The activation of mitogen-activated protein kinases in the optic nerve head in a model of ocular hypertension

Glaucoma is a neurological blinding eye disease, which results from the death of retinal ganglion cells. Although the pathogenesis of glaucoma remains unknown, changes in the tissue microenvironment of the optic nerve head (ONH), where insults are believed to be initiated, will cause signalling alterations in local cells. One important type of signalling involved in the control of cellular functions is protein phosphorylation. This is controlled by the balance between protein kinases and protein phosphatases, which add and remove phosphate groups respectively. One particularly important group of protein kinases is the mitogen-activated protein kinase (MAPK) family, whose activity is known to be altered in neurological diseases. The first aim of this thesis was to determine whether specific MAPK family members (P42/44 MAPK, SAPK/JNK MAPK and P38 MAPK) were altered in a laser-induced ocular hypertension model, used to simulate the pressure elevation often associated with glaucoma. Techniques used for analysis included immunohistochemistry to observe changes in histopathological activation and location, Western immunoblotting to quantify changes in protein level expression, and real time reverse-transcriptase polymerase chain reaction to establish whether there were any changes in MAPK gene expression. Total P42/44 MAPK expression was unaffected after intraocular pressure elevation, but a significant increase in its activation was detected in astrocytes in the ONH after 6-24 hours. Active SAPK/JNK was present throughout treated and untreated RGC axons, but accumulated in the ONH at 6-24 hours after pressure elevation, signifying axon transport disruption. P38 MAPK was expressed by a population of microglial cells throughout the retina, ONH and optic nerve, which were significantly increased in number following elevated intraocular pressure. However, this enzyme was only significantly activated in microglia after more than 3 days and then not in the retina, where it was solely activated in retinal ganglion cell perikarya. These data imply both upregulation and activation of MAPK in the ocular hypertension model, in several distinct locations. Levels of particular phosphoproteins are readily affected by minor perturbations in cellular homeostasis, as will occur when an animal is killed for tissue procurement. Thus, the second aim of this thesis was to identify whether activated MAPKs could be stabilised in procured tissues by perfusing animals with saline containing phosphatase inhibitors before fixation. Immunohistochemical analysis was used to observe differences in specific staining of phosphorylated MAPKs. The addition of phosphatase inhibitors to the perfusate had no significant effect on control animals or animals where there was a robust demonstration of tissue damage, but this procedure significantly reduced variability and improved clarity of outcome in labelling for activated MAPKs in animals with less extensive tissue damage, likely by stabilising levels of these phosphoproteins. These data suggest that phosphatase inhibitors stabilised phosphorylated MAPK levels and enabled a clearer dissemination of the activation of these enzymes, particularly when associated tissue damage was not extensive. Having determined that MAPKs isoenzymes were activated in the ONH after sustained ocular hypertension, future work will concentrate on determining whether manipulation of these enzymes could play a useful role in the management of diseases such as glaucoma.