive immune cells that participate in everything from the defense against microbes to the maintenance of neural function. Static and dynamic imaging studies have revolutionized the field of neuroimmunology by providing detailed maps of CNS immune cells as well as information about how these cells move, organize, and interact during steady state and inflammatory conditions. These studies have also redefined our understanding of neural immune interactions at a cellular level and reshaped our conceptual view of immune privilege in this specialized compartment. This review will focus on insights gained using imaging techniques in the field of neuroimmunology, with an emphasis on anatomy and CNS immune dynamics during homeostasis, infectious diseases, injuries, and aging.

Innate and adaptive immune interactions within the central nervous system (CNS) and surrounding meninges contribute significantly to neural homeostasis as well as a variety of different neurological disorders. Two-photon laser scanning microscopy is a deep tissue imaging technique that provides a means to image immune cell dynamics and interactions in the living CNS with high spatial and temporal resolution. Optical access to the brain and meninges can be achieved through the creation of thinned skull windows, which can be made without inducing damage and inflammation in the underlying tissue. This protocol provides guidance on how to create a thinned skull window without causing CNS injury. We also describe a highly reproducible method to induce a mild traumatic brain injury using the thinned skull approach. © 2018 by John Wiley & Sons, Inc.

Cellular inhibitor of apoptosis proteins (c-IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4-1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c-IAPs to upregulate NF-κB and ERK, and has been implicated in memory T-cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3-inactive c-IAP2 (c-IAP2(H570A)) have impaired signaling downstream of 4-1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c-IAPs were acutely downregulated by c-IAP antagonists, the primary response of c-IAP2(H570A) mice was normal. However, the number of antigen-specific CD8(+) but not CD4(+) T cells declined more rapidly and to a greater extent in c-IAP2(H570A) mice than in WT controls. Studies with T-cell adoptive transfer demonstrated that the enhanced decay of memory cells was T-cell intrinsic. Thus, c-IAP E3 activity is required for 4-1BB coreceptor signaling and maintenance of CD8(+) T-cell memory.

Disseminated candidiasis primarily targets the kidneys and brain in mice and humans. Damage to these critical organs leads to the high mortality associated with such infections, and invasion across the blood-brain barrier can result in fungal meningoencephalitis. Candida albicans can penetrate a brain endothelial cell barrier in vitro through transcellular migration, but this mechanism has not been confirmed in vivo. MRI using the extracellular vascular contrast agent gadolinium diethylenetriaminepentaacetic acid demonstrated that integrity of the blood-brain barrier is lost during C. albicans invasion. Intravital two-photon laser scanning microscopy was used to provide the first real-time demonstration of C. albicans colonizing the living brain, where both yeast and filamentous forms of the pathogen were found. Furthermore, we adapted a previously described method utilizing MRI to monitor inflammatory cell recruitment into infected tissues in mice. Macrophages and other phagocytes were visualized in kidney and brain by the administration of ultrasmall iron oxide particles. In addition to obtaining new insights into the passage of C. albicans across the brain microvasculature, these imaging methods provide useful tools to study further the pathogenesis of C. albicans infections, to define the roles of Candida virulence genes in kidney versus brain infection and to assess new therapeutic measures for drug development.

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330-337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ∼8-fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ∼4-fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2-fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal-to-noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo-damage).

We compared CNS disease following intracerebral injection of SJL mice with Daniel’s (DA) and BeAn 8386 (BeAn) strains of Theiler’s murine encephalomyelitis virus (TMEV). In tissue culture, DA was more virulent then BeAn. There was a higher incidence of demyelination in the spinal cords of SJL/J mice infected with DA as compared to BeAn. However, the extent of demyelination was similar between virus strains when comparing those mice that developed demyelination. Even though BeAn infection resulted in lower incidence of demyelination in the spinal cord, these mice showed significant brain disease similar to that observed with DA. There was approximately 100 times more virus specific RNA in the CNS of DA infected mice as compared to BeAn infected mice. This was reflected by more virus antigen positive cells (macrophages/microglia and oligodendrocytes) in the spinal cord white matter of DA infected mice as compared to BeAn. There was no difference in the brain infiltrating immune cells of DA or BeAn infected mice. However, BeAn infected mice showed higher titers of TMEV specific antibody. Functional deficits as measured by Rotarod were more severe in DA infected versus BeAn infected mice. These findings indicate that the diseases induced by DA or BeAn are distinct.

IFN-gamma is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-gamma, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler's virus model of multiple sclerosis. During viral infections, IFN-gamma is produced by natural killer (NK) cells, CD4(+) and CD8(+) T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-gamma-mediated protection. To determine the lymphocyte subsets that produce IFN-gamma to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-gamma-production. We demonstrate that IFN-gamma production by both CD4(+) and CD8(+) T cell subsets is critical for resistance to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4(+) T cells make a greater contribution to IFN-gamma-mediated protection. To determine the cellular targets of IFN-gamma-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-gamma receptor. We demonstrate that IFN-gamma receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination.