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Ultrastructural and Molecular Characterization of Platelet-derived growth factor Beta-Positive Leptomeningeal Cells in the Adult Rat Brain

  • Tae-Ryong Riew
  • Xuyan Jin
  • Hong Lim Kim
  • Soojin Kim
  • Mun-Yong LeeEmail author
Article
  • 56 Downloads

Abstract

The leptomeninges, referring to the arachnoid and pia mater and their projections into the perivascular compartments in the central nervous system, actively participate in diverse biological processes including fluid homeostasis, immune cell infiltrations, and neurogenesis, yet their detailed cellular and molecular identities remain elusive. This study aimed to characterize platelet-derived growth factor beta (PDGFR-β)-expressing cells in the leptomeninges in the adult rat brain using light and electron microscopy. PDGFR-β+ cells were observed in the inner arachnoid, arachnoid trabeculae, pia mater, and leptomeningeal sheath of the subarachnoid vessels, thereby forming a cellular network throughout the leptomeninges. Leptomeningeal PDGFR-β+ cells were commonly characterized by large euchromatic nuclei, thin branching processes forming web-like network, and the expression of the intermediate filaments nestin and vimentin. These cells were typical of active fibroblasts with a well-developed rough endoplasmic reticulum and close spatial correlation with collagen fibrils. Leptomeningeal PDGFR-β+ cells ensheathing the vasculature in the subarachnoid space joined with pial PDGFR-β+ cells upon entering the cortical parenchyma, yet perivascular PDGFR-β+ cells in these penetrating vessels underwent abrupt changes in their morphological and molecular characteristics: they became more flattened with loss of immunoreactivity for nestin and vimentin and deficient collagen deposition, which was indicative of inactive fibroblasts termed fibrocytes. In the cortical parenchyma, PDGFR-β immunoreactivity was almost exclusively localized to larger caliber vessels, and significantly decreased in capillary-like microvessels. Collectively, our data identify PDGFR-β as a novel cellular marker for leptomeningeal fibroblasts comprising the leptomeninges and perivascular adventitial cells of the subarachnoid and penetrating large-sized cortical vasculatures.

Keywords

Platelet-derived growth factor beta Leptomeninges Perivascular fibroblast Leptomeningeal fibroblast Arachnoid mater Pia mater 

Notes

Funding Information

This research was supported by the grant from the National Research Foundation of Korea (NRF) (grant number NRF-2017R1A2B4002922).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interest.

Ethical Approval

All interventions and animal care provisions were in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Survival Surgery provided by the IACUC (Institutional Animal Care and Use Committee) at the College of Medicine, The Catholic University of Korea (Approval number: CUMS-2017-0321-05).

Supplementary material

12035_2019_1793_Fig10_ESM.png (1.8 mb)
Supplementary Figure 1

Representative images indicating the spatial relationship between PDGFR-β expression and vasculature in the cortical parenchyma. (a–c) Double-labeling for PDGFR-β and the smooth muscle cell marker α–smooth muscle actin (α–SMA), showing that PDGFR-β expression does not colocalize with α–SMA, but is localized along the outer part of smooth muscle cells. Arrows indicate parenchymal PDGFR-β-positive cells with small stellate cells with fine processes. Cell nuclei are stained with DAPI. (d, e) Ultrastructural analysis showing no specific PDGFR-β immunoreactivity in capillaries, which consist of a single layer of endothelial cells (ENs; light blue) and a pericyte (P; yellow). Note that the pericyte and endothelial cell are in direct contract with the glia limitans (magenta). Scale bars = 50 μm for a-c; 1 μm for d; 0.5 μm for e. (PNG 1821 kb)

12035_2019_1793_MOESM1_ESM.tif (4.7 mb)
High Resolution Image (TIF 4804 kb)
12035_2019_1793_Fig11_ESM.png (2.5 mb)
Supplementary Figure 2

Representative images indicating that the 78-kDa glucose-regulated protein (GRP78) expression is associated with nestin positivity in leptomeningeal cells. (a, b) Double-labeling for nestin and GRP78 showing that GRP78 is expressed in both nestin-negative cells with elongated or flattened nuclei (arrows) and nestin-positive cells with large round nuclei (asterisks). Cell nuclei are stained with DAPI. (c, d) Ultrastructural characterization of GRP78-positive cells in the leptomeninges. Notably, GRP78 protein, as indicated by highly electron-dense DAB grains, is specifically localized to the cisternae of the rough endoplasmic reticulum, most of which shows marked dilatation, but not to mitochondria (m) or nucleus (N). Additionally, GRP78-positive cells resemble PDGFR-β-positive cells in that they have euchromatic nuclei and are surrounded by many collagen fibrils (magenta in c). Scale bars = 10 μm for a, b; 1 μm for c; 0.5 μm for d. (PNG 2533 kb)

12035_2019_1793_MOESM2_ESM.tif (6.6 mb)
High Resolution Image (TIF 6745 kb)
12035_2019_1793_Fig12_ESM.png (5.7 mb)
Supplementary Figure 3

Ultrastructural analysis of PDGFR-β-positive cells associated with collagen fibrils in the leptomeninges. (a–f) Higher-magnification views of the boxed areas in Fig. 5a, a2, a3, b, b1, and c2, respectively. PDGFR-β-positive cells are closely associated with bundles of cross-sectioned (open arrows) or longitudinally sectioned (closed arrows) collagen fibrils. Note that collagen fibrils sectioned longitudinally show characteristic cross striation. Scale bars = 1 μm for a-f. (PNG 5857 kb)

12035_2019_1793_MOESM3_ESM.tif (20.5 mb)
High Resolution Image (TIF 20949 kb)

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Copyright information

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Authors and Affiliations

  1. 1.Department of Anatomy, Catholic Neuroscience Institute, College of MedicineThe Catholic University of KoreaSeoulKorea
  2. 2.Department of Biomedicine and Health Sciences, College of MedicineThe Catholic University of KoreaSeoulKorea
  3. 3.Integrative Research Support Center, Laboratory of Electron Microscope, College of MedicineThe Catholic University of KoreaSeoulKorea

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