Fletcher Lab ~ Major Projects

Project 1 - The role of purinergic receptors in normal and diabetic retina.

Intracellular ATP is vital for metabolism. Extracellular ATP, on the other hand, is known to act as a neurotransmitter in some parts of the CNS including the retina. ATP acts on two types of receptors, P2X and P2Y receptors. In addition to their role in mediating neuronal signalling, activation of these receptors causes release of cytokines and changes in inflammatory mediators, such as COX-2. We are particularly interested in understanding how P2 receptors modulate retinal function and whether glial-neuronal communication occurs via these receptors. In addition, we wish to examine whether abnormalities in the release of extracellular ATP are an important factor in retinal diseases such as diabetes or retinal degeneration.

Project 2 - The role of Glial cells in causing neovascularization during diabetic retinopathy.

The retina contains two major classes of glial cells that are integral to the way the retina functions. Müller cells, the main glial cell within the retina, are vital for maintaining the normal health of the retina and have been implicated in many retinal diseases including diabetic retinopathy. Like astrocytes within the CNS, they play an important role in providing metabolic substrates to neurons, deactivation and recycling of neurotransmitters and maintain the ion balance of the retina. In addition, and of particular relevance to diabetes, Müller cells maintain the blood-retinal barrier and express growth factors such as vascular endothelial growth factor (VEGF) a major stimulant for angiogenesis of retinal blood vessels.

The pathogenic factors linking glial dysfunction directly with vascular dysfunction in diabetic retinopathy are unknown. Our major working hypothesis is that there is a link between reactive gliosis and expression of inflammatory mediators such as COX-2. Reactive gliosis of Müller cells is one of the earliest changes in diabetes. Activation of glial purinergic receptors (especially P2 receptors) by extracellular ATP within the CNS and retina causes gliosis. Moreover, this form of activation induces an upregulation of COX-2, an enzyme involved in the inflammatory pathway that converts arachidonic acid to prostaglandins. It is well known that COX-2 is involved in angiogenesis in a variety of conditions including tumour growth. Recent studies in retinopathy of prematurity and proliferative retinal disease suggest that induction of COX-2 plays a role in angiogenesis in the retina. This project will examine the changes in Müller cells and astrocytes that lead to an increase in growth factor expression, and whether new treatments prevent neovascularization in an animal model of Type I diabetes.

Flatmounted rat retinae labeled for the glutamate transporter, EAAT4 and the astrocyte marker glial fibrillary acidic protein (GFAP). The glutamate transporter EAAT4 is localized to astrocytes within the retina.

Vertical sections of non-diabetic and diabetic (mRen2)27 rat retina labeled for the amino acid GABA. In the diabetic retina, GABA labels Müller cell somata and processes indicating that the function of these glial cells is abnormal.

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Project 3 - Aetiology of photoreceptor death in retinal degeneration.

Retinitis pigmentosa is a family of retinal diseases that are characterized by an inability to see at night, tunnel vision and often blindness. Few treatments are available because the causes of these diseases are not fully understood. We are interested in understanding how photoreceptors function and the development of treatments that prevent or slow photoreceptor death.

Our previous studies on the rd/rd mouse (an animal model of RP) have shown that energy metabolism in photoreceptors is higher prior to degeneration. The levels of cyclic nucleotides (cGMP and cAMP) changes depending on light conditions (dark vs light). During retinal degeneration, these switches fail to operate, causing a major burden on metabolic reserves within photoreceptors. We are particularly interested in examining how cAMP levels can be modulated in photoreceptors and the link this has with light levels. We are also interested in examining whether treatments such as dopamine agonists or alpha-adrenergic agonists modulate cAMP in photoreceptors.