European Association for the Study of Diabetic Eye Complications

Annual Meeting Coimbra 27-29th May 2005

Paper Sections 21 - 42

RESULTS OF A RANDOMIZED TRIAL COMPARING THE EFFICACY OF INTRAVITREAL INJECTION OF 2 MG VERSUS 4 MG TRIAMCINOLONE ACETONIDE

Amélie Lecleire-Collet, François Audren, Ali Erginay, Ineta Zundane, John Conrath, Rim Benosman, Pierre- Jean Guillausseau, Pascale Massin. Department of Ophthalmology, Hôpital Lariboisière,Paris , France

Purpose: The optimal dose of triamcinolone acetonide needed to be injected in the vitreous for the treatment of diabetic macular edema remains unknown. The purpose of the study was to prospectively compare the efficacy and safety of a single intravitreal injection of 2 mg versus 4 mg triamcinolone acetonide for diffuse diabetic macular edema refractory to laser photocoagulation.

Methods: Thirty two eyes of 32 patients (5 type 1 diabetics, 27 type 2) with refractory diabetic macular edema were randomly assigned to receive either a 2 mg (in 0.05 ml) (n = 16) or a 4 mg (in 0.1 ml) (n = 16) intravitreal triamcinolone acetonide injection. Outcome measures were central macular thickness measured by Optical Coherence Tomography, best-corrected visual acuity (BCVA) measured by Early Treatment Diabetic Retinopathy Study (ETDRS) scores, intraocular pressure and cataract progression at 4, 12 and 24 weeks.

Results: Before injection, mean central macular thickness ± SD was 528 ± 155 µm in the 2 mg group versus 570 ± 115 µm in the 4 mg group. Four, 12, and 24 weeks after injection, mean central macular thickness ± SD were respectively 263 ± 88 µm (2 mg group) versus 275 ± 80 µm (4 mg group), 285 ± 114µm (2 mg group) versus 262 ± 127 µm (4 mg group), and 397 ± 188 µm (2 mg group) versus 453 ± 145 µm (4 mg group). Before injection, BCVA was 41.8 ± 13.4 ETDRS letters in the 2 mg group versus 37.6 ± 14 in the 4 mg group. At 4 weeks, BCVA was 49.3 ± 13,6 (2 mg group) versus 49.8 ± 14,7 (4 mg group), at 12 weeks, 49.9 ± 13.1 (2 mg group) versus 52.3 ± 11,5 (4 mg group), and at 24 weeks, 45.4 ± 11.3 (2 mg group) versus 42.3 ± 16.5 (4 mg group). In 13 of the 32 injected eyes (7 eyes in the 2 mg group and 6 eyes in the 4 mg group), intraocular pressure exceeded 24 mmHg during the follow-up, but was controlled in all cases by topical medication. Central macular thickness, BCVA, and intraocular pressure were not significantly different between the 2 mg and the 4 mg-groups at any time (p < 0.05).

Conclusion: Intravitreal injection of 2 mg or 4 mg triamcinolone acetonide effectively reduces macular thickening due to diffuse diabetic macular edema in most cases. The differences between the two doses were not statistically significant.

TIMING FOR LASER PHOTOCOAGULATION PRIOR AND AFTER SUCCESSFUL PANCREAS TRANSPLANTATION

Tomas Sosna 1,2, Frantisek Saudek 2, Radka Koznarová 2, Milos Adamec 2 Eye Department of Thomayer Memorial University Hospital, 1 Institute for Clinical and Experimental Medicine, Prague, Czech Republic2

Purpose: Pancreas transplantation is the only option for obtaining long-term normoglycaemia. In spite of relatively optimistic results of our previous studies, we recorded progression of diabetic retinopathy (DR) after successful transplantation. Also we identified some plausible reasons for this progression. We try to summarize proper approaches for avoiding these deteriorations.

Methods: We summarized the results of statistically well analyzable follow-ups of 207 patients who underwent transplantation during the last 19 years. In previous studies, we identified the principal risk factors, which led to progression of DR. It was above all, dramatic decrease of glycosylated hemoglobin (HbAlc), the level of DR before transplantation and the methods and quality of previous laser treatments.

Results: The progression of DR was most often (21 cases) recorded in patients with non-treated severe nonproliferative DR, or with incipient proliferative DR which was not treated or was treated improperly or deficiently. There were 2 cases with fast progression moderate DR as well. We recorded this deterioration in borderline clinically significant macular edema (CSME). Generally, treatable progression was observed between patients with very high level of HbAlc before the transplantation.

Conclusions: A retinal specialist must carefully examine patients prior transplantation. When dramatic decrease of HbAlc is expected and there is imminent danger for transition of DR to severe retinopathy (according to Davis and Murphy´s rules 4-2-1 ETDRS), patients should undergo proper laser treatment. This should be performed even if it causes of a delay in inscription on the waiting list for transplantation. Likewise we must apply the same approaches for treatment of the macular edema. On the other hand, from the diabetological point of view, patients should have close follow-up and treatment of diabetes and all other risk factors. After transplantation, deferral of laser treatment waiting for stabilization of the severe forms of DR and in cases of borderline CSME appears risky.

RETINAL AND RENAL COMPLICATIONS IN MIDD (MATERNALLY INHERITED DIABETES AND DEAFNESS)

M Virally, D Dubois-Laforgue, C Bellanné-Chantelot, J Timsit, M Laloi-Michelin, B Vialettes, M Paques, A Erginay, P Massin, PJean Guillausseau, for the GEDIAM, Mitochondrial Diabetes French Study Group hopital Lariboisière, Paris , France

Purpose: To assess prevalence and determinants of retinal and renal complications of MIDD, which is a systemic disease due to mtDNA 3243 mutation, associating diabetes, neurosensory deafness, pattern dystrophy, and muscular and neurological symptoms.

Methods: Multicenter prospective study. Comparison of 72 patients with MIDD and 139 control patients with either type 1 or type 2 diabetes, matched according to initial presentation of diabetes, age at onset, sex, HbA1c (7.6±1.6 vs 7.8±1.4%), and diabetes duration (12.3 ± 11.3 vs 11.3 ± 11.4 years, NS).

Results: Prevalence of diabetic retinopathy was 4-fold lower in MIDD than controls (5/72, 7% vs 40/139, 29.6%, P<0.0002). In MIDD patients compared to controls, urinary albumin excretion was not significantly different (323 vs 103 mg/24h, P<0.06), creatinine plasma levels were higher (106 [88-125] vs 84[79-87] mmol/l, P<0.0001), creatinine clearance lower (65 [59-71] vs 102[96-107] ml/min, P<0.001, and renal failure (defined as creatinine clearance < 60 ml/min) nearly 6-fold more frequent (28/72, 41% vs 10/139, 7%, P<0.0001). Five patients with MIDD but no diabetic controls presented with end stage renal failure, requiring either renal transplantation or dialysis. Systolic (127±16.6 vs 132±17 mm Hg, P<0.025), but not diastolic blood pressure (77±10.9 vs 75±8.4 mm Hg, NS), was lower in MIDD patients. In univariate analysis, odds-ratio (OR) for retinopathy in MIDD compared with controls was 0.2 [0.1-0.5], P<0.001, and for nephropathy 9.2 [4-21], P<0.001. In multivariate analysis adjusted for diabetes duration, HbA1c, and systolic blood pressure, differences between MIDD and controls for retinopathy were no longer significant (OR 0.3[0.1-1.2], while differences between groups increased for nephropathy : OR 37.9 [9.4-147.6], P<0.0001.

Conclusions: These results suggest that diabetic retinopathy is 4-fold less prevalent in MIDD than in a control diabetes group. This difference appears related rather to better metabolic control and to lower blood pressure levels than to mitochondrial disease per se. By contrast, nephropathy is more frequent in MIDD, when comparisons are ajusted for HbA1c and blood pressure, indicating the presence of a specific mitochondrial kidney disease. A protective role of pattern dystrophy against retinopathy does not seem to be supported by these results.

POSTER PRESENTATIONS RELATION BETWEEN DURATION OF TYPE 1 DIABETES MELLITUS WITH FREQUENCY OF DIABETIC EYE COMPLICATIONS IN WARMIA AND MAZURY REGION , POLAND.

E Bandurska-Stankiewicz(1), J Pieczyski(2), D Wiatr(1), L Surdykowski(1) (1) Endocrinology and Diabetology Ward , General District Hospital , Olsztyn , Poland(2) Ophthalmologic Ward, General District Hospital , Olsztyn , Poland

Purpose: Our study was designed to investigate the relation between duration of type 1 diabetes mellitus (T1DM) and frequency of diabetic eye complications.

Methods: 127 T1DM patients diagnosed using Eurodiab and Dia-Mod methods, 62 female and 67 males, aged 1- 40, with 1–10 years diabetes duration were included in the study. Diabetic eye complications were detected by clinical examination (visual acuity, intraocular pressure, eye adnexae status, fundus examination). The retina was examined using Volk's lens and photographed (2-field colour photos) with Nikon AF 505 fundus camera. Retinopathy was graded according to the Early Treatment Diabetes Retinopathy Study grading system. Additionally, the presence of cataract, glaucoma and optic neuropathy was assessed.

Results: Diabetic eye complications were found in 19 patients (14.9 %), 10 females, 9 males, average age 26.6 +/-9.3 yrs, average duration of diabetes: 6.95 +/- 1.3 yrs. Non-proliferative retinopathy was observed in 10 patients (12.7%)– 18 eyes: 6 females – 11 eyes, and 4 males (3.1%) – 7 eyes (2.7%), aged 24.7 +/-4.6 yrs, 7.5 +/- 1.4 diabetes duration. One female (aged 27, 6 yrs of diabetes duration) was diagnosed with both retinopathy and maculopathy after laser theraphy. Cataract was found in 6 patients (4.72%)(3 females, 3 males), aged 23.5 +/- 2.4, 5.3 years of diabetes duration, while glaucoma in 2 patients: 1 female (aged 16, 6 yrs of diabetes) and 1 male (aged 28, 8 yrs of diabetes)

Conclusion: These data suggest that diabetic eye complications occur soon after the diagnosis of T1DM. T1DM patients should have eye examination as soon as the disease is diagnosed, and should be repeated every year.

INCIDENCE OF LOW VISION IN DIABETIC PATIENTS AS ESTIMATED FROM THE NUMBER OF PATIENTS ADMITTED TO A CENTRE OF VISUAL REHABILITATION.

Johan Elvstam, Gun Olsson Dep. of Ophthalmology, Kalmar County Hospital, Länssjukhuset , Sweden

Purpose: To estimate the incidence of diabetic patients suffering from severe loss of visual acuity.

Methods: A population study of diabetic patients referred to the centre of visual rehabilitation in Kalmar, Sweden. In the study the best-corrected visual acuity (VA), as given from the charts at the time of referral, is used.

Results: The centre of visual rehabilitation in Kalmar serves a geographically defined area with approximately 140 000 inhabitants. The study includes all patients referred to the visual rehabilitation centre during the years 1995 – 2004 with diabetic retinopathy as the main diagnosis and with VA of 0,4 or less (Snellen chart). The total number of diabetic patients admitted yearly varied between 6 and 18, ages 31 – 95 (mean 69 years). Only few (0- 3 patients yearly) had at the time of admittance VA of 0.1 (6/60, 20/200) or worse. The incidence of diabetic patients with low vision as defined as VA 0.1 or worse in the southern county of Kalmar in Sweden is approximately 1 per 100 000 inhabitants per year.

Conclusions: The incidence of diabetic patients with low vision in the southern county of Kalmar in Sweden is approximately 1 per 100 000 inhabitants per year, as estimated from the number of patients admitted to the centre of visual rehabilitation.

SYSTEMIC RISK ASSESSMENT IN A DIABETIC EYE CLINIC

Anju Kadyan(1), Maurice P Headon (1), Baldev Singh (2). Wolverhampton Eye Infirmary(1)and Diabetic Centre (2),Royal Wolverhampton Hospitals NHS Trust, UK.

Purpose: To profile the patients attending the diabetic eye clinic in a district general hospital and to do a systemic risk assessment.

Methods: We prospectively assessed 88 patients randomly chosen from those attending the diabetic eye clinic over a period of 2 months. Data was collected regarding the ocular status, diabetic control and a systemic risk assessment done to calculate the Coronary Heart Disease (CHD) score using the computerised Framingham equation.

Results: Of the 88 patients studied (age 63.8 +/- 11.6 yrs, 62.5% males), 13.6% were type 1 and 86.4% had type 2 diabetes mellitus (59.2% IDDM and 40.8% NIDDM). Mean duration of diabetes was 14.7 +/- 10.2 yrs. Diabetic retinopathy was graded as background in 52%, pre-proliferative in 12.5% and proliferative in 20.5%, while 16% had clinically significant maculopathy. Their HBA1c was 8.63 +/- 1.88% (mean +/- SD). The diabetic control was worse in the type 1 group (>8 in 100%) and type 2 group on insulin treatment (>8 in 64.4%).

Inadequate blood pressure control was noted in 54% patients (systolic pressure >140 mmHg) already on antihypertensive treatment. 3.4% had systolic pressure of >160mm Hg and were not on any antihypertensive treatment. There was established macro vascular disease in 36.4%, persistent smoking in 10%, inadequate foot care in 22%, creatinine >120 in 23% and albumin creatinine ratio >3.5 in 37%. Of those with established macro vascular disease 28% were not on statin and 34.4% were not on aspirin or any other antiplatelet therapy. The mean CHD score calculated for 72/88 (<74 yrs of age) was 17.14 +/- 8.64% with 37.5% having a CHD score of >20%.

Conclusions: Systemic risk assessment in diabetic eye clinics provides a valuable opportunity to detect new and inadequately treated patients and should be encouraged. An integrated care pathway would be useful here.

DIABETIC RETINOPATHY IN A COHORT OF CHILDREN AND ADOLESCENTS FROM FUNEN, DENMARK.

EF Gade (1), ML. Laursen (1), A Green (2), M Hansen (1), AK Sjølie (1) (1) Department of ophthalmology, Odense University Hospital (2) Department of Applied Reserarch and HTA, Odense University Hospital

Purpose: To describe the presence and severity of retinopathy and related risk factors among children and adolescents with diabetes examined at the photographic screening clinic at Odense university Hospital, Denmark.

Methods:From 1999, children and adolescents with diabetes from the county of Funen, Denmark (about 500.000 inhabitants), were included. Fundus Photographs were taken at age 9, 12, 15 and yearly thereafter, and evaluated by retina specialists. Grading of the retinopathy was based on the classification published by Wilkinson et al (Ophthalmology 2003; 110:1677-1682)

Results:147 children, 65 girls (44.2%) and 82 boys (55.8%) were followed from 1999 to 2004. Mean age at diagnosis of diabetes was: 7.4 years (range: 0-16). Mean age at first visit at the screening clinic was 12.6 years (range: 8-17) and mean duration of diabetes was 5.1 years (range: 1-15) and HbA1c was: 8.66 (range: 5.5 – 19.2).Five children had retinopathy (level 1) at the first visit; mean age 16.6 years (range: 15 –17) and mean duration of diabetes was 5.1 years (range: 1-15).Nine children developed some degree of retinopathy. Of these, two patients developed proliferative retinopathy, when they were 19 and 21 years of age, they had a duration of diabetes of 10 and 14 years and a mean value of HbA1c of 11 and 11.2. One of the patients progressed from level 1 to 4 in two years and the other progressed from level 0 to 4 in 3 years.

Conclusions:In our screening clinic we found 14 children with some degree of diabetic retinopathy, out of a total of 147 children followed at the regional photographic screening clinic. Two of these progressed to proliferative retinopathy around the age of twenty. This indicates that it is important to follow young diabetic patients, especially if they have poor glycaemic control.

IS OUR TYPE 1 DIABETIC PATIENT SCREENING SUCCESSFUL?

I Ribeiro, A Lima, R Monteiro, JG. Monteiro, L Costa, P Rodrigues, M Bilhoto Ophthalmology Department - Hospital Pedro Hispano, Matosinhos, Portugal

Purpose: To describe the screening, prevalence and management of eye disease in Type 1 diabetic patients observed in our ophthalmology department.

Methods: Data was collected from the S.E.E. database of W.H.O. and analysed using the Epi Info program.

Results: One hundred forty two Type 1 diabetic patients were evaluated (age range 7-69 years, mean 34+/-15; 57% females) with mean diabetes duration of 19+/-12 years (range 1-56). Corrected visual acuity in the best eye ranged from light perception to 10/10 (mean 0.8+/-0.3). 62 patients did not have diabetic retinopathy (DR). 41 patients (29%) had nonproliferative DR, described as incipient in 18 patients (13%), moderate in 20 (14%) and severe in 3 (2%). Thirty four patients (24%) had proliferative DR, from which 32 were photocoagulated and 2 were referred for photocoagulation. Five patients (3,5%) had advanced disease. The mean age and the mean duration of the disease for the patients without DR and with nonproliferative and proliferative DR were respectively: 23+/-10 and 10+/-6 for the first group, 42+/-15 and 25+/-11 for the second group, 43+/-12 and 27+/-10 for the third. Macular edema was found in 37 patients (26%) and 16 patients (11%) had previous macular edema, successfully treated. The mean age and the mean duration of the disease of the group with macular edema were respectively 41+/-12 and 25+/-11 years. Argon laser panretinal photocoagulation was performed in 87 eyes and grid pattern photocoagulation in 102 eyes (82 eyes underwent both treatments). 49 eyes had nonoperated cataract and cataract surgeries were performed in 11 eyes. 25 eyes (14 patients) had glaucoma. Four patients were found to be legally blind.

Conclusions: The prevalence of nonproliferative and proliferative DR matches with the group characteristics. We therefore conclude that the screening program is being successful.

DIABETIC RETINOPATHY IN TYPE 1 DIABETES: FROM 1985 TO 2004

J. Figueira, T. Torrent, L. Ribeiro, M. Luz, R Silva, J.R. Faria de Abreu, J. G. Cunha-Vaz Ophthalmology Department. Hospitais da Universidade de Coimbra. Coimbra. Portugal, AIBILI. Coimbra. Portugal

Purpose: To study the value of Vitreous Fluorophotometry (PR) in identifying type 1 diabetic adolescents in risk of developing diabetic retinopathy.

Methods: This is a still-going prospective study started in 1985 with a mean follow-up of 11,4 years (in 2004) in which 80 diabetics were enrolled (41 males, 39 females). Initial value of PR was that at puberty, age at diagnostis was 8,5±4,2 years old (Mean ± SD), age in 2004 was 24,5±4,8. All patients underwent several ophthalmological exams (fundus photograph, Fluorescein Angiography, Vitreous Fluorophotometry) as well as biological evaluation (CBC and HbA1c).

Results:After this follow-up 29 patients (34%) showed no retinopathy (ODR), 41 (48%) showed non proliferative forms (NPDR) and 15 (18%) proliferative forms (PDR) Duration HbA1c ODR 14,5±4,7 8,8±2,4 NPDR 17,4±5,3 9,1±1.8 PDR 18,0±5,5 9,1± 2 Patients without retinopathy had a shorter duration of the disease (p=0,02) but there were no statistically significant differences in terms of HbA1c. PR at puberty was higher in patients with PDR, with 3,5 being a cut-off value.

Conclusion: Deterioration of the blood retina barrier at puberty may be important in the development of Diabetic Retinopathy.

AUTOMATIC ESTABLISHMENT OF MACULAR COORDINATES

Pedro Baptista(1), Rui Bernardes (1), Jorge Dias (2), José Cunha-Vaz(1,3) 1 Association for Innovation and Biomedical Research on Light and Image (AIBILI/CNTM), Coimbra, 2 Institute of Systems and Robotics (ISR), Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal, 3 Institute of Biomedical Research on Light and Image (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Purpose: To automatically establish retinal coordinates on high-resolution fundus images.

Methods: High-resolution color fundus images (50 degree retinographies) were taken using a Zeiss fundus camera (Carl Zeiss, Germany), with a Hiko h10 digital camera of 3192x2656 pixels. A template matching algorithm allows to determine the center of the fovea on a multi-resolution approach. A first step to determine the optic disk (OD) center and contour consists in determining a point inside the OD. This is also achieved using a template matching algorithm. Afterwards, the brightness along a set of radial lines crossing at this point are analyzed to determine the location of the OD contour and an ellipse is fitted to that set of points that also acts as a starting point to the active contour algorithm. Based on this set of points along the OD contour, a new center of the OD is computed and a line connecting it to the center of the fovea establishes the x-axis of the macular coordinates system. This process allows to extract the scaling factor as defined by a 2 disc diameter line from the center of the fovea to the OD contour.

Results: These procedures were tested in a series of both normal and diseased eyes. The fovea was completely detected in all cases, while the OD detection failed in 8,7% of the cases. OD contour showed was unsuccessfull in 13% of the cases while for the remaning 78,3% the detection was sucessfull. For the sucessfull cases, the RMS error was 16,9 pixels when compared to the contour drawn by manual grading on a computer monitor.

Conclusions: The procedure reported herein allows for automatic determination of the major retinal features and thus to establish absolute macular coordinates corrected for differences in scaling.

RETINAL THICKNESS MEASUREMENTS: RTA II VS. STRATUS OCT IN HEALTHY SUBJECTS.

Sandrina Nunes(1), Rui Bernardes (1), Pedro Baptista (1), José Cunha-Vaz (1,2) (1) Association for Innovation and Biomedical Research on Light and Image (AIBILI/CNTM), Coimbra; 2 Institute of Biomedical Research on Light and Image (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Purpose: To compare retinal thickness in eyes of healthy volunteers using the Retinal Thickness Analyzer (RTA II, Talia Technology, Israel) and the Optical Coherence Tomography (Stratus OCT, Humphrey Instruments, USA).

Methods: Thirty-four eyes from 20 healthy volunteers (14 females/6 males), aged from 40 to 60 years-old (mean±SD: 51.5±5.5), were examined in the same session using the RTA II and the Stratus OCT. Using a proprietary software, a new retinal thickness map based on the RTA II data was developed to produce Stratus OCT-like retinal thickness maps (9 areas in all, with a 1 mm diameter central disc area and 8 retinal quadrants (papillo-macular, superior, temporal and inferior) between 1 and 3 mm radii and between 3 and 6 mm radii). For both maps (RTA and OCT) the mean, SD and 95% CI of the mean were computed for each of the 9 areas. Absolute agreement was considered when both techniques showed the same behaviour (increase or decrease for the 95% CI of the mean or both on the normal range). Absolute disagreement was considered when both techniques showed opposite behaviour (increase/decrease or decrease/increase).

Results: All areas for both instruments presented a normal distribution and a statistical significant difference was found between them (p<0.001). The Stratus OCT presents retinal thickness values higher than the RTA II for all areas (21% to 34% higher). Absolute agreement was found in 56% of the cases (areas/eyes) while 8% of the cases showed absolute disagreement. As expected, good agreement was found for the central area (k=0.686,p<0.001) and moderate to poor agreement was found for the remaining areas.

Conclusions: The Stratus OCT presents retinal thickness values higher than the RTA II for all of the 9 areas and the best agreement was found to be in central area, where the Stratus OCT has a higher density of retinal thickness measurements.

EARMARKING RETINAL CHANGES IN A SEQUENCE OF DIGITAL COLOR FUNDUS PHOTOGRAPHS.

João Ferreira(1), Rui Bernardes(1), Pedro Baptista(1), José Cunha-Vaz(1,2). Association for Innovation and Biomedical Research on Light and Image (AIBILI/CNTM), Coimbra (2) Institute of Biomedical Research on Light and Image (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Purpose: The aim of the work presented here is to help graders detect changes on digital color photographs time- sequences of the human fundus.

Methods: A sequence of digital color fundus photographs taken every six-months during a two-year follow-up period was available. All images were taken using a Zeiss FF450 fundus camera, using a 3CCD detector to produce RGB color images of 768 x 576 pixels resolution of the central 50 degrees centered on the macula. All images were pre-processed and converted into the HSV color-space. The V-channel was then normalized to correct differences in intensity and non-uniform illumination and the retinal vascular tree detected by means of differential geometry. Vessel bifurcations and intersections are then used as landmarks for image registration using a perspective transformation. Following the registration, the differences between the normalized V- channels of each image in the sequence and their counterpart of the baseline image were computed in a total of 4 difference images per eye sequence. To achieve better representation it is possible to consider each difference as a component of a hyperspectral image, compute the respective principal component analysis (PCA) and therefore represent the projection that shows in the best manner the differences that occurred along the sequence. In order to earmark the differences found in the reference image, the resulting PCA image was projected onto the baseline fundus image by modifying its H-channel to show a light blue shade over the areas where differences were found.

Results: A fully automated procedure was developed to allow mapping of the changes detected over a time- sequence of color fundus images onto a reference image.

Conclusions: This system helps graders in the assessment of retinal changes occurring in a follow-up study by leading their attention to the areas where those changes have occurred.

REGISTRATION OF FUNCTIONAL AND MORPHOLOGICAL IMAGING MODALITIES.

Rui Bernardes (1), Pedro Baptista (1), Jorge Dias (2), José Cunha-Vaz (1,3) 1 Association for Innovation and Biomedical Research on Light and Image (AIBILI/CNTM), Coimbra; 2 Institute of Systems and Robotics (ISR), Faculty of Science and Technology, University of Coimbra, Coimbra; 3 Institute of Biomedical Research on Light and Image (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Purpose: The aim of this work is to present the registration of two different and complementary imaging modalities of the human fundus.

Methods: The retinography modality herewith considered, consists on a digitally taken RGB color fundus photography, 50 degrees field–of–view, centered on the fovea and with a resolution of 768x576 pixels. This image is taken by a 3CCD camera mounted on a Zeiss Fundus Camera system, model FF450. On the other hand, the blood-retinal barrier (BRB) functional image, also known as the retinal leakage analyzer (RLA), is obtained from a stack of 32 confocal planes, each one read in a raster–scan mode by an Heidelberg Retina Angiograph (HRA). Due to this raster–scan mode of acquisition and the 1.6 seconds acquisition time, where both voluntary and non–voluntary saccadic eye movements might occur, it is expected that a rigid registration may not produce results with the required accuracy. The image resolution for this modality is limited to 256x256 pixels (maximum). For this purpose, a three–step approach was taken consisting of: 1. rigid registration; 2. perspective registration, and; 3. non-rigid registration.

Results: For the first time, it was possible to register the BRB functional imaging of the human eye to a color fundus photography of the same eye in a fully automated way. It is important to note not only the multimodality involved, but also the differences in resolution, field–of–view, and the inclusion of an area with low level of morphological information from the point–of–view of the signal.

Conclusions: The fusion of complementary information on a clinical practice basis opens new perspectives for the understanding of the changes occurring in the macular area of the human eye, thus improving our knowledge on the retinal changes occurring in health and disease.

HYPERGLYCEMIA AFFECTS THE PURINERGIC SYSTEM IN CULTURED RAT RETINAL CELLS

T Pereira (1),A Neto (1),A Cristóvão (1,2), A Ambrósio(3) and P Santos(1,2) 1Center for Neuroscience and Cell Biology, 2Dept. of Zoology and 3 Fac. of Medicine, University of Coimbra.

Purpose: Diabetic retinopathy (DR) is the leading cause of blindness in the working-age group. It has been traditionally regarded as a vascular disorder. However, an increasing amount of evidence suggests that it is also a neurodegenerative process. Our work intends to assess whether ATP and P2 receptors can have a significant role on the neurodegeneration of the retina associated with DR. We are interested in evaluating how hyperglycemia affects extracellular ATP levels and P2 receptors expression and function.

Methods: We used cultures of retina cells, isolated from 3-5 days newborn Wistar rats. The cultures were incubated on the 3rd day after isolation with 30 mM glucose (HG cells) or 30 mM mannitol, and used for experimentation on the 9th day of culture. Protein expression was evaluated by Western Blotting for the P2X2,3,4 and P2X7 receptor subunits. Changes in [Ca2+]i were monitored by Single Cell Imaging. The extracellular levels of ATP were quantified by HPLC or by the luciferin/luciferase assay.

Results: We have observed that extracellular levels of ATP from stimulated retinal cells are higher in hyperglycemic conditions than in controls, and that added ATP is degraded at a lower rate in those conditions. We have observed that all the subunits studied (P2X2,3,4 and 7) are expressed and that, in HG cells, P2X4 expression is considerably decreased. However, expression of the P2X3 and P2X7 subunits are not significantly different from control cells. Calcium imaging studies have shown that stimulation of retinal cells with ATP caused an increase in [Ca2+]i that was prevented by PPADS. Also, the percentage of responsive cells is different in control or HG cells.

Conclusion: The results clearly show that culture of retina cells in hyperglycemic conditions affects the extracellular levels of ATP and the expression and function of some P2X receptors, suggesting that the ATP- purinergic signaling system may play a role in diabetic retinopathy. Supported by FCT (POCTI/CBO/38545/01 and POCTI/SAU-NEU/59003/2004)

OXIDATIVE STRESS UPREGULATES UBIQUITIN PROTEASOME PATHWAY (UPP) IN RETINAL ENDOTHELIAL CELLS

Fernandes R., Ramalho J. and Pereira P. Center Ophthalmology, IBILI, University of Coimbra (Portugal)

Purpose: Oxidative stress has been implicated in the development of diabetic complications. The purpose of this study is to establish whether oxidative stress up-regulates the UPP leading to increased protein degradation in diabetes.

Methods: Retinal endothelial cells were exposed to 40 or 100 uM of hydrogen peroxide. Endogenous ubiquitin conjugates were detected by Western Blotting. The ubiquitin conjugating activity assays was determined using radiolabeled ubiquitin or a-lactalbumin. The 20S proteasome activity was determined by degradation of the fluorogenic substrate, Z-Leu-Leu-Leu-AMC. Levels of mRNA were determined by radioactive Northern Blot.

Results: The exposure of endothelial cells to physiological concentrations of hydrogen peroxide leads to an increase on ubiquitin conjugating activity to endogenous and exogenous substrates. Moreover, levels of mRNA for a particular ubiquitin gene (UBA) are increased in response to oxidative stress, whereas proteolytic activity (20S proteasome) decreases by about 31%.

Conclusions: Oxidative stress up-regulates the UPP by increasing expression of UBA and leads to proteasome inhibition. Impaired proteolytic activities may account for retinal endothelial cell damage associated with diabetes or other pathologies involving increased production of oxidants

HEME OXYGENASE-1: A DEFENSE AGAINST STRESS CONDITIONS IN RETINAL ENDOTHELIAL CELLS

Á Castilho,C Aveleira,EC.Leal,A Serra,C Fernandes,R Meirinhos,AF.Ambrósio Center of Ophtalmology of Coimbra, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Purpose: Oxidative stress is believed to play a significant role in the development of Diabetic Retinopathy. Since the expression of heme oxygenase-1 (HO-1), a protective enzyme, may be induced by stress conditions, we investigated the effect of high glucose and oxidative stress on HO-1 immunoreactivity in retinal endothelial cells. We also investigated the potential protective role of HO-1 in retinal endothelial cells exposed to oxidative stress conditions.

Methods: Rat retinal capillary endothelial cells (TR-iBRB2 cell line) were exposed to D-glucose (30 mM), H2O2 or NOC-18 in the absence or in the presence of a HO-1 inhibitor, SnPPIX , in a time-dependent manner. HO-1 immunoreactivity was evaluated by Western Blotting and immunocytochemistry, and cell viability was accessed by MTT assay.

Results: High glucose and oxidative stress conditions (H2O2 and NO) decreased cell viability and increased HO- 1 immunoreactivity. The viability of endothelial cells exposed to H2O2 or NOC-18, in the presence of SnPPIX, was lower than in cells exposed to H2O2 or NOC-18, in the absence of SnPPIX. . Also, the protein levels of HO- 1 in cells exposed to H2O2 or NOC-18 in the presence of SnPPIX were higher than those observed in cells exposed to H2O2 or NOC-18 alone.

Conclusions: These results demonstrate that high glucose and oxidative stress increase HO-1 levels and decrease retinal endothelial cell viability. The results also suggest that HO-1 might exert a protective role against oxidative stress in endothelial cells, which upregulate HO-1 protein levels when the enzyme is inhibited. Supported by FCT – POCTI/CBO/38545/2001; SFRH/BD/9686/2002 and FEDER.

INTERLEUKIN-1 BETA SYNTHESIS AND INTERLEUKIN-1 RECEPTOR TYPE I EXPRESSION IN RETINAL CELLS: EFFECT OF HIGH GLUCOSE AND INTERLEUKIN-1 BETA

C Aveleira (1),Á Castilho(1),E Leal(1),A Serra(1), A Álvaro(2), C Fernandes(1),R Meirinhos(1),AF. Ambrósio (2) 1 Center Ophthalmology of Coimbra, IBILI, Faculty Medicine, University Coimbra; 2 Center for Neurosciences and Cell Biology, Coimbra, Portugal

Purpose: Diabetic Retinopathy (DR) has been considered a low-grade chronic inflammatory disease, and the pro- inflammatory cytokine interleukin-1 beta (IL-1beta) plays an important role in the breakdown of blood-retinal barrier. Since IL-1beta can be synthesized and may affect different cell types, we intended to identify retinal cells that synthesize IL-1beta and express the interleukin-1 type 1 receptor (IL-1RI). We also investigated the effect of high glucose and IL-1beta on the expression of IL-1RI in retinal cells.

Methods: Rat retinal capillary endothelial cell line (TR-iBRB2) and rat retinal primary neural cultures were exposed to high concentrations of glucose (30mM) or mannitol, the osmotic control, for 7 days. The cells were also exposed in a time-dependent manner (1, 3, 6, 12, 24h) to glucose, mannitol and IL-1beta (10ng/ml). IL- 1beta and IL-1RI immunoreactivity was evaluated by Western Blotting and immunocytochemistry.

Results: We observed that rat retinal neurons, endothelial and glial cells synthesize IL-1beta and express its receptor, IL-1RI. Long-term exposure of endothelial cells to high glucose (7 days) significantly decreased the protein content of IL-1RI. Similar results were obtained in the presence of mannitol. However, the expression of IL-1RI was not altered in retinal neural cultures exposed to chronic high glucose. In short-time exposure to high glucose or IL-1beta (1-24h) a time-dependent downregulation of IL-1RI protein was observed in endothelial cells and retina neural cultures.

Conclusions: Several types of retinal cells are able to synthesize IL-beta and also express the IL-1beta receptor, IL-1RI, and, therefore, they may be directly affected by IL-1beta. These results also indicate that high glucose, probably due to osmotic stress, and IL-1beta downregulate the protein expression of IL-1RI in retinal cells. The downregulation of IL-1RI may have some implications in IL-1beta signaling in retinal cells. (Support: FCT - POCTI/CBO/38545/2001, SFRH/BD/9686/2002, SFRH/BD/18827/2004 and FEDER)

HIGH GLUCOSE ALTERS CA2+-PERMEABLE AMPA RECEPTORS AND CALCIUM HOMEOSTASIS IN RETINAL NEURAL CELLS

Ana R. Santiago (1,2), Susana C. Rosa (1), Paulo F. Santos (1), Armando J. Cristóvão (1), Alistair J. Barber (3), António F. Ambrósio (1,2) 1Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 2Center of Ophthalmology of Coimbra, IBILI, Faculty of Medicine, University of Coimbra, Portugal; 3Penn State Retina Research Group, Penn State College of Medicine, Hershey, USA.

Purpose: Diabetes leads to abnormal retinal function and increases apoptosis in retinal neurons, probably due to glutamate excitotoxicity. We investigated the effect of elevated extracellular glucose on AMPA receptor permeability and calcium homeostasis in retinal neural cells.

Methods: Primary cultures of rat retinal cells and R28 cells were grown in media with normal (5 mM) or 30 mM or 20 mM glucose, respectively, or mannitol. Cobalt staining was used to identify cells with Ca2+-permeable AMPA receptors. Neuronal morphology was assessed by MAP-2 immunocytochemistry. GluR2 subunit levels were analysed by western blot. The [Ca2+]i changes evoked by KCl or kainate were quantified in individual cells by confocal microscopy with the calcium-sensitive dye Fluo-4.

Results: MAP-2 immunocytochemistry revealed no significant changes in neuronal morphology in overall population due to osmolarity changes, but the number of cell bodies and the length of cell processes stained with Co2+ significantly decreased in high glucose-treated cells, indicating a reduction in the Ca2+ permeability of AMPA receptors, and confirmed by increased levels of GluR2 subunit in glucose-treated cells. The KCl-evoked increase in [Ca2+]i was significantly higher in cells grown in high glucose, and did not recover to baseline during the course of the experiment. Similar results were obtained in primary cultures stimulated with kainate. In Na+ free medium, kainate-evoked [Ca2+]i changes decreased in high-glucose treated cells compared to control, further indicating a decrease in Ca2+-permeable AMPA receptors. In all cases, mannitol did not cause significant differences compared to control.

Conclusions: The results demonstrate that Ca2+ homeostasis and AMPA receptors can be altered by high concentrations of extracellular glucose. These findings offer a potential mechanism for the loss of neural function and apoptosis in diabetes. Support: FCT (Portugal) and FEDER.

PATTERNS OF RETINOPATHY PROGRESSION IN A FIVE-YEAR FOLLOW-UP OF MILD NONPROLIFERATIVE RETINOPATHY IN DIABETES TYPE 2: DEVELOPMENT OF CLINICALLY SIGNIFICANT MACULAR EDEMA

L Ribeiro,L Duarte,S Nunes,R Bernardes, C Lobo1, J.Figueira1, J Cunha-Vaz1. Center of Ophthalmology of Coimbra, IBILI, Faculty of Medicine, University of Coimbra, Portugal

Purpose: To characterize retinopathy progression and development of clinically significant macular edema (CSME) in the initial stages of retinal disease in diabetes type 2.

Methods: Fifty seven eyes of 57 patients with type 2 diabetes mellitus and mild nonproliferative retinopathy were classified into three different retinopathy progression phenotypes (A, B and C) after an initial two year follow-up period, involving eye examinations every 6 months. Thereafter, they were examined at yearly intervals for another 3 years, completing a five-year follow-up period. All patients were examined by fundus photography, fluorescein angiography and had retinal leakage and retinal thickness measurements performed.

Results: Of the 37 eyes (65% of the total) showing progression pattern A, in the initial two-year period of follow-up, only 1 developed CSME (3%) at the last examination of the five year follow-up. Of the 12 eyes (21%) showing pattern B, 4 eyes (30%) developed CSME, 2 at the three-year examination and the other 2 at the five-year examination. Of the 8 eyes (14%) identified with pattern C, 4 eyes (50%) developed CSME; 3, at the three-year examination and 1 at the four-year examination. Increases in leakage and thickness are prominent in pattern B whereas increases in capillary closure characterize pattern C.

Conclusions: The development of CSME over a five-year period of follow-up in eyes with mild nonproliferative retinopathy and diabetes type 2 is rare in the more common progression pattern A. This is in contrast with progression patterns B and C where there is frequent development of CSME.

VISUAL PROGNOSIS AFTER PANRETINAL PHOTOCOAGULATION FOR PROLIFERATIVE DIABETIC RETINOPATHY

Toke Bek1 and Mogens Erlandsen2 1Department of Ophthalmology, Århus University Hospital 2Department of Biostatistics, Århus University

Purpose: Proliferative diabetic retinopathy is treated with panretinal photocoagulation which improves the visual prognosis of this complication considerably. The visual acuity and grade of retinopathy before treatment are known indicators of the visual prognosis after treatment, but the prognostic value of other clinical background and treatment parameters is unknown.

Methods: The study reports predictors for visual outcome identified among retrospective clinical background data and treatment parameters from 4422 panretinal photocoagulation sessions for proliferative diabetic retinopathy in 1013 eyes from 601 patients performed at the Department of Ophthalmology, Århus University Hospital between 1985 and 2002.

Results: Pre-treatment VA was a strong predictor of post-treatment VA, p < 0.0001, and there was a significant worsening of the visual prognosis with increasing age, p < 0.0001 which was independent of diabetes type (p = 0.7851), diabetes duration (p = 0.1234), and calendar year (p = 0.0812). Visual prognosis was inversely related to the number of panretinal treatment sessions and the number of vitrectomies, but were unrelated to any of the other clinical background and treatment parameters studied.

Conclusions: Pre-treatment visual acuity and the number of panretinal treatment sessions and vitrectomies necessary to halt the disease are strong indicators of visual prognosis after panretinal laser photocoagulation for proliferative diabetic retinopathy.

SUGAR CRACKS : PATHOPHYSIOLOGY OF CATARACT FORMATION IN DIABETES.

G.J.M. Tangelder(presenting author), M. Dubbelman, P.J. Ringens VU University Medical Center, Amsterdam , The Netherlands

Purpose: Presentation of a unique case of sudden onset, reversible, bilateral osmotic cataract immediately after initiation of metformine therapy in a diabetes mellitus patient with severe hyperglycaemia. We introduce the term ‘Sugar cracks'.

Methods: Observational case report.

Results: Two days after initiation of metformine 750 mg b.i.d., the patient developed blurred vision ODS. Blood glucose level prior to medication was 21.8 mmol/l , decreased to 14.6 mmol/l during the first four days and to 8.7 in the following week. Scheimpflug images, at the time of presentation, show crack shaped cavities in the crystalline lens of both eyes. VOD was 20/80 and VOS was 20/33. No retinal pathology was present. These cracks had resolved on follow-up examination 4 months later and visual acuity had recovered to 20/20 ODS.

Conclusion: Given the characteristic morphology and the relationship in time of the crack shaped defects with blood glucose regulation, these sugar cracks may be considered as a hitherto unknown form of true diabetic cataract. An explanation for the development of these sugar cracks will be presented, based on our observations and on present theories concerning the pathophysiology of sugar cataract (i.e. true diabetic and galactosaemic cataract) formation.

VISUAL FIELDS,VISUAL ACUITY AND VASCUALR CHANGES IN DIABETIC RETINOPATHY

Elisabet Agardh, Boel Bengtsson, Anders Heijl Department of Clinical Sciences, Ophthalmology, Malmö University Hospital Malmö, Sweden

Purpose: To compare outcome of perimetric and visual acuity tests in patients with diabetic retinopathy.

Methods: Fifty-nine diabetic patients with different degrees of retinopathy were subjected to stereo fundus photography according to the Early Treatment Diabetic Retinopathy Study (ETDRS), and fluorescein angiography. Conventional White-White-Perimetry (WWP) and blue-on-yellow perimetry, also called Short- Wavelength-Automated-Perimetry (SWAP), were performed and analyzed with reference to normal values. Visual acuity (VA) was measured with ETDRS charts.

Results: Regression analyses revealed that both VA and degree of visual field loss were significantly associated with increasing severity of retinopathy according to the ETDRS scale. VA decreased by 0.02 LogMar/ETDRS step (p=0.03). Perimetric sensitivity decreased by 0.44 dB/ETDRS step (p=0.0001) using WWP, and by -0.40 dB/ETDRS step (p=0.04) with SWAP. Area of the foveal avascular zone (FAZ) and adjacent perifoveal intercapillary areas (PIAs) also affected the central visual field as obtained both by WWP, –2.6 dB/ mm2 (p=0.03), and by the SWAP, –7.9dB/ mm2 (p=0.002), but not VA (p=0.08). The regression model fit for peripheral retinopathy according to the ETDRS scale was better using WWP than SWAP or VA, while SWAP testing was superior to both WWP and VA when measuring effects caused by enlarged FAZ and PIAs

Conclusion: The correlation between conventional WWP and severity of diabetic retinopathy, and between SWAP and area of FAZ and PIAs suggest that perimetry can be useful in detection of functional loss in diabetic retinopathy, particularly when the perifoveal capillary network is damaged.


 




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