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C. Lupien, P. Horner; Stat3 Pathway in Glaucoma. A Study of the DBA/2J Mouse Model. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5902.
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Gliosis, the proliferation and hypertrophy of glial cells such as astrocytes and microglia, occurs in glaucoma. Glaucoma, the second leading cause of blindness worldwide, is actually a family of ocular diseases with a common endpoint, the progressive loss of vision as a result of optic nerve neuropathy. A dramatic hypertrophy of astrocytes and Müller cells endfeet, with upregulation of glial fibrillary acidic protein (GFAP) has been observed in the glaucomatous retina. The signaling pathways associated with this reactive gliosis remain to be elucidated but recent evidence implicates the JAK/STAT pathway. We hypothesize that gliosis is mediated by an early stress response in glaucoma and that chronic glial reactivity leads to neuronal vulnerability and dysfunction. The goal of this study is to determine the partners of the JAK/STAT (STAT3) pathway implicated in the retinal glial cells of a DBA/2J mouse, an inherited model of glaucoma.
To determine the implication of astrocytes or Müller cells in the JAK/STAT pathway, we have isolated retinal layers (inner nuclear layer (INL) and ganglion cells layer (GCL)) by laser capture, from mice at 3,5,9 and 16 months of age (Control C57BL/6J). Different sets of primers were designed for JAK/STAT partners (STAT3, JAK-1, JAK-2, IL-6, CNTF, LIF, gp-130, IL6-receptor, CNTF-receptor and LIF-receptor). Primers were used for RT-PCR and Q-PCR analyses. Retinal protein extracts from pooled animals, young (4 month), middle (8 month) and old (14, 16 and 17 month) were used for Western blot analyses with STAT3 and pSTAT3 antibodies. Fresh retinal tissue was used to determine the colocalization of STAT3 by immunofluorescence with specific antibodies for Müller cells and astrocytes.
By RT-PCR, we have determined that STAT3 RNA is expressed at all ages in INL and GCL layers of the DBA/2J mouse. The Q-PCR results demonstrated that at 3 month in the GCL layer, STAT3 RNA expression is at the highest level followed by a decrease at 5 months. STAT3 protein expression increases with age and pSTAT3 is only detected in the oldest animals. STAT3 protein was immuno-colocalized with astrocytes in the GCL and increased with age.
By this study we have demonstrated that STAT3 pathway is activated in the oldest DBA/2J mice. The STAT3 protein colocalized with the GCL layer in fresh retinal tissue. We could thus conclude that STAT3 is primarily expressed in the GCL layer (astrocytes) and despite decreased RNA level, protein production is increased with age.
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