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Bioinformatic and statistical analysis of the optic nerve head in a primate model of ocular hypertension.

Kompass KS, Agapova OA, Li W, Kaufman PL, Rasmussen CA, Hernandez MR - BMC Neurosci (2008)

Bottom Line: Laser scarification of the trabecular meshwork of cynomolgus macaques produced elevated intraocular pressure that was monitored over time and led to varying degrees of damage in different samples.We validated a subset of differentially expressed genes using qRT-PCR, immunohistochemistry, and immuno-enriched astrocytes from healthy and glaucomatous human donors.These genes have previously defined roles in axonal outgrowth, immune response, cell motility, neuroprotection, and extracellular matrix remodeling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St, Louis, MO 63110, USA. kompass@vision.wustl.edu

ABSTRACT

Background: The nonhuman primate model of glaucomatous optic neuropathy most faithfully reproduces the human disease. We used high-density oligonucleotide arrays to investigate whole genome transcriptional changes occurring at the optic nerve head during primate experimental glaucoma.

Results: Laser scarification of the trabecular meshwork of cynomolgus macaques produced elevated intraocular pressure that was monitored over time and led to varying degrees of damage in different samples. The macaques were examined clinically before enucleation and the myelinated optic nerves were processed post-mortem to determine the degree of neuronal loss. Global gene expression was examined in dissected optic nerve heads with Affymetrix GeneChip microarrays. We validated a subset of differentially expressed genes using qRT-PCR, immunohistochemistry, and immuno-enriched astrocytes from healthy and glaucomatous human donors. These genes have previously defined roles in axonal outgrowth, immune response, cell motility, neuroprotection, and extracellular matrix remodeling.

Conclusion: Our findings show that glaucoma is associated with increased expression of genes that mediate axonal outgrowth, immune response, cell motility, neuroprotection, and ECM remodeling. These studies also reveal that, as glaucoma progresses, retinal ganglion cell axons may make a regenerative attempt to restore lost nerve cell contact.

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Expression of GPNMB in experimental glaucoma in sagittal sections of the ONH (Macaque #3; Supplemental Table 3). (A) Low expression of GPNMB (red) in the retinal ganglion cell layer (GCL) and nerve fiber layer (NFL) in control monkey eye. Granular staining of GPNMB is present in the area adjacent to the inner limiting membrane (ILM). Very low staining for GFAP (green) is observed in the normal nerve fiber layer. Nuclei are stained with DAPI. (B) Upregulation of GPNMB in retinal ganglion cells (GCL) and in axons and retinal astrocytes in the nerve fiber layer in ExpG. Notice marked increase in GFAP staining in the nerve fiber layer. A, B: Magnification bar = 25 μm. (C) Low expression of GPMNB in astrocytes and axons in the normal monkey optic nerve head. (D) Marked upregulation of GPNMB in axons in the nerve bundles (NB) and in GFAP+ astrocytes (green) in the lamina cribrosa (LC) and glial columns (GC). Note that GPNMB appears to localize to the nucleus and cytoplasm in astrocytes in ExpG. C, D: Magnification bar = 40 μm. (E and F) Low expression of GPNMB associated with astrocytes and axons in the post laminar optic nerve. (E, Control; F, ExpG). E, F: Magnification bar = 40 μm.
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Figure 3: Expression of GPNMB in experimental glaucoma in sagittal sections of the ONH (Macaque #3; Supplemental Table 3). (A) Low expression of GPNMB (red) in the retinal ganglion cell layer (GCL) and nerve fiber layer (NFL) in control monkey eye. Granular staining of GPNMB is present in the area adjacent to the inner limiting membrane (ILM). Very low staining for GFAP (green) is observed in the normal nerve fiber layer. Nuclei are stained with DAPI. (B) Upregulation of GPNMB in retinal ganglion cells (GCL) and in axons and retinal astrocytes in the nerve fiber layer in ExpG. Notice marked increase in GFAP staining in the nerve fiber layer. A, B: Magnification bar = 25 μm. (C) Low expression of GPMNB in astrocytes and axons in the normal monkey optic nerve head. (D) Marked upregulation of GPNMB in axons in the nerve bundles (NB) and in GFAP+ astrocytes (green) in the lamina cribrosa (LC) and glial columns (GC). Note that GPNMB appears to localize to the nucleus and cytoplasm in astrocytes in ExpG. C, D: Magnification bar = 40 μm. (E and F) Low expression of GPNMB associated with astrocytes and axons in the post laminar optic nerve. (E, Control; F, ExpG). E, F: Magnification bar = 40 μm.

Mentions: GPNMB immunohistochemistry is shown in Figure 3. In control samples, GPNMB protein was detected in the retinal ganglion cell and nerve fiber layers of the retina (Figure 3A). In ExpG samples, GPNMB protein was sharply upregulated in retinal ganglion cells and their axons, and in retinal astrocytes in the retinal nerve fiber layer (Figure 3B). Within the optic nerve head, healthy samples had baseline expression levels of GPNMB protein in astrocytes and retinal ganglion cell axons (Figure 3C). In ExpG samples, GPNMB was sharply upregulated in retinal ganglion cells and their axons, as well as optic nerve head astrocytes (Figure 3D). GPNMB staining appeared to localize to the membranes of GFAP+ astrocytes. In contrast, GFAP+ astrocytes in the lamina cribrosa exhibited strong GPNMB staining where, at higher magnification, GPNMB protein could be seen around and over the nucleus in ExpG. Abundant GPNMB was also present in axons in the nerve bundles at the level of the lamina cribrosa (Figure 3D). Low levels of GPNMB were present in astrocytes, oligodendrocytes and nerve fibers in the myelinated post laminar optic nerve in normal and in ExpG (Figure 3E, F)


Bioinformatic and statistical analysis of the optic nerve head in a primate model of ocular hypertension.

Kompass KS, Agapova OA, Li W, Kaufman PL, Rasmussen CA, Hernandez MR - BMC Neurosci (2008)

Expression of GPNMB in experimental glaucoma in sagittal sections of the ONH (Macaque #3; Supplemental Table 3). (A) Low expression of GPNMB (red) in the retinal ganglion cell layer (GCL) and nerve fiber layer (NFL) in control monkey eye. Granular staining of GPNMB is present in the area adjacent to the inner limiting membrane (ILM). Very low staining for GFAP (green) is observed in the normal nerve fiber layer. Nuclei are stained with DAPI. (B) Upregulation of GPNMB in retinal ganglion cells (GCL) and in axons and retinal astrocytes in the nerve fiber layer in ExpG. Notice marked increase in GFAP staining in the nerve fiber layer. A, B: Magnification bar = 25 μm. (C) Low expression of GPMNB in astrocytes and axons in the normal monkey optic nerve head. (D) Marked upregulation of GPNMB in axons in the nerve bundles (NB) and in GFAP+ astrocytes (green) in the lamina cribrosa (LC) and glial columns (GC). Note that GPNMB appears to localize to the nucleus and cytoplasm in astrocytes in ExpG. C, D: Magnification bar = 40 μm. (E and F) Low expression of GPNMB associated with astrocytes and axons in the post laminar optic nerve. (E, Control; F, ExpG). E, F: Magnification bar = 40 μm.
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Figure 3: Expression of GPNMB in experimental glaucoma in sagittal sections of the ONH (Macaque #3; Supplemental Table 3). (A) Low expression of GPNMB (red) in the retinal ganglion cell layer (GCL) and nerve fiber layer (NFL) in control monkey eye. Granular staining of GPNMB is present in the area adjacent to the inner limiting membrane (ILM). Very low staining for GFAP (green) is observed in the normal nerve fiber layer. Nuclei are stained with DAPI. (B) Upregulation of GPNMB in retinal ganglion cells (GCL) and in axons and retinal astrocytes in the nerve fiber layer in ExpG. Notice marked increase in GFAP staining in the nerve fiber layer. A, B: Magnification bar = 25 μm. (C) Low expression of GPMNB in astrocytes and axons in the normal monkey optic nerve head. (D) Marked upregulation of GPNMB in axons in the nerve bundles (NB) and in GFAP+ astrocytes (green) in the lamina cribrosa (LC) and glial columns (GC). Note that GPNMB appears to localize to the nucleus and cytoplasm in astrocytes in ExpG. C, D: Magnification bar = 40 μm. (E and F) Low expression of GPNMB associated with astrocytes and axons in the post laminar optic nerve. (E, Control; F, ExpG). E, F: Magnification bar = 40 μm.
Mentions: GPNMB immunohistochemistry is shown in Figure 3. In control samples, GPNMB protein was detected in the retinal ganglion cell and nerve fiber layers of the retina (Figure 3A). In ExpG samples, GPNMB protein was sharply upregulated in retinal ganglion cells and their axons, and in retinal astrocytes in the retinal nerve fiber layer (Figure 3B). Within the optic nerve head, healthy samples had baseline expression levels of GPNMB protein in astrocytes and retinal ganglion cell axons (Figure 3C). In ExpG samples, GPNMB was sharply upregulated in retinal ganglion cells and their axons, as well as optic nerve head astrocytes (Figure 3D). GPNMB staining appeared to localize to the membranes of GFAP+ astrocytes. In contrast, GFAP+ astrocytes in the lamina cribrosa exhibited strong GPNMB staining where, at higher magnification, GPNMB protein could be seen around and over the nucleus in ExpG. Abundant GPNMB was also present in axons in the nerve bundles at the level of the lamina cribrosa (Figure 3D). Low levels of GPNMB were present in astrocytes, oligodendrocytes and nerve fibers in the myelinated post laminar optic nerve in normal and in ExpG (Figure 3E, F)

Bottom Line: Laser scarification of the trabecular meshwork of cynomolgus macaques produced elevated intraocular pressure that was monitored over time and led to varying degrees of damage in different samples.We validated a subset of differentially expressed genes using qRT-PCR, immunohistochemistry, and immuno-enriched astrocytes from healthy and glaucomatous human donors.These genes have previously defined roles in axonal outgrowth, immune response, cell motility, neuroprotection, and extracellular matrix remodeling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St, Louis, MO 63110, USA. kompass@vision.wustl.edu

ABSTRACT

Background: The nonhuman primate model of glaucomatous optic neuropathy most faithfully reproduces the human disease. We used high-density oligonucleotide arrays to investigate whole genome transcriptional changes occurring at the optic nerve head during primate experimental glaucoma.

Results: Laser scarification of the trabecular meshwork of cynomolgus macaques produced elevated intraocular pressure that was monitored over time and led to varying degrees of damage in different samples. The macaques were examined clinically before enucleation and the myelinated optic nerves were processed post-mortem to determine the degree of neuronal loss. Global gene expression was examined in dissected optic nerve heads with Affymetrix GeneChip microarrays. We validated a subset of differentially expressed genes using qRT-PCR, immunohistochemistry, and immuno-enriched astrocytes from healthy and glaucomatous human donors. These genes have previously defined roles in axonal outgrowth, immune response, cell motility, neuroprotection, and extracellular matrix remodeling.

Conclusion: Our findings show that glaucoma is associated with increased expression of genes that mediate axonal outgrowth, immune response, cell motility, neuroprotection, and ECM remodeling. These studies also reveal that, as glaucoma progresses, retinal ganglion cell axons may make a regenerative attempt to restore lost nerve cell contact.

Show MeSH
Related in: MedlinePlus