European Association for the Study of Diabetic Eye Complications

Annual Meeting Coimbra 27-29th May 2007

Poster Abstract 10 - 19

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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