Laser Photocoagulation Ocular Research And Therapy In Diabetic Retinopathy

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Caroline E. Graham*, Nicolette Binz*, Wei-Yong Shen*, Ian J. Constable*, and Elizabeth P. Rakoczy*

1. INTRODUCTION

Diabetic retinopathy is a severe complication of diabetes leading to some degree of vision impairment in long-term diabetes sufferers. Currently, the most successful treatment available for diabetic retinopathy is laser photocoagulation, a therapy that destroys part of the retina to save central vision. The principal aim of laser photocoagulation in the treatment of diabetic retinopathy is to effect regression of abnormal vessels, reduce oxygen tension and reverse angiogenesis in the retina. Although laser photocoagulation has been employed for more than 30 years, its underlying molecular mechanisms remain unknown. Research is now focused on identifying and understanding these factors, to ultimately develop therapies to protect against the initiation and progression of neovascularisation.

2. DIABETIC RETINOPATHY

Diabetic retinopathy involves changes in the retinal microvasculature and is believed to be a result of long-term hyperglycaemia and possibly hypertension (Porta and Allione, 2004). Early changes are seen in the blood flow of the retina along with thickening of the basement membrane and/or pericyte loss. Non-proliferative diabetic retinopathy progresses with damage to the endothelial cells lining the capillaries, resulting in a breakdown in the blood-retina barrier. This stage is characterised by the presence of microaneurysms, intra-retinal haemorrhages, macular oedema, cotton wool spots and deposits of hard exudates formed from precipitating blood products. Capillary damage causes decreased oxygenation in part of the retina and this localised ischemia provides the stimulus for upregulation of angiogenic factors such as VEGF, thus worsening these microvascular changes.

* Lions Eye Institute and Centre for Ophthalmology and Visual Science, The University of Western Australia, 2 Verdun Street, Nedlands, Australia 6009. [email protected].

Proliferative diabetic retinopathy is characterised by the presence of abnormal vessels arising from the optic disk or retina. New vessels develop as fine, leaky structures and bleed easily into the retina and vitreous, leading to the development of haemorrhages that can cause sudden vision loss. This is accompanied with thickening of the extracellular matrix that instigates contraction of the fibrotic component of vessels, resulting in retinal detachment. Central vision can also be affected as these retinal changes move towards the macula. In severe cases, proliferating vessels obscure normal retinal blood flow and can result in the development of neovascular glaucoma, ultimately leading to severe loss of vision or permanent blindness.

3. LASER PHOTOCOAGULATION THERAPY

Laser photocoagulation of the retina is a non-invasive laser treatment and remains the primary therapy for proliferative retinopathies such as diabetic retinopathy. Current photocoagulation treatment is based on the original developments of ruby, argon and krypton lasers first utilised in the late 1960's [Reviewed in Petrovic and Bhistkul (1999)]. Tunable dye and diode lasers were subsequently introduced, making available a range of useful techniques for the treatment of proliferative retinopathies.

Laser photocoagulation acts by focusing laser energy on the retinal pigment epithelium (RPE), damaging the outer retinal layers whilst leaving Bruch's membrane intact. The main site of energy absorption is the melanin within the RPE and choroid as it has an absorption spectrum of between 400-700 nm. The different types of lasers used in photocoagulation include argon (emission at 488nm, blue/green; 514nm, green), Nd:YAG (532nm, green) krypton (647nm, red) diode (810nm, infrared) and tuneable dyes such as rhodamine that emit over a selected range of wavelengths. Retinal damage caused by laser photocoagulation can be reduced by decreasing the wavelength, spot size, irradiance and exposure duration (Mainster, 1999). Argon laser is strongly absorbed by melanin and haemoglobin and therefore makes an excellent source for direct targeting of vessels. Krypton laser is less absorbed by melanin and produces deeper and more painful chorioretinal lesions and is therefore less often used in treating retinopathies where induction of choroidal neovascu-larisation should be avoided.

3.1. Histological Changes

The observed histopathological changes following laser treatment, are a result of the heat transfer out of the absorbing RPE and choroid and subsequent denaturation of the surrounding tissue (Roider et al., 1998). The use of short wavelength lasers such as argon minimises damage to the choroid and therefore is less likely to induce choroidal neovascu-larisation, detrimental to diabetic retinopathy treatment. The major sites of damage following argon laser photocoagulation are the RPE and outer retinal layers and within the lasered site, the outer segments of the photoreceptors are destroyed (Figure 29.1B). An initial inflammatory response occurs and coagulated cells in the lesion core are removed by phagocytosis. The RPE monolayer can lift to form a gap at the site where photoreceptor outer segments were. This is followed by RPE cell migration and proliferation into multiple layers. Müller cells also proliferate and migrate into areas of damage and interdigitate into RPE cells forming a glial scar (Lewis et al., 1992).

Figure 29.1. Histology of control and lasered mouse retinae at 3 days and 90 days post-argon laser photocoagulation. A, Control retina and B, lasered retina at 3 days post-treatment. C, Control retina and D, lasered retina at 90 days post-treatment. C, choroid; RPE, retinal pigment epithelium; POS, photoreceptor outer segments; PIS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Magnification 40x. Scale bars represent 10 mm.

Figure 29.1. Histology of control and lasered mouse retinae at 3 days and 90 days post-argon laser photocoagulation. A, Control retina and B, lasered retina at 3 days post-treatment. C, Control retina and D, lasered retina at 90 days post-treatment. C, choroid; RPE, retinal pigment epithelium; POS, photoreceptor outer segments; PIS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Magnification 40x. Scale bars represent 10 mm.

Late changes in the retina following laser photocoagulation include further extension of glial processes and denser areas of glial scars. The outer nuclear layer reduces in thickness over time (Figure 29.1D) and the number of apoptotic cells present in the laser lesion decreases. Interestingly, photoreceptors adjacent to laser lesions appear to have increased survival compared to photoreceptors in normal retinae as shown by an increase in basic fibroblast growth factor (bFGF)-immuno-reactive cells in this area (Xiao et al., 1998, 1999). The mechanism contributing to the increased survival of these photoreceptors may be due to the increase in bFGF, suppressing apoptosis in these cells.

The benefits of laser photocoagulation therapy are not without risks. Potentially dangerous complications such as haemorrhaging, corneal burns and the development of cataracts are risks in laser therapy. Ultimately, understanding the underlying molecular mechanisms which produce the beneficial effects of laser photocoagulation could lead to the development of non-destructive treatments, thereby circumventing these potential hazards.

3.2. Changes in Gene Expression

3.2.1. Early Changes

High oxygen demand in the diabetic retina is thought to play a role in the initiation and progression of microvascular changes. The development of areas of hypoxia stimulates the up-regulation of angiogenic factors such as vascular endothelial growth factor (VEGF) and angiopoietins, potentiating neovascularisation (Oh et al., 1999, Park et al., 2003, Yancopoulos et al., 2000). Laser therapy decreases the oxygen demand of the tissue by localised destruction of the photoreceptors and the consequential development of glial scars facilitates the diffusion of oxygen through the retina. It is also thought to diminish the stimulation of angiogenesis by photocoagulation of normal and abnormal vessels and/or stimulate the expression of anti-angiogenic factors.

Many studies have been conducted to examine the effects of laser photocoagulation on specific factors in the rat and mouse. These studies clearly demonstrated that laser photocoagulation does not only destroy oxygen-demanding photoreceptor cells within the laser lesions, but does have a very important and significant effect on the expression of genes within the retina. These include bFGF/FGF2, epithelial growth factor (EGF), transforming growth factor alpha and beta (TGFa, TGFb), insulin growth factor I (IGF-I), glial fibrillary acidic protein (GFAP), platelet derived growth factor (PDGF) and VEGF among others (Humphrey et al., 1997; Xiao et al., 1999). However, these factors are mainly associated with wound healing, an early response to the treatment.

With the advent of array-based gene expression studies, we can now examine the entire gene expression profile of any given tissue, identifying many novel associations and functions for both known and unknown genes. A previous study in our laboratory aimed to identify those genes that were affected by laser photocoagulation (Wilson et al., 2003). This study demonstrated the effect on gene expression of a normal mouse eye three days postargon laser photocoagulation. Angiogenic factors such as FGF14 and FGF16 were found to be down-regulated whilst angiotensin II type 2 receptor, a potent inhibitor of VEGF and VEGF-induced angiogenesis, was significantly up-regulated. As expected, proteins implicated in tissue remodelling and wound healing were also differentially expressed.

3.2.2. Late Changes

Few studies have followed the expression of factors past the initial wound healing response. Xiao et al. (1998) demonstrated a sustained increase (up to 180 days) in bFGF immuno-reactive photoreceptor cells adjacent to the laser lesions. GFAP-positive glial cells were also evident for more than 30 days post-treatment. Zhang et al. (1993) reported that RPE cells became aFGF and bFGF-positive while losing their CRALBP-immuno-reactivity for up to 80 days post laser treatment. These studies demonstrated some of the changes that occurred following laser photocoagulation and indicated that cells within these damaged areas can change the expression of factors controlling angiogenesis.

We sought to extend our earlier study to measure the changes in gene expression after laser photocoagulation long-term. The focus was on identifying genes that had a known functional relationship with angiogenesis. Ultimately, this study aimed to identify novel targets for diabetic retinopathy therapy whereby a directed change in expression would lead to a reduction in neovascularisation without the need to use lasers. Changes in gene expres sion at 90 days post-laser photocoagulation in the normal mouse were measured by micro-array analysis and further examined by real-time PCR (Binz et al., 2005). At 90 days post laser photocoagulation, 107 genes were identified as differentially expressed compared to unlasered controls. Of these 107 genes, 34 had previously been identified as differentially expressed at three days post-treatment. Therefore, these genes demonstrated a true long-term change in expression due to laser photocoagulation.

Inducers of angiogenesis such as VEGF, PDGF, or bFGF were not differentially expressed, indicating that beneficial effects of laser photocoagulation do not stem from long-term changes to this pathway. However, this study identified genes previously associated with cytoskeletal and structural remodelling as differentially expressed at 90 days post-treatment and the level of protein measured corroborated this large increase. Interestingly, some of these genes were not differentially expressed at three days, suggesting there was a late stage of remodelling in the retina following initial wound healing and scar formation. Alternatively, these gene products could be functioning via a novel mechanism contributing to the beneficial effects of laser photocoagulation.

4. CONCLUSIONS

The goal of prevention and treatment of diabetic retinopathy requires the knowledge of factors and events that reduce or prevent neovascularisation. One approach to achieve this goal is to identify genes differentially expressed following successful laser photocoagulation. With the identification of genes initially affected by laser treatment and those whose expression remains changed long-term, we can now apply this knowledge to the diabetic retina. Ultimately, this will enable the development of therapeutic targets for long-term protection and prevention of vision impairment caused by chronic conditions such as diabetes.

5. ACKNOWLEDGEMENTS

We thank the Foundation for Fighting Blindness for C.E. Graham's Young Investigator Award to attend the RD2004 Conference. The authors gratefully acknowledge financial support from the Juvenile Diabetes Research Foundation International, the Australian National Health and Medical Research Council and Westpac Foundation. This work is part of the research effort of the Diabetic Retinopathy Consortium, Perth, Western Australia.

6. REFERENCES

Binz, N., Graham, C. E., Simpson, K., Lai, Y. K. Y., Shen, W.-Y., Lai, C.-M., Speed, T. P. and Rakoczy, P. E., 2005, Long-term effect of therapeutic laser photocoagulation on gene expression in the eye, EMBO:Submitted.

Humphrey, M. F., Chu, Y., Mann, K. and Rakoczy, P., 1997, Retinal GFAP and bFGF expression after multiple argon laser photocoagulation injuries assessed by both immunoreactivity and mRNA levels, Exp Eye Res. 64:361-9.

Lewis, G. P., Erickson, P. A., Guerin, C. J., Anderson, D. H. and Fisher, S. K., 1992, Basic fibroblast growth factor: a potential regulator of proliferation and intermediate filament expression in the retina, J Neurosci. 12:396878.

Mainster, M. A., 1999, Decreasing retinal photocoagulation damage: principles and techniques, Semin Ophthal-mol. 14:200-9.

Oh, H., Takagi, H., Suzuma, K., Otani, A., Matsumura, M. and Honda, Y., 1999, Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells, J Biol Chem. 274:15732-9.

Park, Y. S., Kim, N. H. and Jo, I., 2003, Hypoxia and vascular endothelial growth factor acutely up-regulate angiopoietin-1 and Tie2 mRNA in bovine retinal pericytes, Microvasc Res. 65:125-31.

Petrovic, V and Bhisitkul, R. B., 1999, Lasers and diabetic retinopathy: the art of gentle destruction, Diabetes Technol Ther. 1:177-87.

Porta, M. and Allione, A., 2004, Current approaches and perspectives in the medical treatment of diabetic retinopathy, Pharmacology & Therapeutics. 103:167-77.

Roider, J., El Hifnawi, E. S. and Birngruber, R., 1998, Bubble formation as primary interaction mechanism in retinal laser exposure with 200-ns laser pulses, Lasers Surg Med. 22:240-8.

Wilson, A. S., Hobbs, B. G., Shen, W.-Y., Speed, T. P., Schmidt, U., Begley, C. G. and Rakoczy, P. E., 2003, Argon Laser Photocoagulation-Induced Modification of Gene Expression in the Retina, Invest Ophthalmol Vis Sci. 44:1426-34.

Xiao, M., McLeod, D., Cranley, J., Williams, G. and Boulton, M., 1999, Growth factor staining patterns in the pig retina following retinal laser photocoagulation, Br J Ophthalmol. 83:728-36.

Xiao, M., Sastry, S. M., Li, Z. Y., Possin, D. E., Chang, J. H., Klock, I. B. and Milam, A. H., 1998, Effects of retinal laser photocoagulation on photoreceptor basic fibroblast growth factor and survival, Invest Ophthalmol Vis Sci. 39:618-30.

Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J. and Holash, J., 2000, Vascular-specific growth factors and blood vessel formation, Nature. 407:242-8.

Zhang, N. L., Samadani, E. E. and Frank, R. N., 1993, Mitogenesis and retinal pigment epithelial cell antigen expression in the rat after krypton laser photocoagulation, Invest Ophthalmol Vis Sci. 34:2412-24.

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