Abstract
purpose. The DBA/2J mouse is a model for secondary angle-closure glaucoma, due to iris atrophy and pigment dispersion, which ultimately lead to increased intraocular pressure (IOP). The study was undertaken to correlate changes in retinal gene expression with IOP elevation by performing microarray analysis of retinal RNA from DBA/2J mice at 3 months before disease onset and at 8 months after IOP elevation.
methods. IOP was monitored monthly in DBA/2J animals, and animals with normal (3 months) or elevated IOP (8 months) were identified. RNA was prepared from three individual retinas at each age, and the RNA was amplified and used to generate biotin-labeled probe for high-density mouse gene microarrays (U430.2; Affymetrix, Santa Clara, CA). A subset of genes was selected for confirmation by quantitative RT-PCR, by using independent retina samples from DBA/2J animals at 3, 5, and 8 months of age and compared to retinas from C57BL/6J control animals at 3 and 8 months.
results. There were changes in expression of 68 genes, with 32 genes increasing and 36 genes decreasing at 8 months versus 3 months. Upregulated genes were associated with immune response, glial activation, signaling, and gene expression, whereas downregulated genes included multiple crystallin genes. Significant changes in nine upregulated genes and two downregulated genes were confirmed by quantitative RT-PCR, with some showing changes in expression by 5 months.
conclusions. DBA/2J retina shows evidence of glial activation and an immune-related response after IOP elevation, similar to what has been reported after acute elevation of IOP in other models.
Glaucoma is a progressive eye disease that leads to blindness due to loss of retinal ganglion cell (RGC) viability and degeneration of the optic nerve.
1 2 3 4 5 Elevation in intraocular pressure (IOP) is a significant risk factor for glaucoma and can lead to optic nerve damage.
6 7 However, although sensitivity to IOP can be a significant initiating event, other factors must contribute to neurodegeneration in this disease. For example, not all patients with elevated IOP have glaucoma. Conversely, there are also patients with normal IOP who exhibit optic nerve disease and vision loss characteristic of glaucoma.
8 Furthermore, in the DBA/2J mouse model of glaucoma in which IOP increases with age due to pigment dispersion from the iris and obstruction of the trabecular meshwork, high-dose radiation followed by syngeneic bone marrow transfer almost completely rescues optic nerve damage and RGC loss, without altering the course of IOP elevation.
9 This raises the question of whether there are factors intrinsic to the retina that contribute to RGC disease as glaucoma progresses.
2
Animal models are critical for obtaining a detailed molecular analysis of retinal changes in glaucoma. For example, IOP can be experimentally elevated in the rat or monkey by injecting hypertonic saline into the episcleral vein or by laser photocoagulation of the trabecular meshwork, and this elevation is associated with optic nerve damage, loss of RGC viability, and blindness.
6 10 These acute models of glaucoma mimic many aspects of the disease and have been used to analyze both cellular and molecular changes associated with increased IOP. Significant changes in the optic nerve correlate with induced elevations in IOP. These include disruption in retrograde axonal transport along the optic nerve
11 ; blockage of both BDNF and TrkB transport, which may result in neurotrophin deprivation at the soma
3 4 ; and remodeling of the optic nerve head, which mimics the cupping of the optic nerve head in humans.
6 10 Within the retina, RGCs undergo apoptotic death,
12 13 14 15 and there are changes in other retinal cell populations as well. For example, there is evidence that Müller glia and retinal astrocytes become activated after IOP elevation,
16 17 18 19 and microglial activation has been reported in association with degenerating RGCs.
20 Molecular analysis of the retina using microarrays or quantitative reverse transcription polymerase chain reaction (RT-PCR) has shown that IOP elevation elicits changes in the expression of multiple genes, including those involved in iron regulation, glial activation and an immune response.
16 21 22
Together, the studies just described reveal a complex retinal response to the insult of elevated IOP. This response raises the question of how gene expression changes in retinal tissue during the progressive development of glaucoma, as occurs in humans, and whether these changes provide insight into the molecular events underlying the loss of RGCs. The inbred DBA/2J mouse strain has emerged as a useful model of secondary, angle-closure glaucoma. This mouse strain carries mutations in two genes,
Tyrp1 and
Gpnmb, that trigger an immune response in the iris. The immune response leads to iris atrophy and pigment dispersion,
23 24 25 which blocks aqueous humor drainage and ultimately causes increased IOP that worsens over time in an age-dependent manner, mimicking the progressive time course associated with human glaucoma.
23 24 25 The DBA/2J is becoming increasingly relevant in light of recent advances in mouse genetics that have made it possible to manipulate gene expression and directly test the role candidate genes play in glaucoma progression. However, to date there has not been an analysis of retinal gene expression in the DBA/2J and an assessment of whether particular molecular changes are associated with IOP elevation in this mouse. Thus, we have undertaken a microarray analysis of whole retina RNA from DBA/2J animals to obtain an initial survey of gene expression changes associated with IOP elevation. We also have used quantitative RT-PCR to compare expression of certain genes identified with the array at early and later time points after IOP elevation. The pattern of gene expression we describe is consistent with a glial response and upregulation of immune-related genes, including complement components, suggesting that these genes represent a response to elevated IOP in the DBA/2J retina. These changes in gene expression bear similarity to gene array results from other glaucoma models
16 22 and also highlight parallels with other neurodegenerative diseases of the central nervous system.
Whole retinas were removed from 3-, 5-, and 8-month-old animals and rinsed in ice-cold 0.1 M phosphate-buffered saline to remove blood. Individual retinas were homogenized in buffer (RLT; Qiagen,Valencia, CA) by using a micropestle and were pulled five times through a 22-gauge needle and flash frozen in liquid nitrogen. Total RNA was then isolated (RNeasy Kit; Qiagen). The quantity and quality of the RNA was assessed with a spectrophotometer (ND1000; NanoDrop Technologies, Rockland, DE) and a bioanalyzer (model 2100; Agilent, Palo Alto, CA). For the 3- and 8-month samples, 20 ng of each retinal RNA sample was amplified, fractionated, and labeled with a biotin kit (Ovation; NuGen Technologies, San Carlos, CA) to generate probes for Affymetrix (Santa Clara, CA) microarray analysis.