Advances in Intravital Non-Linear Optical Imaging of the Central Nervous System in Rodents

Abstract

Purpose of review: Highly coordinated cellular interactions occur in the healthy or pathologic adult rodent central nervous system (CNS). Until recently, technical challenges have restricted the analysis of these events to largely static modes of study such as immuno-fluorescence and electron microscopy on fixed tissues. The development of intravital imaging with subcellular resolution is required to probe the dynamics of these events in their natural context, the living brain.Recent findings: This review focuses on the recently developed live non-linear optical imaging modalities, the core principles involved, the identified technical challenges that limit their use and the scope of their applications. We highlight some practical applications for these modalities with a specific attention given to Experimental Autoimmune Encephalomyelitis (EAE), a rodent model of a chronic inflammatory disease of the CNS characterized by the formation of disseminated demyelinating lesions accompanied by axonal degeneration.Summary: We conclude that label-free nonlinear optical imaging combined to two photon imaging will continue to contribute richly to comprehend brain function and pathogenesis and to develop effective therapeutic strategies.

Mechanisms of antigen-induced reversal of CNS inflammation in experimental demyelinating disease

“…Autoimmune demyelination has been thought to involve different APCs, such as CD11c + dendritic cells (DCs) that prime myelin-reactive T cells, microglial cells that phagocytose myelin debris, and perivascular macrophages that interact with effector T cells in the leptomeninges (45)(46)(47). The cellular distribution of IV protein antigen therapies in real time has not been previously established but is now possible using intravital two-photon microscopy (IVM), which has been used to document cell trafficking in EAE (48)(49)(50). Therefore, we examined the fate of R-MMPt and R-OVA by IVM using implanted spinal cord window chambers.…”

In Vivo Single-Molecule Detection of Nanoparticles for Multiphoton Fluorescence Correlation Spectroscopy to Quantify Cerebral Blood Flow

“…The complex microvascular network plays a fundamental role within the CNS, where neuronal activity regulates the flow of oxygen and nutrients. , Understanding blood flow dynamics with high spatiotemporal resolution is essential for unraveling the role of vascular dysfunction in a variety of pathological processes. , Ideally, flow rates could be quantified with subcapillary resolution, providing the capability to map flow rate cross-sections within individual vessels in order to distinguish between flow within the center vessel compared to that in the vicinity of the wall of the vessel. Existing methods for blood flow measurements using multiphoton laser-scanning microscopy (MPLSM) provide the capability to measure changes over time by utilizing high concentrations of fluorescent dyes in order to visualize vessels and produce a contrast with red blood cells (RBCs). − However, multiphoton blood flow measurements have not achieved the same spatial and temporal resolution available for multiphoton imaging. , This is in part because they are limited by the scan rate of the microscope and the requirement to track RBCs over a segment of the vessel, rather than providing a pixel-level readout of the flow rate. , …”

“…4−6 However, multiphoton blood flow measurements have not achieved the same spatial and temporal resolution available for multiphoton imaging. 7,8 This is in part because they are limited by the scan rate of the microscope and the requirement to track RBCs over a segment of the vessel, rather than providing a pixel-level readout of the flow rate. 4,9 To date, several reports have demonstrated the use of fluorescence correlation spectroscopy (FCS) to measure the flow profiles and determine flow rates in microfluidic devices with high sensitivity.…”