'A picture is worth a thousand words': The use of microscopy for imaging neuroinflammation

Abstract

Since the first studies of the nervous system by the Nobel laureates Camillo Golgi and Santiago Ramon y Cajal using simple dyes and conventional light microscopes, microscopy has come a long way to the most recent techniques that make it possible to perform images in live cells and animals in health and disease. Many pathological conditions of the central nervous system have already been linked to inflammatory responses. In this scenario, several available markers and techniques can help imaging and unveil the neuroinflammatory process. Moreover, microscopy imaging techniques have become even more necessary to validate the large quantity of data generated in the era of 'omics'. This review aims to highlight how to assess neuroinflammation by using microscopy as a tool to provide specific details about the cell's architecture during neuroinflammatory conditions. First, we describe specific markers that have been used in light microscopy studies and that are widely applied to unravel and describe neuroinflammatory mechanisms in distinct conditions. Then, we discuss some important methodologies that facilitate the imaging of these markers, such as immunohistochemistry and immunofluorescence techniques. Emphasis will be given to studies using two-photon microscopy, an approach that revolutionized the real-time assessment of neuroinflammatory processes. Finally, some studies integrating omics with microscopy will be presented. The fusion of these techniques is developing, but the high amount of data generated from these applications will certainly improve comprehension of the molecular mechanisms involved in neuroinflammation.

Keywords: immunofluorescence; immunohistochemistry; microscopy; nervous system; neuroinflammation.

FIGURE 1

Neuroinflammation is characterized by several cellular and molecular processes leading to different cell phenotypes, mainly in astrocytes and microglia. These neuroinflammatory features (production and release of proinflammatory mediators and BBB leakage) can drive morphological changes in glial cells, which can be assessed through histological techniques with different markers (see the main text for details). BBB = blood–brain barrier

FIGURE 2

Simplified illustration of Sholl analysis of cell arborization. (a) In this technique, the cell can be imaged using immunofluorescence or immunohistochemistry. (b) Concentric circles are drawn over the cell, with the body at the center. (c) Two diagonal lines can be drawn to establish two lateral quadrants and two central quadrants. (d) Analyzed parameters can include: the number of branches starting from the soma; the number of times branches cross the circles in each quadrant; the length of the longest branch. Parameters can be reported in total or by quadrant [128, 130, 131].

FIGURE 3

In vivo microscopy can be performed in cell cultures or tissue slices. The microscope is coupled to an incubation system that maintains controlled conditions such as temperature, atmospheric gases (O₂, CO₂), pH, and osmolarity to keep cells/tissue alive for as long as possible.

FIGURE 4

Two‐photon versus one‐photon microscopy. Two‐photon microscopy uses the simultaneous excitation of a fluorophore by two photons. The excitation wavelength is longer than the emission wavelength. Longer wavelengths are lower energy, reducing sample damage caused by phototoxicity. In traditional one‐photon fluorescence microscopy shorter excitation wavelengths are used (for example, ultraviolet light), reducing cell/tissue viability.

FIGURE 5

Omics analyses are a range of approaches that examine the totality or a broad selection of features in a biological sample, for example, genomics, transcriptomics, proteomics and metabolomics identifying, respectively, genes, mRNA, proteins, and metabolites. However, omics analysis uses techniques that require the cells to be removed from the original tissue or site, losing crucial information related to the spatial distribution and heterogeneity of cells. The fusion of microscopy and omics allows the analysis of a large amount of data without losing spatial information, which is extremely important in the study of neuroinflammation.