Authors
Hongyan Han, Wang Jun
Published in
Acta histochemica. Volume 128. Issue 4. Pages 152369. Sep 05, 2026. Epub Sep 05, 2026.
Abstract
Cytotoxic brain edema is a life-threatening condition arising from pathological water accumulation within brain parenchymal cells, primarily astrocytes, following metabolic or toxic insults. Aquaporin-4 (AQP4), the predominant water channel in the central nervous system, is highly enriched in the perivascular endfeet of astrocytes and is considered a key facilitator of bidirectional water flux. Despite extensive molecular and light-microscopic studies, the precise nanoscale distribution of AQP4 within endfoot membranes and its spatial reorganization during the development of cytotoxic edema remain insufficiently characterized. We employed a high-resolution post-embedding immunogold electron microscopy protocol on brain tissue from an established murine model of water-intoxication-induced cytotoxic edema. Adult C57BL/6 J mice were subjected to acute systemic hypo-osmolality by intraperitoneal water loading, and perfused at defined time points. Ultrathin sections of frontoparietal cortex embedded in hydrophilic resin were labeled with primary anti-AQP4 antibodies and secondary antibodies conjugated to ultra-small (1.4 nm) or 10 nm gold particles, followed by silver enhancement for size-based discrimination of two gold particle populations. Quantitative analyses included gold particle density mapping along astrocytic plasma membrane domains, nearest-neighbour distance measurements, polarization indices, and double-label colocalization studies with the inwardly rectifying potassium channel Kir4.1. In control brains, AQP4 immunoreactivity was strikingly polarized, with a 12.7-fold higher gold particle density on perivascular endfoot membranes (62.4 ± 4.8 particles/µm) compared to the parenchymal-facing membranes of the same cells (4.9 ± 1.2 particles/µm; p < 0.001). Double immunogold labeling revealed that 73.5% of AQP4-positive clusters in endfeet colocalized with Kir4.1 signals. Following induction of cytotoxic edema, the density of AQP4 gold particles on endfoot membranes decreased by 41% within 15 min and by 64% at 60 min (p < 0.001), accompanied by a reciprocal increase in labeling on non-endfoot membranes. The polarization index collapsed from 12.7 to 2.1 at 60 min. Morphometric analysis documented a 3.2-fold increase in endfoot cross-sectional area and a progressive loss of orthogonal array-like particle clusters. Across pooled groups, AQP4 polarization was inversely associated with endfoot cross-sectional area; however, within-group analyses indicated that this association was largely attributable to treatment-group differences. This study provides a time-resolved quantitative ultrastructural analysis of AQP4 labeling across astrocytic membrane domains during the evolution of cytotoxic edema. These findings demonstrate a marked time-dependent loss of perivascular AQP4 polarization during acute cytotoxic edema and provide a quantitative framework for future studies examining the mechanisms underlying changes in AQP4 membrane organization.
PMID:
42702136
Bibliographic data and abstract were imported from PubMed on 07 Sep 2026.
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