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


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.


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


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

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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)

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High Resolution Image (TIF 4804 kb)
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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)

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High Resolution Image (TIF 6745 kb)
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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)


  1. 1.
    Funa K, Sasahara M (2014) The roles of PDGF in development and during neurogenesis in the normal and diseased nervous system. J Neuroimmune Pharmacol : the official journal of the Society on NeuroImmune Pharmacology 9(2):168–181. CrossRefGoogle Scholar
  2. 2.
    Sil S, Periyasamy P, Thangaraj A, Chivero ET, Buch S (2018) PDGF/PDGFR axis in the neural systems. Mol Asp Med 62:63–74. CrossRefGoogle Scholar
  3. 3.
    Shim AH, Liu H, Focia PJ, Chen X, Lin PC, He X (2010) Structures of a platelet-derived growth factor/propeptide complex and a platelet-derived growth factor/receptor complex. Proc Natl Acad Sci U S A 107(25):11307–11312. CrossRefPubMedGoogle Scholar
  4. 4.
    Heldin CH (2012) Autocrine PDGF stimulation in malignancies. Ups J Med Sci 117(2):83–91. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Lindahl P, Johansson BR, Leveen P, Betsholtz C (1997) Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science (New York, NY) 277(5323):242–245CrossRefGoogle Scholar
  6. 6.
    Winkler EA, Bell RD, Zlokovic BV (2010) Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling. Mol Neurodegener 5:32. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Armulik A, Genove G, Betsholtz C (2011) Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev Cell 21(2):193–215. CrossRefPubMedGoogle Scholar
  8. 8.
    Hartmann DA, Underly RG, Grant RI, Watson AN, Lindner V, Shih AY (2015) Pericyte structure and distribution in the cerebral cortex revealed by high-resolution imaging of transgenic mice. Neurophotonics 2(4):041402. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sweeney MD, Ayyadurai S, Zlokovic BV (2016) Pericytes of the neurovascular unit: key functions and signaling pathways. Nat Neurosci 19(6):771–783. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Wang J, Zhang D, Fu X, Yu L, Lu Z, Gao Y, Liu X, Man J et al (2018) Carbon monoxide-releasing molecule-3 protects against ischemic stroke by suppressing neuroinflammation and alleviating blood-brain barrier disruption. J Neuroinflammation 15(1):188. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Milesi S, Boussadia B, Plaud C, Catteau M, Rousset MC, De Bock F, Schaeffer M, Lerner-Natoli M et al (2014) Redistribution of PDGFRbeta cells and NG2DsRed pericytes at the cerebrovasculature after status epilepticus. Neurobiol Dis 71:151–158. CrossRefPubMedGoogle Scholar
  12. 12.
    Garbelli R, de Bock F, Medici V, Rousset MC, Villani F, Boussadia B, Arango-Lievano M, Jeanneteau F et al (2015) PDGFRbeta(+) cells in human and experimental neuro-vascular dysplasia and seizures. Neuroscience 306:18–27. CrossRefPubMedGoogle Scholar
  13. 13.
    Kyyriainen J, Ekolle Ndode-Ekane X, Pitkanen A (2017) Dynamics of PDGFRbeta expression in different cell types after brain injury. Glia 65(2):322–341. CrossRefPubMedGoogle Scholar
  14. 14.
    Iihara K, Sasahara M, Hashimoto N, Hazama F (1996) Induction of platelet-derived growth factor beta-receptor in focal ischemia of rat brain. J Cereb Blood Flow Metab : official journal of the International Society of Cerebral Blood Flow and Metabolism 16(5):941–949. CrossRefGoogle Scholar
  15. 15.
    Vasefi MS, Kruk JS, Liu H, Heikkila JJ, Beazely MA (2012) Activation of 5-HT7 receptors increases neuronal platelet-derived growth factor beta receptor expression. Neurosci Lett 511(2):65–69. CrossRefPubMedGoogle Scholar
  16. 16.
    Vasefi MS, Kruk JS, Heikkila JJ, Beazely MA (2013) 5-Hydroxytryptamine type 7 receptor neuroprotection against NMDA-induced excitotoxicity is PDGFbeta receptor dependent. J Neurochem 125(1):26–36. CrossRefPubMedGoogle Scholar
  17. 17.
    Riew TR, Choi JH, Kim HL, Jin X, Lee MY (2018) PDGFR-beta-positive perivascular adventitial cells expressing nestin contribute to fibrotic scar formation in the striatum of 3-NP intoxicated rats. Front Mol Neurosci 11:402. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Oda Y, Nakanishi I (1984) Ultrastructure of the mouse leptomeninx. J Comp Neurol 225(3):448–457. CrossRefPubMedGoogle Scholar
  19. 19.
    Alcolado R, Weller RO, Parrish EP, Garrod D (1988) The cranial arachnoid and pia mater in man: anatomical and ultrastructural observations. Neuropathol Appl Neurobiol 14(1):1–17CrossRefGoogle Scholar
  20. 20.
    Zhang ET, Inman CB, Weller RO (1990) Interrelationships of the pia mater and the perivascular (Virchow-Robin) spaces in the human cerebrum. J Anat 170:111–123PubMedPubMedCentralGoogle Scholar
  21. 21.
    Peters A, Palay S, Webster H (1991) The fine structure of the nervous system: the neurons and supporting cells, 3rd edn. Oxford University Press, New YorkGoogle Scholar
  22. 22.
    Lam MA, Hemley SJ, Najafi E, Vella NGF, Bilston LE, Stoodley MA (2017) The ultrastructure of spinal cord perivascular spaces: implications for the circulation of cerebrospinal fluid. Sci Rep 7(1):12924. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Nakagomi T, Taguchi A, Fujimori Y, Saino O, Nakano-Doi A, Kubo S, Gotoh A, Soma T et al (2009) Isolation and characterization of neural stem/progenitor cells from post-stroke cerebral cortex in mice. Eur J Neurosci 29(9):1842–1852. CrossRefPubMedGoogle Scholar
  24. 24.
    Nakano-Doi A, Nakagomi T, Fujikawa M, Nakagomi N, Kubo S, Lu S, Yoshikawa H, Soma T et al (2010) Bone marrow mononuclear cells promote proliferation of endogenous neural stem cells through vascular niches after cerebral infarction. Stem cells (Dayton, Ohio) 28(7):1292–1302. CrossRefGoogle Scholar
  25. 25.
    Nakayama D, Matsuyama T, Ishibashi-Ueda H, Nakagomi T, Kasahara Y, Hirose H, Kikuchi-Taura A, Stern DM et al (2010) Injury-induced neural stem/progenitor cells in post-stroke human cerebral cortex. Eur J Neurosci 31(1):90–98CrossRefGoogle Scholar
  26. 26.
    Nakagomi T, Molnar Z, Nakano-Doi A, Taguchi A, Saino O, Kubo S, Clausen M, Yoshikawa H et al (2011) Ischemia-induced neural stem/progenitor cells in the pia mater following cortical infarction. Stem Cells Dev 20(12):2037–2051. CrossRefPubMedGoogle Scholar
  27. 27.
    Bifari F, Decimo I, Pino A, Llorens-Bobadilla E, Zhao S, Lange C, Panuccio G, Boeckx B et al (2017) Neurogenic radial glia-like cells in meninges migrate and differentiate into functionally integrated neurons in the neonatal cortex. Cell Stem Cell 20(3):360–373.e367. CrossRefPubMedGoogle Scholar
  28. 28.
    Paxinos G, Watson C (2013) The rat brain in stereotaxic coordinates, 7th Edition edn. Elsevier Academic Press, San DiegoGoogle Scholar
  29. 29.
    Riew TR, Kim HL, Choi JH, Jin X, Shin YJ, Lee MY (2017) Progressive accumulation of autofluorescent granules in macrophages in rat striatum after systemic 3-nitropropionic acid: a correlative light- and electron-microscopic study. Histochem Cell Biol 148(5):517–528. CrossRefPubMedGoogle Scholar
  30. 30.
    Lawrenson JG, Reid AR, Finn TM, Orte C, Allt G (1999) Cerebral and pial microvessels: differential expression of gamma-glutamyl transpeptidase and alkaline phosphatase. Anat Embryol (Berl) 199(1):29–34CrossRefGoogle Scholar
  31. 31.
    Decimo I, Fumagalli G, Berton V, Krampera M, Bifari F (2012) Meninges: from protective membrane to stem cell niche. Am J Stem Cells 1(2):92–105PubMedPubMedCentralGoogle Scholar
  32. 32.
    Saboori P, Sadegh A (2015) Histology and morphology of the brain subarachnoid trabeculae. Anat Res Int 2015:279814. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Weller RO, Sharp MM, Christodoulides M, Carare RO, Mollgard K (2018) The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS. Acta Neuropathol 135(3):363–385. CrossRefPubMedGoogle Scholar
  34. 34.
    Bifari F, Decimo I, Chiamulera C, Bersan E, Malpeli G, Johansson J, Lisi V, Bonetti B et al (2009) Novel stem/progenitor cells with neuronal differentiation potential reside in the leptomeningeal niche. J Cell Mol Med 13(9b):3195–3208. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Decimo I, Bifari F, Rodriguez FJ, Malpeli G, Dolci S, Lavarini V, Pretto S, Vasquez S et al (2011) Nestin- and doublecortin-positive cells reside in adult spinal cord meninges and participate in injury-induced parenchymal reaction. Stem Cells (Dayton, Ohio) 29(12):2062–2076. CrossRefGoogle Scholar
  36. 36.
    Bifari F, Berton V, Pino A, Kusalo M, Malpeli G, Di Chio M, Bersan E, Amato E et al (2015) Meninges harbor cells expressing neural precursor markers during development and adulthood. Front Cell Neurosci 9:383. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Gerdes J, Schwab U, Lemke H, Stein H (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 31(1):13–20CrossRefGoogle Scholar
  38. 38.
    Scholzen T, Gerdes J (2000) The Ki-67 protein: from the known and the unknown. J Cell Physiol 182(3):311–322.<311::Aid-jcp1>3.0.Co;2-9 CrossRefPubMedGoogle Scholar
  39. 39.
    Fernandez-Klett F, Potas JR, Hilpert D, Blazej K, Radke J, Huck J, Engel O, Stenzel W et al (2013) Early loss of pericytes and perivascular stromal cell-induced scar formation after stroke. J Cereb Blood Flow Metab : official journal of the International Society of Cerebral Blood Flow and Metabolism 33(3):428–439. CrossRefGoogle Scholar
  40. 40.
    Himango WA, Low FN (1971) The fine structure of a lateral recess of the subarachnoid space in the rat. Anat Rec 171(1):1–19. CrossRefPubMedGoogle Scholar
  41. 41.
    Frederickson RG (1991) The subdural space interpreted as a cellular layer of meninges. Anat Rec 230(1):38–51. CrossRefPubMedGoogle Scholar
  42. 42.
    Leduc C, Etienne-Manneville S (2015) Intermediate filaments in cell migration and invasion: the unusual suspects. Curr Opin Cell Biol 32:102–112. CrossRefPubMedGoogle Scholar
  43. 43.
    Lowery J, Kuczmarski ER, Herrmann H, Goldman RD (2015) Intermediate filaments play a pivotal role in regulating cell architecture and function. J Biol Chem 290(28):17145–17153. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60(4):585–595CrossRefGoogle Scholar
  45. 45.
    Wiese C, Rolletschek A, Kania G, Blyszczuk P, Tarasov KV, Tarasova Y, Wersto RP, Boheler KR et al (2004) Nestin expression--a property of multi-lineage progenitor cells? Cell Mol Life Sci : CMLS 61(19-20):2510–2522. CrossRefPubMedGoogle Scholar
  46. 46.
    Kishaba Y, Matsubara D, Niki T (2010) Heterogeneous expression of nestin in myofibroblasts of various human tissues. Pathol Int 60(5):378–385. CrossRefPubMedGoogle Scholar
  47. 47.
    Park D, Xiang AP, Mao FF, Zhang L, Di CG, Liu XM, Shao Y, Ma BF et al (2010) Nestin is required for the proper self-renewal of neural stem cells. Stem Cells (Dayton, Ohio) 28(12):2162–2171. CrossRefGoogle Scholar
  48. 48.
    Calderone A (2012) Nestin+ cells and healing the infarcted heart. Am J Phys Heart Circ Phys 302(1):H1–H9. CrossRefGoogle Scholar
  49. 49.
    Mercier F, Hatton GI (2000) Immunocytochemical basis for a meningeo-glial network. J Comp Neurol 420(4):445–465CrossRefGoogle Scholar
  50. 50.
    Krisch B, Leonhardt H, Oksche A (1984) Compartments and perivascular arrangement of the meninges covering the cerebral cortex of the rat. Cell Tissue Res 238(3):459–474CrossRefGoogle Scholar
  51. 51.
    Hutchings M, Weller RO (1986) Anatomical relationships of the pia mater to cerebral blood vessels in man. J Neurosurg 65(3):316–325. CrossRefPubMedGoogle Scholar
  52. 52.
    Mercier F, Hatton GI (2003) Meninges and perivasculature as mediators of CNS plasticity. In: Advances in Molecular and Cell Biology, vol 31. Elsevier, pp. 215–253. Google Scholar
  53. 53.
    Hannocks MJ, Pizzo ME, Huppert J, Deshpande T, Abbott NJ, Thorne RG, Sorokin L (2018) Molecular characterization of perivascular drainage pathways in the murine brain. J Cereb Blood Flow Metab : official journal of the International Society of Cerebral Blood Flow and Metabolism 38(4):669–686. CrossRefGoogle Scholar
  54. 54.
    Krisch B (1988) Ultrastructure of the meninges at the site of penetration of veins through the dura mater, with particular reference to Pacchionian granulations. Investigations in the rat and two species of New-World monkeys (Cebus apella, Callitrix jacchus). Cell Tissue Res 251(3):621–631CrossRefGoogle Scholar
  55. 55.
    Brinker T, Stopa E, Morrison J, Klinge P (2014) A new look at cerebrospinal fluid circulation. Fluids Barriers CNS 11:10. CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Abbott NJ, Pizzo ME, Preston JE, Janigro D, Thorne RG (2018) The role of brain barriers in fluid movement in the CNS: is there a ‘glymphatic’ system? Acta Neuropathol 135(3):387–407. CrossRefPubMedGoogle Scholar
  57. 57.
    Liu S, Lam MA, Sial A, Hemley SJ, Bilston LE, Stoodley MA (2018) Fluid outflow in the rat spinal cord: the role of perivascular and paravascular pathways. Fluids Barriers CNS 15(1):13. CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Pizzo ME, Wolak DJ, Kumar NN, Brunette E, Brunnquell CL, Hannocks MJ, Abbott NJ, Meyerand ME et al (2018) Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery. J Physiol 596(3):445–475. CrossRefGoogle Scholar
  59. 59.
    Albargothy NJ, Johnston DA, MacGregor-Sharp M, Weller RO, Verma A, Hawkes CA, Carare RO (2018) Convective influx/glymphatic system: tracers injected into the CSF enter and leave the brain along separate periarterial basement membrane pathways. Acta Neuropathol 136(1):139–152. CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

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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