The outcomes of this thesis are two-fold, and both centre on the core theme of inflammation in relation to AMD. First, my thesis focuses on identifying and understanding the problem by examining the different techniques that illustrate the changes resulting from inflammation-mediated photoreceptor cell death in a photo-oxidative damage model. The focus of my thesis then shifts to investigating a solution by targeting inflammation through the use of corticosteroids.
It is well known that inflammation is implicated in the pathogenesis of AMD (as reviewed in [5, 99]), and in association with death of RPE cells, atrophy of photoreceptors, and dysfunction of the choriocapillaris [6]. The photo-oxidative damage model has been extensively used to investigate a number of features of geographic atrophy in the context of inflammation [127, 140, 141]. The present thesis builds on prior work in this area by incorporating modern methods through the utility of OCT to track photo-oxidative damage to the retina. In addition to comparing the efficacy of corticosteroids to manage inflammatory signaling by retinal cells, and the progression of the photic lesion, with strong potential as a preclinical model to test therapeutic means to slow lesion expansion in geographic atrophy.
Corticosteroids have been identified in the literature as a successful therapeutic in the treatment of disease (as reviewed in [185]). Traditionally, glucocorticoids are known for their anti-inflammatory properties [188, 189], while mineralocorticoids maintain electrolyte homeostasis and fluid balance [189]. This thesis demonstrates the anti-inflammatory properties of glucocorticoids and mineralocorticoids in Müller cells in vitro, showing TA reduces inflammatory markers by approximately 2/3 in vitro, and that FA can return expression of key chemokines such as CCL2, IL-6 and IL-8 to baseline levels. Although there has been evidence of the use of TA in the treatment of other inflammatory conditions such as wet AMD [222-230], one of the key outcomes of this work is that it indicates an anti- inflammatory effect of glucocorticoids in a model for atrophic AMD. More significantly, the
data suggest that FA may be a more potent anti-inflammatory agent than TA, particularly in the presence of IL-1β-mediated inflammation. This is of particular significance as IL-1β has been shown to promote chemokine expression by Müller cells in retinal degeneration [342].
This thesis demonstrates that the glucocorticoid receptor is the key receptor which mediates the effects of both TA and FA with respect to modifying the expression of key chemokines and cytokines by Müller cells that are implicated in retinal degeneration, such as
CCL2, IL-6 and IL-8 [216]. It is known that Müller cells promote immune cell recruitment and mediate inflammation, therefore suppression of these cytokines by corticosteroids in Müller cells may be an efficacious strategy in treating inflammation. In particular, blocking of the glucocortiocoid receptor almost completely abrogated the ant-inflammatory of FA in vitro, and partially ablated the effects of TA administration. One outstanding question, however, are the specific signaling pathways downstream of the glucocorticoid receptor that are most pertinent to the powerful inflammatory responses elicited following PD. While this was outside of the scope of these investigations, it is certainly an important consideration for study in future investigations.
Blocking the mineralocorticoid, estrogen and androgen receptors had no impact on the efficacy of TA exerting its anti-inflammatory properties, suggesting that involvement of a receptor outside the steroid hormone receptor family partially mediates the effects of TA. While it has been postulated that TA may be binding to a combination of mineralocorticoid and glucocorticoid receptors, this work challenges that hypothesis. Further investigation is required to ascertain potential receptors and pathways involved.
I further investigated the use of TA and FA in vivo to monitor the impact of intravitreal administration of TA and FA in the photo-oxidative damage model. FA was found to effectively reduce photoreceptor cell death and preserve retinal function, and to be more effective in these respects than TA. Interestingly, the anti-inflammatory properties of FA in
the photooxidative damage model were found to be dependent on the delivery vehicle, with the Suspension vehicle being superior to Cyclodextrin. This contributes to the literature supporting the notion that delivery vehicles are a key consideration in the development of pharmaceuticals [253]. In this instance, while the Suspension vehicle was identified as superior to Cylcodextrin, it also exhibited slight retinal toxicity. The findings of this study emphasise the need for continual development of more vehicle formulations that strike a better balance between maintaining efficacy of the drug whilst not being cytotoxic to the retina. Overall while these results are promising, further testing in other animal models of AMD would be useful in ascertaining the broader implications of these steroid/vehicle combinations. One such model could be NaIO3 toxicity, which induces RPE degeneration in the rodent retina [137]. Another outstanding question beyond the scope of this work is whether the in vitro results in relation to mechanism of action would be observed in vivo. A follow-up study may involve testing the effect of antagonists such as Mifepristone (RU486) in vivo using the PD model, in combination with either TA or FA administration.
The therapeutic potential of FA raises questions regarding how it may be used clinically. A common theme in the treatment of a suite of retinal degenerative diseases and other ocular diseases is the need for frequent follow up injections as part of the treatment plan, as observed in the bi-monthly and monthly anti-VEGF treatments for wet AMD (as reviewed in [106]). Given that FA has shown promise in reducing inflammation, the question remains if it was to be tested clinically, would it have a long-lasting effect or would frequent injections be needed to sustain the effect. The advantage of corticosteroids is that they come in powder form and by nature cannot dissolve and stay in the eye for an extended period of time because crystalline powder is insoluble (as reviewed in [189]). In contrast, the disadvantage is that corticosteroids are not soluble in water, and this poses a challenge to find an effective, non-cytotoxic delivery vehicle. Given that FA was found to be more potent than
TA, perhaps it has the potential to be longer lasting. A future research direction could be to undertake a more detailed pharmokinetic analysis to better understand corticosteroid behavior in the eye. This warrants further investigation using an animal model that mimics the facets of AMD and facilitates a long, protracted time course such as the rat photo-oxidative model used in Chapter 3 [141].
Overall this thesis contributes valuable information to both the understanding of inflammation-mediated photoreceptor cell death, particularly the role of Muller cells, and the refinement of corticosteroid-based approaches for thwarting these deleterious responses. These data shed light on an unmet need for continued investigation into the refinement of steroid-based treatments for ocular inflammatory conditions, which remain potent and versatile therapeutic candidates for potentially improving outcomes in AMD.