Cell Proliferation and Interleukin-6–Type Cytokine Signaling Are Implicated by Gene Expression Responses in Early Optic Nerve Head Injury in Rat Glaucoma

Experimentally elevated IOP in animals produces glaucoma-like retinal ganglion cell (RGC) and optic nerve axonal loss and a characteristic remodeling of the optic nerve head (ONH) that includes the rearrangement of glial cells and the deposition of extracellular matrix (ECM) proteins. Although experimental glaucoma models have been produced in a number of species, primate and rodent models are the most extensively used. In both human and experimental glaucoma, there is a general consensus that the ONH is the primary site of initial glaucomatous injury to RGC axons.1,2 Recently, sophisticated 3-D histomorphometric and biomechanical studies have been used to examine some of the earliest morphologic and structural ONH responses in experimental glaucoma attributed to elevated IOP in primates.3–11 However, relatively few studies have examined the early cellular responses of the ONH to elevated IOP exposure. Such studies should help us understand how the ONH cells respond to an environment altered by elevated IOP and how these responses might affect axonal integrity. To examine the response to acute IOP elevation, the anterior chamber can be cannulated to allow IOP to be elevated for up to a few hours without altering retinal perfusion. After these acute elevations, abnormalities in axonal transport, including the accumulation of mitochondria, dynein, neurotrophins and their receptors, and the distribution of axonal and astrocytic proteins have been observed.12–17 For longer IOP elevation, a number of techniques are available to obstruct aqueous outflow and produce glaucoma-like optic nerve degeneration.18–23 In initial studies using our chronic, hypertonic, saline-induced rat glaucoma model and immunohistochemistry, we found characteristic glaucoma-like changes in the rat ONH ECM. As early as 11 days after IOP elevation, we observed depositions of collagen IV, collagen VI, and laminin.24 Next, we used immunohistochemistry to study the chronology of morphologic and protein distribution changes in the ONH. At 3 days after IOP elevation, we observed decreased labeling in ONH glial columns for gap junction protein alpha 1 (connexin 43, Gja1) and increases in a cell proliferation marker.25 These changes were followed at 1 week by decreased labeling for neurotrophins and Gfap, increased vascular labeling of collagen VI, and the appearance of swollen, degenerating axons in the ONH and focal lesions in optic nerve cross-sections. In a separate study using laser-induced IOP elevation, an accumulation of the retrograde transport motor dynein was observed in the ONH at approximately the same duration of IOP elevation.26 More recently, we have used our glaucoma model to determine gene expression changes in extensively injured ONH.27 As part of that study, we also reported expression changes in a few specific genes in ONH from eyes with focal optic nerve injuries (<50% axon degeneration). These genes had all been found to be highly regulated in ONH with extensive nerve injury. Among these, we found significant upregulation of tissue inhibitor of metalloproteinases 1 (Timp1), fibulin 2 (Fbln2), and tenascin c (Tnc) as well as downregulation of aquaporin 4 (Aqp4) in these focally injured ONH. In the study presented here, we greatly expand these initial findings by using microarray analysis and quantitative polymerase chain reaction (QPCR) to identify the gene expression changes in glaucoma model ONH from eyes with <15% optic nerve axon degeneration attributed to elevated IOP exposure.