Ventriculomegaly associated with ependymal gliosis and declines in barrier integrity in the aging human and mouse brain

Abstract

Age-associated ventriculomegaly is typically attributed to neurodegeneration; however, additional factors might initiate or contribute to progressive ventricular expansion. By directly linking postmortem human MRI sequences with histological features of periventricular tissue, we show that substantial lateral ventricle surface gliosis is associated with ventriculomegaly. To examine whether loss of ependymal cell coverage resulting in ventricle surface glial scarring can lead directly to ventricle enlargement independent of any other injury or degenerative loss, we modeled in mice the glial scarring found along the lateral ventricle surface in aged humans. Neuraminidase, which cleaves glycosidic linkages of apical adherens junction proteins, was administered intracerebroventricularly to denude areas of ependymal cells. Substantial ependymal cell loss resulted in reactive gliosis rather than stem cell-mediated regenerative repair of the ventricle lining, and the gliotic regions showed morphologic and phenotypic characteristics similar to those found in aged humans. Increased levels of aquaporin-4, indicative of edema, observed in regions of periventricular gliosis in human tissue were also replicated in our mouse model. 3D modeling together with volume measurements revealed that mice with ventricle surface scarring developed expanded ventricles, independent of neurodegeneration. Through a comprehensive, comparative analysis of the lateral ventricles and associated periventricular tissue in aged humans and mouse, followed by modeling of surface gliosis in mice, we have demonstrated a direct link between lateral ventricle surface gliosis and ventricle enlargement. These studies highlight the importance of maintaining an intact ependymal cell lining throughout aging.

Keywords: aging; ependymal cells; human; injury; lateral ventricle; mouse; neural stem cells; ventriculomegaly.

Figure 1

Increased ventricle volume and periventricular gliosis are typically associated with age in humans. (A) Representative MRI-based 3D reconstructions of the lateral ventricle from humans of different age groups. (B) A cross-sectional sampling of MRI scans from the Open Access Series of Imaging Studies data set (nondemented) demonstrates that increased lateral ventricle volume is associated with age. (Subject 1 and Subject 2 volumes are superimposed and denoted with red dots). (C) Longitudinal data show that expansion typically occurs over time in both males and females. (D) MRI-based lateral ventricle reconstructions show example of regional expansion (red) for two subsequent time periods. (E, F) From aged tissue (56 years old), periventricular, coronal sections stained with H&E and whole-mount preparations of the lateral wall of the lateral ventricle show regions where an intact ependymal is present. (G) H&E staining of representative coronal sections of aged tissue (77 years old) clearly show areas devoid of an intact ependymal layer (bracket). (H) Enlarged region denoted in (G) shows regions lacking ependymal cells, and (I) immunohistochemical analysis reveals that areas without an ependymal monolayer have GFAP⁺ processes at the ventricle surface and a thick hypocellular region. (J) Whole-mount preparations (tissue from 62 years old) revealed areas of intact ependyma (K) and large expanses of GFAP⁺ processes (L, M), magnification of contiguous sheets of ependymal cells (K), ependymal cells with small GFAP⁺ clusters (L), and regions of extensive gliosis (M). Scale bars, 25 μm (F); 500 μm (G, I) and 100 μm (H, K); and 200 μm (J).

Figure 2

Large ventricular volume is associated with widespread gliosis at the ventricle surface in humans. (A) MRI-based 3D reconstructions of the lateral ventricle for Subject 1. (B) H&E staining of periventricular tissue revealed a compromised, attenuated ependymal cell lining of mixed cell composition, including areas devoid of ependymal cell coverage (arrows indicate separation between nuclei of ependymal cells). (C) Representative regional images from extensive immunohistochemical analysis of the ventricle surface revealed that while some areas with normal ependymal cell coverage were present (*, β-catenin indicates cell borders), large expanses of gliotic scarring at the ventricle surface (GFAP⁺ astrocyte processes) predominated. (D) Coded schematic of entire lateral wall of the lateral ventricle, with red indicating areas of astrocytic gliosis, yellow indicating a compromised ependymal lacking a distinct layer of cuboid ependymal cells, and green indicating intact ependyma. Scale bars, 100 μm (B); 40 μm, confocal image (C); 1 mm, schematic (C).

Figure 3

An intact ependyma is found along the entire ventricle surface in the elderly subject with a small volume ventricle. (A) MRI-based 3D reconstructions of the lateral ventricle for Subject 2. (B) H&E staining revealed a robust ependymal monolayer, and (C) immunohistochemistry of whole-mount preparations showed uninterrupted ependymal cell coverage with no surface gliosis. Scale bars, 100 μm (B); 40 μm, confocal image (C); 1 mm, schematic (C).

Figure 4

Throughout aging, mice maintain an intact ependyma, and lateral ventricle volume does not change. (A) Representative confocal image from a whole-mount preparation of lateral ventricle lateral wall and its cartoon representation. β-catenin labels adherens junctions at ependymal cell borders (green outline of cells), and γ-tubulin labels basal bodies of cilia within the apical portion of ependymal cell cytoplasm (small dots within each cell). (B) The number and size of ependymal cells lining the anterior/ventral (AV, see schematic) lateral ventricle is similar in 3-month- and 2-year-old mice. A coronal section from 2-year-old mice stained for S100β reveals a dense ependymal cell monolayer at the ventricle surface (arrows indicate ependymal cell nuclei). (C) In the region posterior/dorsal (PD) to the lateral wall adhesion (red dot on schematic), ependymal cells have a larger surface area in 2-year- vs. 3-month-old mice. Coronal section from 2-year-olds stained for S100β reveals stretched ependymal cells (arrows indicate ependymal cell nuclei). (D) Regional quantification of ependymal cell surface area (*P < 0.05, students t-test). (E) Following a 6-week EdU chase, newly generated ependymal-like cells (EdU⁺/S100β⁺) were observed in the ependyma of 2-year-old mice. (F) Quantification of EdU⁺ ependymal cells at different regions along ventricle surface reveals a significant increase at the PD region (−0.01 to −0.6 mm from Bregma). (G) No age-related change was detected in lateral ventricle volume for any segmented region. (AD, anterior/dorsal; AV, anterior/ventral; PD, posterior/dorsal; Mid, middle) Scale bars, 20 μm (A); 30 μm (B); 15 μm (E). Data are means; error bars are SEM.

Figure 5

In mice, neuraminidase-induced ependymal cell denudation leads to gliosis at the ventricle surface and upregulation of AQP4, similar to AQP4 labeling in glial scars found along the ventricle wall in humans. (A) Two weeks after intraventricular injection of neuraminidase (10 ng μL⁻¹), focal areas of GFAP⁺ processes were observed at the ventricle surface (arrows). (γ-tubulin marks cilia; β-catenin outlines apical membranes of ependymal cells). (B–D) Following the scheme outlined, a dense GFAP⁺ band (bracket, C), magnified in (D), containing many BrdU⁺ cells was observed in regions devoid of ependymal cells (absence of s100β). (V, ventricle) (E) Following intraventricular injection of neuraminidase (50 mU), areas of gliosis (GFAP⁺, denoted by arrows and demarcated by the dotted line) show increased expression of AQP4. Ependymal cells (*) show low levels of AQP4 staining in nonscarred regions. (F) Similarly, in human tissue, areas of surface gliosis (arrows, demarcated by the dotted line) in human tissue show increased expression of AQP4, whereas areas of ependymal cell coverage (*) are marked by β-catenin and low level expression of AQP4. AQP4 imaging was overexposed in regions of scar to show very low levels of AQP4 in intact ependymal cell layer. Scale bars, 100 μm (A, C, F); 40 μm (D); 50 μm (E).

Figure 6

Extensive ventricle surface gliosis results in lateral ventricle enlargement in mice. (A) Two months after intraventricular injection of neuraminidase, lateral ventricles are significantly larger (V, ventricle). (B) Contours traced around the ventricles were complied to create 3D reconstructions to determine lateral ventricle volumes (* denotes adhesion between lateral and medial walls). (C) Ventricle volumes at 2 months postinjection were larger in mice that received neuraminidase compared with mice injected with saline or the uninjected littermates (n = 5 for neuraminidase (NA) group, n = 3 for saline and uninjected controls, *P < 0.03; Student’s t-test). (D) Immunohistochemistry confirmed gliosis and increased levels of AQP4 at the ventricle surface (bracket) and showed that it persisted 2 months after neuraminidase injection. Data are means; error bars are SEM.