Activation of P42/44 MAPK in the optic nerve head in an experimental rat model of glaucoma

Teresa Mammone, Glyn Chidlow, Robert J. Casson, John P. Wood

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Purpose: To identify whether a specific mitogen-activated protein kinase (MAPK), P42/44 MAPK, is activated in the optic nerve head (ONH) of eyes subjected to chronic ocular hypertension-induced experimental glaucoma and to determine whether this activation is associated with retinal ganglion cell (RGC) death.

Methods: A well established laser-induced procedure was used to cause a chronic intraocular pressure (IOP) elevation in Sprague Dawley rats. Animals were killed at the following time points after pressure elevation: 1, 3, 6, and 24 hours and also 3, 7 and 14 days. The expression of P42/44 MAPK in the region of the optic nerve head was investigated by using immunohistochemistry with antibodies directed against both total P42/44 MAPK and phosphorylated-P42/44 MAPK (the activated form). Actual protein levels were determined by Western immunoblot, again identifying both total and phosphorylated-P42/44 MAPK. mRNA was also extracted from ONH tissue and gene expression using real-time reverse-transcription polymerase chain reaction (RT-PCR) was determined.

Results: Immunohistochemistry confirmed an activation of P42/44 MAPK at the ONH with weak immunoreactivity for active, phosphorylated-P42/44 MAPK present after 3hr and increasingly robust immunoreactivity detectable by 24hr. Furthermore, accumulating immunoreactivity was detectable further into the optic nerve after 7 and 14d, as compared to controls. Immunofluorescence identified that active, phosphorylated-P42/44 MAPK co-localised to optic nerve (head) astrocytes in experiment eyes. Furthermore, western immunoblot analyses suggested distinct increases over time in levels of activated P42/44MAPK in ONH samples, with statistical analysis revealing significant differences at time points as early as 1 day. Finally, RT-PCR confirmed that activation state changes of this enzyme were unrelated to gene expression, since mRNA levels were unchanged in eyes with elevated pressure, as compared with controls.

Conclusions: These results demonstrated that activation of P42/44 MAPK occurred soon after elevation of IOP in experimental eyes. In addition, this activation increased over time and progressed further along the optic nerve to the brain. These results may have significance in the search for an understanding of the mechanisms and possible treatments for glaucoma, resulting from chronic hypertension .
Original languageEnglish
Article number2446
JournalInvestigative Ophthalmology and Visual Science
Issue number7
Publication statusPublished - Jun 2015


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