Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases

Retinal neurodegenerative diseases like age-related macular degeneration, glaucoma, diabetic retinopathy and retinitis pigmentosa each have a different etiology and pathogenesis. However, at the cellular and molecular level, the response to retinal injury is similar in all of them, and results in morphological and functional impairment of retinal cells. This retinal degeneration may be triggered by gene defects, increased intraocular pressure, high levels of blood glucose, other types of stress or aging, but they all frequently induce a set of cell signals that lead to well-established and similar morphological and functional changes, including controlled cell death and retinal remodeling. Interestingly, an inflammatory response, oxidative stress and activation of apoptotic pathways are common features in all these diseases. Furthermore, it is important to note the relevant role of glial cells, including astrocytes, Müller cells and microglia, because their response to injury is decisive for maintaining the health of the retina or its degeneration. Several therapeutic approaches have been developed to preserve retinal function or restore eyesight in pathological conditions. In this context, neuroprotective compounds, gene therapy, cell transplantation or artificial devices should be applied at the appropriate stage of retinal degeneration to obtain successful results. This review provides an overview of the common and distinctive features of retinal neurodegenerative diseases, including the molecular, anatomical and functional changes caused by the cellular response to damage, in order to establish appropriate treatments for these pathologies.

Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys

Vascular endothelial growth factor (VEGF) is one of the major mediators of retinal ischemia-associated neovascularization. We have shown here that adeno-associated virus (AAV)-mediated expression of sFIt-1, a soluble form of the Flt-1 VEGF receptor, was maintained for up to 8 and 17 months postinjection in mice and in monkeys, respectively. The expression of sFIt-1 was associated with the long-term (8 months) regression of neovascular vessels in 85% of trVEGF029 eyes. In addition, it resulted in the maintenance of retinal morphology, as the majority of the treated trVEGF029 eyes (75%) retained high numbers of photoreceptors, and in retinal function as measured by electroretinography. AAV-mediated expression of sFIt-1 prevented the development of laser photocoagulation-incluced choroidal neovascularization in all treated monkey eyes. There were no clinically or histologically detectable signs of toxicity present in either animal model following AAV.sFlt injection. These results suggest that AAV-mediated secretion gene therapy could be considered for treatment of retinal and choroidal neovascularizations.

A GH receptor antisense oligonucleotide inhibits hepatic GH receptor expression, lGF-I production and body weight gain in normal mice

Diabetic retinopathy and acromegaly are diseases associated with excess action of GH and its effector IGF-1, and there is a need for improved therapies. We have designed all optimised 2'-O-(2-methoxyethyl)-modified phosphorothioate oligodeoxynucleotide, ATL 227446, and demonstrated its ability to Suppress GH receptor mRNA in vitro. Subcutaneous injections of ATL 227446 reduced GH receptor mRNA levels, GH binding activity and serum IGF-1 levels in mice after seven days of closing. The reduction in serum IGF-1 could be sustained for over tell weeks of dosing at therapeutically relevant levels, during which there was also a significant decrease in body weight gain in antisense-treated mice relative to saline and mismatch control-treated mice. The findings indicate that administration of an antisense oligonucleotide to the GH receptor may be applicable to human diseases in which suppression of GH action provides therapeutic benefit.

Nutritional supplementation for type 2 diabetes: a systematic review

The role of nutritional supplementation is of increasing interest with regard to ocular disease. Randomised controlled trials have demonstrated the effectiveness of supplementation for age-related macular degeneration, and formulations are now being developed for use by people with diabetes and diabetic retinopathy. The aim of this review was to synthesise the evidence for use of nutritional supplementation in type 2 diabetes. MEDLINE and EMBASE databases were searched using a systematic approach. Only double-masked randomised controlled trials were selected. A total of 50 trials were identified as suitable for inclusion. The potential role of alpha-lipoic acid, chromium, folic acid, isoflavones, magnesium, Pycnogenol®, selenium, vitamin C, vitamin E, and zinc in the treatment of type 2 diabetes is discussed. The review of trials identifies positive effects of these nutrients on various outcome measures relating to insulin resistance and cardiovascular factors. Chromium was the most studied supplement, accounting for 16 of the 50 trials. A majority of the trials found a positive effect of chromium on fasting plasma glucose. Isoflavones were found to have a positive effect on insulin resistance and cardiovascular outcome measures, but only when combined with soy proteins. Vitamin E is reported to reduce oxidative stress at levels of 200 mg day-1 or more.

Methylglyoxal, glyoxalses and cell proliferation

The metabolic function of the glyoxalase system was investigated in (a) the differentiation and proliferation of human tumour cells in vitro, (b) the cell-free assembly of microtubules and (c) in the red blood cells during hyperglycaemia associated with Diabetes Mellitus. Chemically-induced differentiation of human promyelocytic HL60 leukaemia cells to neutrophils, and K562 erythroleukaemia cells, was accompanied by a decrease and an increase in the activity of glyoxalase I, respectively. Growth-arrest of Burkitt's lymphoma Raji cells and GM892 lymphoblastoid cells was accompanied by an increase and a decrease in the activity of glyoxalase I respectively. However, differentiation and growth arrest generally proceeded with an increase in the activity of glyoxalase II. Glyoxalase I activity did not consistently correlate with cell differentiation or proliferation status; hence, it is unlikely that glyoxalase I activity is either an indicator or a regulator of cell differentiation or proliferation. Conversely, glyoxalase II activity consistently increased during cell differentiation and growth-arrest and may be both an indicator and regulator of cell differentiation or proliferation. This may be related to the control of cellular microtubule assembly. S-D-Lactoylglutathione potentiated the cell-free, GTP-promoted assembly of microtubules. The effect was dose-related and was inhibited by glyoxalase II. During assembly, S-D-lactoylglutathione was consumed. This suggests that the glyoxalase system, through the influence of S-D-lactoylglutathione, may regulate the assembly of microtubules in cellular systems The whole blood concentrations of methylglyoxal and S-D-lactoylglutathione were increased in Diabetes Mellitus. There was no significant difference between red blood cell glyoxalase activities in diabetics, compared to healthy controls. However, insulin-dependent diabetic patients with retinopathy had a significantly higher glyoxalase I activity and a lower glyoxalase II activity, than patients without retinopathy. Diabetic retinopathy correlated with high glyoxalase I activity and low glyoxalase II activity and suggests the glyoxalase system may be involved in the development of diabetic complications.

Pain response and follow-up of patients undergoing panretinal laser photocoagulation with reduced exposure times

Purpose - We performed a study of laser panretinal photocoagulation in 20 patients with proliferative retinopathy. We compared short exposure, high-energy laser settings with conventional settings, using a 532?nm, frequency doubled, Neodymium–Yag laser and assessed the patients in terms of pain experienced and effectiveness of treatment. Methods - Twenty patients having panretinal photocoagulation for the first time underwent random allocation to treatment of the superior and inferior hemi-retina. Treatment A used ‘conventional’ parameters: exposure time 0.1?s, power sufficient to produce a visible grey-white burns, spot size 300?µm. The other hemi- retina was treated with treatment B using exposure 0.02?s, 300?µm and sufficient power to have similar endpoint. All patients were asked to evaluate severity of pain on a visual analogue scale. (0=no pain, 10=most severe pain). All patients were masked as to the type of treatment and the order of carrying out the treatment on each patient was randomised. Patients underwent fundus photography and were followed up for 6–45 months. Results - Seventeen patients had proliferative diabetic retinopathy, two had ischaemic central retinal vein occlusion and one had ocular ischaemic syndrome. The mean response to treatment A was 5.11, compared to 1.40 treatment B, on the visual analogue scale, which was statistically significant (P=0.001). All patients preferred treatment B. Further treatments, if required, were performed with treatment B parameters and long-term follow-up has shown no evidence of undertreatment. Conclusions - Shortening exposure time of retinal laser is significantly less painful but equally effective as conventional parameters.

Ranibizumab versus Bevacizumab to treat neovascular age-related macular degeneration:one-year findings from the IVAN Randomized Trial

PURPOSE: To compare the efficacy and safety of ranibizumab and bevacizumab intravitreal injections to treat neovascular age-related macular degeneration (nAMD). DESIGN: Multicenter, noninferiority factorial trial with equal allocation to groups. The noninferiority limit was 3.5 letters. This trial is registered (ISRCTN92166560). PARTICIPANTS: People >50 years of age with untreated nAMD in the study eye who read =25 letters on the Early Treatment Diabetic Retinopathy Study chart. METHODS: We randomized participants to 4 groups: ranibizumab or bevacizumab, given either every month (continuous) or as needed (discontinuous), with monthly review. MAIN OUTCOME MEASURES: The primary outcome is at 2 years; this paper reports a prespecified interim analysis at 1 year. The primary efficacy and safety outcome measures are distance visual acuity and arteriothrombotic events or heart failure. Other outcome measures are health-related quality of life, contrast sensitivity, near visual acuity, reading index, lesion morphology, serum vascular endothelial growth factor (VEGF) levels, and costs. RESULTS: Between March 27, 2008 and October 15, 2010, we randomized and treated 610 participants. One year after randomization, the comparison between bevacizumab and ranibizumab was inconclusive (bevacizumab minus ranibizumab -1.99 letters, 95% confidence interval [CI], -4.04 to 0.06). Discontinuous treatment was equivalent to continuous treatment (discontinuous minus continuous -0.35 letters; 95% CI, -2.40 to 1.70). Foveal total thickness did not differ by drug, but was 9% less with continuous treatment (geometric mean ratio [GMR], 0.91; 95% CI, 0.86 to 0.97; P = 0.005). Fewer participants receiving bevacizumab had an arteriothrombotic event or heart failure (odds ratio [OR], 0.23; 95% CI, 0.05 to 1.07; P = 0.03). There was no difference between drugs in the proportion experiencing a serious systemic adverse event (OR, 1.35; 95% CI, 0.80 to 2.27; P = 0.25). Serum VEGF was lower with bevacizumab (GMR, 0.47; 95% CI, 0.41 to 0.54; P

Multispectral imaging of the ocular fundus using light emitting diode illumination

We present an imaging system based on light emitting diode (LED) illumination that produces multispectral optical images of the human ocular fundus. It uses a conventional fundus camera equipped with a high power LED light source and a highly sensitive electron-multiplying charge coupled device camera. It is able to take pictures at a series of wavelengths in rapid succession at short exposure times, thereby eliminating the image shift introduced by natural eye movements (saccades). In contrast with snapshot systems the images retain full spatial resolution. The system is not suitable for applications where the full spectral resolution is required as it uses discrete wavebands for illumination. This is not a problem in retinal imaging where the use of selected wavelengths is common. The modular nature of the light source allows new wavelengths to be introduced easily and at low cost. The use of wavelength-specific LEDs as a source is preferable to white light illumination and subsequent filtering of the remitted light as it minimizes the total light exposure of the subject. The system is controlled via a graphical user interface that enables flexible control of intensity, duration, and sequencing of sources in synchrony with the camera. Our initial experiments indicate that the system can acquire multispectral image sequences of the human retina at exposure times of 0.05 s in the range of 500-620 nm with mean signal to noise ratio of 17 dB (min 11, std 4.5), making it suitable for quantitative analysis with application to the diagnosis and screening of eye diseases such as diabetic retinopathy and age-related macular degeneration.

How accurate are photographic surrogate markers used to detect macular oedema in the english national Screening Programme?

Introduction: Macular oedema is not directly visible on digital photographs used in screening. Photographic surrogate markers are used to detect patients who may have macular oedema. Evidence suggests that only around 10% of patients with these surrogate markers referred to an ophthalmologist have macular oedema when examined by slit-lamp biomicroscopy. Purpose: The purpose of this audit was to determine how many patients with surrogate markers were truly identified by optical coherence tomography (OCT) as having macular oedema. Method: Data were collected from patients attending digital diabetic retinopathy screening. Patients who presented with surrogate markers for macular oedema also had an OCT scan. The fast macula scan on the Stratus OCT was used and an ophthalmologist reviewed the scans to determine whether macular oedema was present. Results: Out of 66 patients with maculopathy defined as haemorrhages or microaneurysms within one optic disc diameter (DD) of the fovea and visual acuity (VA) worse than 6/9 11 (17%) showed thickening on the OCT, only 4 (6%) had macular oedema. None required laser. Out of 155 patients with maculopathy defined as any exudate within one DD of the fovea or circinate within two DD 45 (29%) showed thickening on the OCT of these 27% required laser. Conclusion: OCT is a useful tool in screening to help identify those who need a true referral to ophthalmology for maculopathy. If exudate is present the chance of having macular oedema and requiring laser treatment is greater than the presence of microaneurysms within one DD and reduced VA.

Rapid access wet AmD referrals using teleophthalmology and a central retinal grading centre

Introduction: We have adapted the existing , optometry diabetic retinopathy screening pathway and software , so that it can be used for wet AMD fast track referral. Purpose: To compare the conventional, optometry wet AMD fast track referral service using FAX transmission, with a teleophthalmology service using colour fundus photography transmitted to a central retinal grading centre. Method: 40 optometry practices involved in diabetic retinopathy screening were enrolled and had modified computer software installed. Referrals were made by conventional fast track FAX to the macular clinic, and patients were photographed by the optometrist and images transmitted to a central grading centre Results of the two pathways were compared in terms of 1)speed of diagnosis and 2)sensitivity and specificity of diagnosis of wet AMD. Results: Over a ten month period, 62 consecutive patients were referred. The mean time for conventional pathway was 20.8 days (range 3-34),and for new teleophthalmology pathway was 6.9 days (range 1-13). Sensitivity of technician grading of images was 96%, Specificity 53%, and consultant ophthalmologist was sensitivity 96%, specificiity 87%. The technician showed a learning effect with specificity increasing from 30.7% for first 31 patient cohort, to 70.6% for the second cohort. One patient had images that could not be graded. Conclusion: Rapid referral of wet AMD cases by optometrists using modified diabetic retinopathy screening software, allows fast and accurate diagnosis, and may reduce unnecessary referrals. Retinal grading technicians can be trained to grade wet AMD images.

OCT evaluation of visible and subthreshold retinal photocoagulation using 532nm laser

Presentation Purpose:We conducted a study to determine if the spectral domain optical coherence tomography (SD-OCT) could be used as a tool to assess effective delivery of threshold and subthreshold laser burns created using 532nm green wavelength laser. Methods:10 patients planned for panretinal photocoagulation (PRP) for proliferative diabetic retinopathy were included in this study. Before initiating the full PRP, a row of moderately white laser burns as used for conventional PRP was created using 532 nm laser set at threshold power for 0.1 second with 300 microns spot size. Further rows of laser burns were created by altering the duration and power settings on the laser device. The area of the retina irradiated with laser was imaged using the Topcon SD-OCT within a few minutes of laser treatment. Results:Laser burns created using threshold power were seen on the OCT scan in all cases as a homogenous diffuse increase in reflectivity extending across the full thickness of retina (Fig 1). Retinal burns created by lowering the duration of laser pulse to 0.01s were barely visible ophthalmoscopically but were clearly detectable on the OCT scan as a localised, well-defined area of increased tissue reflectivity (Fig 2). Conclusions:OCT is a useful to tool to assess the delivery of laser burns created using the 532 nm green laser. Burns of a subthreshold intensity that may not be visible ophthalmoscopically result in retinal changes that are clearly detectable on OCT imaging. Further studies would be needed to assess the clinical effectiveness of subthreshold laser treatment for retinal vascular diseases using the 532 nm green laser.

Optic disc haemorrhage audit

Poster section INTRODUCTION. Retrospective Analysis PURPOSE. To evaluate the morphology and location of optic disc haemorrhages (ODH) identified at diabetic retinopathy (DR) screening to establish whether particular ODH are predictive of ocular disease (e.g. glaucoma). METHODS. Retrospective analysis of 77 patients who presented with ODH at DR screening in the Birmingham and Black Country screening programme between June 2009-March 2010. Mean age was 71 years (range 39-89). Cup/disc ratio (CDR), location and morphology of the haemorrhage were recorded. The outcome of the referral and the status of the ODH were followed up a year later. RESULTS. Of the 77 referred, 34 patients were unassessed for possible glaucoma. Of the 43 patients that were assessed in the hospital eye service for glaucoma, 11 (26%) were diagnosed with glaucoma. These glaucoma patients mostly presented with flame haemorrhages (64%) and blot haemorrhages (36%). Haemorrhages tended to adjoin the margin of the OD (64%), and were more commonly flame shaped (64%). They less commonly occurred in the optic disc itself (36%), and were all blot shaped. The OD Cup/disc ratio (CDR) of the patients with glaucoma (n=11) ranged from 0.33-0.57. It is interesting to note the highest CDR was 0.68 in the 77 patients referred. 32 patients were confirmed as not having glaucoma. 24 (75%) of these patients presented with an ODH adjoining the margin, of which 20 (83%) were flame, and 4 blot (17%) shaped. Only 8 (25%) presented with an ODH in the OD, of which 6 (75%) were blot shaped. One year follow up of the 77 referred cases revealed that the ODH resolved in 45 (57%) patients while 10 (13%) still had an ODH present. 15 (21%) were still under ophthalmology hence digital retinal photos were not available for assessment. Six patients (8%) (age range 71-91 years) died within the year, and one lost to follow up. CONCLUSIONS. The results suggest that a significant number of patients with ODH have glaucoma and that the differing morphology of the haemorrhage is not a major predictor i.e. blot versus flame shaped, adjoining or in the optic disc. The cup/disc ratio did not predict glaucoma either.

Retinal thickness in pregnant women with diabetes

Poster section Design. Retrospective study. Purpose. To assess whether there are changes in foveal thickness (FT) and total macular volume (TMV) in pregnancy in diabetic subjects. Methods. The audit consisted of pregnant women with diabetes, with no maculopathy, who completed their antenatal care at Birmingham Heartlands Hospital. The Zeiss Stratus Optical coherence tomography (OCT) was performed on patients attending diabetic retinopathy (DR) screening at intervals throughout their pregnancy. To be included in the audit patients had to have at least one OCT scan during their pregnancy. Results. Altogether there were 8 type 1 and 22 type 2 patients with mean diabetes duration of 6 years (range 1-20). Mean gestation at DR screening with OCT during the first trimester was 9.7 weeks (6-13) (n=22). The mean and standard deviation for FT for the right was 179.1 µm ± 21.49 and for the left eye was 187.3 µm ± 23.55. The mean TMV was right 6.43 µm ± 0.35 and left 6.50 µm ± 0.39. The mean gestation at DR screening with OCT during the second trimester was 23.4 weeks (18-26) (n=25). The mean FT for the right was 191.4 µm ± 22.70 and the left 195.6 µm ± 24.77. The mean TMV was right 6.74 µm ± 0.45 and left 6.91 µm ± 0.35. The gestation of DR screening with OCT during the third trimester was 31.1 weeks (27-36) (n=15). The mean FT for the right was 181.5 µm ± 24.84 and for the left 193.1 µm ± 28.55. The mean TMV was right 6.80 µm ± 0.40 and left 6.84 µm ± 0.31. There were no significant differences in FT over the 3 trimesters. The TMV showed a significant difference when comparing the first and second trimesters (p<0.05). However, there was no significant statistical difference in TMV in the second and third trimesters. None of the patients showed any macula edema on the OCT. Conclusions. The results suggest there is no significant change in foveal thickness in pregnancy in diabetic subjects. There was a significant statistical difference in total macular volume in the second trimester; however, this would not be clinically significant. This is an important observation proven by the OCT which has not been previously studied.

How accurate are photographic surrogate markers used to detect macular edema in the English national screening programme?

DESIGN. Retrospective analysis PURPOSE. Macular oedema is not directly visible on two dimensional digital photographs such that surrogate markers need to be used. In the English National Screening Programme these are exudate within one optic disc diameter (DD) of the fovea, group of exudates within two DD of the fovea and haemorrhages or microaneurysms (HMA) within one DD of the fovea with best corrected visual acuity (VA) worse than 6/9. All patients who present with any of these surrogate markers at screening are referred to an ophthalmology clinic for slit lamp examination. The purpose of this audit was to determine how many patients with positive maculopathy diagnosis on photography were truly identified by optical coherence tomography (OCT) with macular oedema. METHODS. Data was collected from patients attending digital diabetic retinopathy screening. Patients who presented with surrogate markers for macular oedema also had an OCT scan. The fast macula scan on the Stratus OCT was used and an ophthalmologist reviewed the scans to determine whether macular oedema was present. RESULTS. Maculopathy by exudates: Of 155 patients 45 (29%) showed thickening on the OCT of these 12 required laser. Those who also had pre-proliferative retinopathy (n=20) were more likely to have macular oedema (75%) than those with background diabetic retinopathy. Maculopathy by HMA and VA worse than 6/9: Of 66 patients 11 (16.7%) showed thickening on the OCT. 5 (7.6%) of these had macular oedema, 5 (7.6%) epi-retinal membrane, and 1 (1.5%) age related macular degeneration. None of these patients required laser. CONCLUSIONS. The likelihood of the presence of macular oedema and requiring laser treatment is greater with macular exudation than HMA within one DD and reduced VA. Overall the surrogate markers used show low specificity for macular oedema, however combining OCT with photography does identify those with macular oedema who require a true referral for an ophthalmological slit lamp examination.