Isolation and characterization of human optic nerve head astrocytes and lamina cribrosa cells

Highlights

• Dissection of human optic nerve head (ONH).

• A technique to isolate human ONH astrocytes and lamina cribrosa cells from a single ONH explant.

• Characterization of ONH cells.

Abstract

The lamina cribrosa is the initial site of glaucomatous injury. Pathological changes to the lamina cribrosa include posterior displacement of the lamina cribrosa, loss of trophic support, and remodeling of the extracellular matrix. Optic nerve head (ONH) astrocytes and lamina cribrosa cells synthesize extracellular matrix proteins to support and maintain the lamina cribrosa under physiological conditions. During glaucoma, these cells respond to mechanical strain and other stimuli, which leads to pathological remodeling of the ONH. Although ONH astrocytes and lamina cribrosa cells have been previously cultured, there is no well-accepted, straightforward technique to isolate both cell types from a single dissected human ONH. To better understand the pathophysiology of glaucoma, we obtained and cultured lamina cribrosa explants from human donor eyes. Initially, cells that grew out from the explant were ONH astrocytes and lamina cribrosa cells. Using a specialized medium, we isolated pure populations of lamina cribrosa cells and ONH astrocytes. ONH astrocytes expressed glial fibrillary acidic protein (GFAP). Lamina cribrosa cells expressed alpha-smooth muscle actin (α-SMA), but were negative for GFAP. This method of ONH cell isolation and cell-culture will provide a technique to better understand the molecular and cell-specific changes in glaucomatous damage to the ONH.

Introduction

The optic nerve head (ONH) is an opening in the posterior sclera, where more than a million unmyelinated retinal ganglion cell axons converge at the optic disc and exit the eye to form the optic nerve. Histological analysis of the ONH shows distinct regional differences recognized as the surface nerve fiber layer, pre-laminar, lamina cribrosa, and post-laminar myelinated optic nerve. For all types of glaucoma, the primary site of injury is the lamina cribrosa region of the ONH (Quigley and Addicks, 1981; Quigley et al., 1983). Cells in the lamina cribrosa actively respond to physiological and pathophysiological conditions; therefore, isolating and culturing these cells may help understand normal lamina cribrosa function and glaucoma pathophysiology.

Cells of the human ONH include five cell types: astrocytes, lamina cribrosa cells, microglia, vascular endothelial cells, and vascular pericytes (Hernandez, 2000; Hernandez et al., 1988). The lamina cribrosa explant yields two primary cell types, ONH astrocytes and lamina cribrosa cells (Hernandez et al., 1988). ONH astrocytes are glial fibrillary acidic protein (GFAP) positive and the major glial cell type found throughout the ONH, which mechanically supports and provides neurotrophic support to retinal ganglion cell axons (Hernandez et al., 2008; Lambert et al., 2001; Tovar-Vidales et al., 2016; Vecino et al., 2016; Yang and Hernandez, 2003). Lamina cribrosa cells are broad, polygonal-shaped cells, localized within and between connective tissue plates in the lamina cribrosa (Hernandez et al., 1988; Tovar-Vidales et al., 2016). Lamina cribrosa cells express alpha-smooth muscle actin (α-SMA), and similar to ONH astrocytes, these cells synthesize extracellular matrix proteins that form the cribriform network (Hernandez et al., 1988; Lambert et al., 2001; Zode et al., 2011). Glaucomatous ONH astrocytes and lamina cribrosa cells show increased expression of growth factors and fibrotic genes; these differences are likely responsible for the fibrotic remodeling of the glaucomatous lamina cribrosa (Hernandez, 2000; Hernandez et al., 2002; Kirwan et al., 2009; Pena et al., 1999; Schneider and Fuchshofer, 2016; Wallace and O'Brien, 2016; Zode et al., 2011).

Our lab aims to gain a better understanding of the role of ONH cells and remodeling of the extracellular matrix. Previous methods have been described to isolate astrocytes and lamina cribrosa cells from human ONH tissue (Hernandez et al., 1988; Lambert et al., 2001; Rogers et al., 2012a, 2012b; Yang and Hernandez, 2003). Here, we describe a modified method where both lamina cribrosa cells and ONH astrocytes can be isolated from a single ONH explant from human donor eyes. We characterize lamina cribrosa cells and ONH astrocytes by cell morphology, intracellular, and extracellular matrix markers.