Fletcher Lab - Major Projects
1. The role of purinergic receptors in retinal degeneration
Photoreceptor death is associated with 50% of all cases of blindness in Australia, being a major contributor to visual impairment from dry Age-Related Macular Degeneration (AMD), and hereditary retinal degenerations including Retinitis Pigmentosa (RP) [1]. There are currently no treatments for RP or dry AMD. A clearer understanding of the underlying cause of photoreceptor death is crucial for the development of agents that preserve vision. Moreover, an understanding of the mechanism(s) of photoreceptor death has broader implications for understanding neural loss in other diseases affecting the CNS.
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. P2X receptors in particular, are highly calcium permeable, and have been implicated in neuronal death in the CNS. Following injury to the spinal cord or cortical ischaemia, significant levels of ATP are released into the extracellular space causing over stimulation of P2 receptors on neighbouring cells, calcium influx and ultimately neuronal apoptosis. Blocking the action of P2X receptors prevents neuronal death in the injured spinal cord and ischaemic cortex. We are particularly interested in examining whether overstimulation of P2X receptors on retinal photoreceptors contributes to their death. We want to examine how extracellular ATP kills photoreceptors in rodents, examine whether antagonists to P2 receptor prevents photoreceptor death in animal models of retinal degeneration. This project involves measuring visual function in animal models of retinal degeneration, detailed structural anlaysis and also some molecular biology.
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Figure: A vertical section of rat retina labeled for the P2X7 receptor. Fine puncta can bee seen in both the outer plexiform and inner plexiform layers of the retina, suggesting that this receptor is localized within synapses in the retina. |
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Figure: (A) normal rat retina (B) rat retina that was injected with ATP 5 days before. There is a noticeable loss of photoreceptors (red arrow) following ATP exposure. |
2. The role of microglia in Age-Related macular Degeneration
Age-Related Macular Degeneration (AMD) is a major cause of vision loss in the older community. There are two major questions that we are interested in addressing. First, whether functional changes in the retina of an animal model of AMD occur before the earliest visible signs of disease appear. This information is important for developing clinical tools that are predictive of disease progression. Secondly, we are evaluating the role that dendritic cells, especially microglia play in the development of drusen, a key contributor to early disease formation. This work involves examining the structure and function of an animal model of AMD.
3. The role of glial cells in retinal vascular disease.
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 if 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 increase in growth factor expression, and whether new treatments prevent neovascularization in an animal model of type I diabetes.
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Figure: 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. |
4. The role of glial cells in retinopathy of prematurity
Retinopathy of Prematurity (ROP) is a major cause of visual impairment in children born prematurely. Indeed, approximately 68% of children born weighing less that 1250g are at risk of developing ROP, and around 50% of these children have long term visual problems. It is well known that ROP is a vascular disease, caused by excessive growth of blood vessels on the surface of the retina in response to the combined effects of extreme immaturity of the retina and high levels of oxygen used for critical care of neonates. Currently, treatment targets the pathological angiogenesis. Despite treatment, many children suffer ongoing vision impairment.
Our recent work using a well established rat model of ROP [referred to here as Oxygen-induced retinopathy (OIR)] challenges this conventional view of ROP. We found that OIR was not just a disease characterized by aberrant blood vessel growth. Rather, there was pathological vessel growth into the vitreous at the expense of growth of vessels into the deeper layers of the retina; the peripheral retina was rendered avascular. In addition, there was significant neuronal loss, especially in the neural circuits subserving vision at low light levels, which we attribute to the lack of blood vessels.
We think that the key to understanding aberrant vessel growth and neuronal dysfunction in OIR rests in understanding the change(s) that occur in retinal glial cells. Glial cells, which in the retina include astrocytes and Müller cells, are supporting cells that are crucial for the development and maintenance of the vasculature. Our recent work in a rat model of OIR shows that substantial astrocyte loss occurs within the peripheral retina, and that pathological angiogenesis occurs because of the loss of this glial template . Importantly, we have found that treatment with an antagonist of the angiotensin receptor 1, valsartan, prevented astrocyte death, and promoted normal vessel development into the deeper layers of the retina.
In this project we will address several key unresolved questions related to the effects angiotensin II on glial cells during and the consequences of glial dysfunction for vessel growth and neuronal dysfunction. This project involves examining animal models of ROP to correlate neuronal dysfunction with vascular and glial abnormalities. In addition we will be using calcium imaging and Laser Capture Microscopy to assess the upregulation of angiogenic growth factors in the retina during ROP.
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Figure: Blood vessels on the surface of the retina during ROP |
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Figure: Vertical sections through a retina that has been exposed to high light levels and immunolabelled for the glial cell markers GFAP and Glutamine synthetase. As the photoreceptors die, a glial scar forms around the dying photoreceptors. |