Bursting at the Seams: Molecular Mechanisms Mediating Astrocyte Swelling

Abstract

Brain swelling is one of the most robust predictors of outcome following brain injury, including ischemic, traumatic, hemorrhagic, metabolic or other injury. Depending on the specific type of insult, brain swelling can arise from the combined space-occupying effects of extravasated blood, extracellular edema fluid, cellular swelling, vascular engorgement and hydrocephalus. Of these, arguably the least well appreciated is cellular swelling. Here, we explore current knowledge regarding swelling of astrocytes, the most abundant cell type in the brain, and the one most likely to contribute to pathological brain swelling. We review the major molecular mechanisms identified to date that contribute to or mitigate astrocyte swelling via ion transport, and we touch upon the implications of astrocyte swelling in health and disease.

Keywords: Kir4.1; NKCC; Na+/K+-ATPase; SUR1-TRPM4; TRPV4; VRAC; aquaporin; astrocyte; gap junction channels; swelling.

Figure 1

Astrocyte swelling and volume regulation involve multiple complex processes. (A) Illustration depicting the major sites associated with movement of osmotically active molecules into and out of astrocytes influencing astrocyte swelling. Astrocytic end feet at both the glial limitans and surrounding parenchymal blood vessels have been well documented to play a role in astrocyte volume change via movement of osmolytes and water. Molecules also move through the panglial syncytium through contacts with both oligodendrocytes at the paranode and with neighboring astrocytes. Uptake of osmolytes at synapses is also involved in alterations in astrocyte volume. The potential localization of channels reported to regulate astrocyte swelling and/or volume decrease are also depicted; however, exact subcellular localization for many of these channels remains to be determined. (B) Summary of channels mediating astrocyte swelling (red channels) or regulated volume decrease (RVD; blue channels). Yellow channels are involved in both swelling and RVD. During astrocyte swelling, the channels colored red are involved in mediating the influx of ions and osmotically active molecules (red arrows). Swelling involves K⁺ ions moving into the cell via Cx43 gap junctions and hemichannels, Kir4.1 and Na⁺/K⁺-ATPase. The NKCC1 and SUR1-TRPM4 channels allow the influx of multiple ions, including K⁺, Na⁺ and Cl⁻. Glutamate movement into astrocytes through transporters and Ca²⁺ influx through TRPV4 channels also increases the osmotic gradient leading to water movement into swelling astrocytes through AQP4 channels. Following swelling, the channels colored blue are involved in reducing astrocytic volume and expelling osmolytes (blue arrows). Upon RVD, K⁺ moves out of individual astrocytes via Cx43, and Kir4.1 channels. Both ClC-2 and LRRC8/VRAC channels remove Cl⁻ from astrocytes resulting in water movement out of astrocytes through AQP4 channels. The TRPV4 and NKCC1 channels also might play roles in mediating astrocyte volume decrease, however, the mechanisms by which this happens are not yet understood. It is important to note that astrocyte swelling and RVD are complex processes with multiple players that may or may not act together in any given situation and/or following any particular pathological event and that our knowledge regarding many of these mechanisms is still limited, therefore parts of this figure are speculative.

Figure 2

TRPM4 mediates astrocyte swelling after diffuse TBI. (A) Bar graph depicting the average area of the somatic cytoplasm surrounding the nucleus. TRPM4 expression nearly doubled the astrocyte cytoplasmic area, compared with TRPM4 -negative astrocytes. (B) Representative electron micrograph of astrocytes from the hippocampal gray matter 4 weeks post–central fluid percussion injury labeled against TRPM4 (white arrows; red pseudo color). The arrows indicate immunoreactivity against TRPM4. The astrocyte in the middle of the electron micrograph (black arrow head; blue pseudo color) is a TRPM4-negative astrocyte located between two TRPM4-positive astrocytes. N indicates the nucleus of each cell. Traumatic brain injury (TBI), three animals TRPM4-negative, n = 57 cells; TRPM4-positive, n = 110 cells. Analysis of variance; error bars represent standard error of the mean. * p < 0.05. Scale bar 5 µm. From [38].

Figure 3

TRPM4 mediates astrocyte swelling after cerebellar cold injury. (A) Image processing pipeline for astrocyte volume quantification; DiI-stained (red), GFAP-positive (green) astrocytes were imaged; 3D region growing of DiI image outputs a binary image segmentation (Seg) of intracellular voxels (white) from extracellular voxels (black); overlay image of segmentation with DiI/GFAP image demonstrates full coverage of astrocyte arborization (B) Montage of micrographs of murine cerebellum with granule cell layer defined with DAPI (dense dotted lines) and co-labeled for GFAP (red) and TUNEL (white) showing TUNEL-positive granule cell layer tissues do not overlap (sparse dotted line) with GFAP-positive granule cell layer tissues; the results shown are representative of n = 4 mice. (C) Slices of segmented binary images of cerebellar granule cell layer astrocytes from wild-type (WT) and TRPM4^−/− mice submitted to control sham surgery (CTR) or cerebellar cold injury (Cryo) showing that in WT mice, granule cell layer astrocytes in cold injured cerebellum exhibited swelling of somata and processes, whereas astrocytes from TRPM4^−/− mice were protected from astrocyte swelling after cerebellar cold injury; the results shown are representative of n > 15 cells from 3 independent mice. (D) Quantification of granule cell layer astrocytic volume in WT and TRPM4^−/− mice submitted to sham surgery (CTR) or cerebellar cold injury (Cryo) showing that after cerebellar cold injury, WT astrocytes increased in volume from 8.86 × 10⁴ μm³ to 22.47 × 10⁴ μm³; TRPM4^−/− astrocytes increased in volume from 7.5 × 10⁴ μm³ to only 10.2 × 10⁴ μm³; TRPM4 knockout led to significant reduction in astrocyte swelling after cold injury; * p < 0.05 in ANOVA with Tukey tests between groups denoted with brackets; n.s. = non-significant; n > 15 cells from 3 different mice. From [23].