RETINOPATHY IS A FREQUENT

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1 CLINICAL SCIENCES Three-Year Follow-up Study of Blood-Retinal Barrier and Retinal Thickness Alterations in Patients With Type 2 Diabetes Mellitus and Mild Nonproliferative Diabetic Retinopathy Conceição L. Lobo, MD, PhD; Rui C. Bernardes, MSc; João P. Figueira, MD; José R. Faria de Abreu, MD, PhD; José G. Cunha-Vaz, MD, PhD Objective: To examine the 3-year alterations of the blood-retinal barrier and changes in retinal thickness occurring in the macular region in 14 eyes of 14 patients with type 2 diabetes mellitus (DM) and mild nonproliferative diabetic retinopathy. Methods: We classified 14 eyes of 14 patients with type 2 DM and mild nonproliferative diabetic retinopathy, as having disease levels 20 (microaneurysms only) or 35 (microaneurysm plus retinal hemorrhage[s] and/or hard exudates) of Wisconsin Card-Sorting Test grading, by using 7-field stereoscopic fundus photographs. We examined them 7 times at 6-month intervals, using fundus photography, fluorescein sodium angiography, the retinal leakage analyzer (RLA) modified confocal scanning laser ophthalmoscope, and the retinal thickness analyzer. The retinal leakage and retinal thickness maps were aligned and integrated into 1 image. Data from the group of individuals with type 2 DM were compared with those of a healthy control population (n=14; mean age, 48 years; age range, years) to establish reference maps for the RLA and retinal thickness analyzers. Results: Areas of abnormally increased fluorescein leakage were detected in all eyes examined at baseline. The sites of increased fluorescein leakage reached values as high as 483% above normal levels, but in 20 of the total 95 examinations performed, fluorescein leakage returned to normal levels. Every eye that showed reversal to normal levels of fluorescein leakage showed stabilization or a decrease in glycosylated hemoglobin A 1c values at the same visit. When comparing the RLA-leaking sites among the 7 examinations, they remained, in general, in the same locations, but there was a clear fluctuation in the percentage of increases. No clear correlation was observed among the location of areas of increased retinal thickness and RLA-leaking sites, the number of microaneurysms, or the glycosylated hemoglobin A 1c values. Microaneurysms on fundus photographs showed different cumulative incidences throughout the follow-up period in the different eyes. Associations between these different abnormalities suggest specific patterns of evolution of type 2 DM related retinal disease. Conclusions: The dominant alteration in the retina of patients with type 2 DM and mild nonproliferative retinopathy is the presence of RLA-leaking sites. This damage seems to be reversible and directly associated with variations in glycemic metabolic control. Together with the intensity and persistence of RLAleaking sites, the rates of microaneurysm accumulation and alterations of the foveal avascular zone may characterize different genetically based phenotypes of diabetic retinopathy. Arch Ophthalmol. 2004;122: From the Centre of Ophthalmology, University Hospital and Institute of Biomedical Research on Light and Image, Faculty of Medicine, University of Coimbra (Drs Lobo, Figueira, de Abreu, and Cunha-Vaz), and the Centre of New Technologies for Medicine, Association for Innovation and Biomedical Research on Light and Image (Dr Bernardes), Coimbra, Portugal. The authors have no relevant financial interest in this article. RETINOPATHY IS A FREQUENT microvascular complication of diabetes mellitus (DM) that remains a major therapeutic challenge. 1 To prevent and improve the treatment of diabetic retinopathy, it is fundamental that we know the evolution of the earliest changes that occur in the retina affected by DM and how these changes relate to the progression of retinopathy. The accepted methods of assessing retinopathy are based on changes in visual acuity and on findings noted on fundus photographs. Examination of the initial alterations in retinal disease caused by DM must involve other methods, such as measuring alterations of the blood-retinal barrier (BRB) and changes in retinal thickness. 2 Methods of multimodal imaging of the ocular fundus have recently been developed by combining information obtained from different sources, such as fundus photographs and fluorescein sodium angiography, with measurements obtained with instrumentation such as the retinal leakage analyzer (RLA) and the retinal thickness analyzer (RTA). 3,4 To our knowledge, we are providing the results of the first 3-year follow-up study of changes occurring in the macular region of eyes affected by type 2 DM and mild 211

2 Table 1. Patient and Eye Characterization Patient No./ Eye/Age, y Duration of Type 2 DM, y Macular HbA 1c Level, % Wisconsin Card-Sorting No. of Microaneurysms, Accumulated V0 V6 V12 V18 V24 V30 V36 Test Grade* V0 V6 V12 V18 V24 V30 V36 Change in FAZ Contour 1/L/ /R/ /L/ /R/ /L/ /L/ /L/ NA NA 8/R/ /L/ /L/ /L/ /L/ /R/ /R/ Abbreviations: DM, diabetes mellitus; FAZ, foveal avascular zone; HbA 1c, glycosylated hemoglobin A 1c ; NA, not applicable; V, visit;, normal; +, abnormal. *Wisconsin Card-Sorting Test grades are defined as follows: 20 indicates that the patient s eye had microaneurysms only; 35, microaneurysms plus retinal hemorrhage(s) and/or hard exudates. Patient had clinically significant macular edema. nonproliferative diabetic retinopathy using new methods of multimodal macula mapping. METHODS PATIENTS Fourteen eyes from 14 patients (5 men and 9 women), aged 44 to 68 years (mean [SD], 55 [5] years), with type 2 DM and mild nonproliferative diabetic retinopathy were studied. Characterized on 7-field stereoscopic fundus photographs, these 14 eyes met the inclusionary criteria of having either level 20 (microaneurysms only) or level 35 (microaneurysm plus retinal hemorrhage[s] and/or hard exudates) disease according to the Wisconsin Card-Sorting Test grading. We examined these 14 eyes 7 times using stereoscopic fundus photography, fluorescein sodium angiography, RLA (Carl Zeiss International, Oberkochen, Germany), and RTA (Talia Technology Ltd, Neve Ilan Zion, Israel) at 6-month intervals during a 3-year period. All patients had corrected visual acuity of 20/20. When both eyes met the inclusionary criteria, the right eye was chosen as the study eye. An abridged version of the Early Treatment Diabetic Retinopathy Study (ETDRS) scale was used to characterize the fundus lesions.. The patients were followed-up by the same diabetologist and remained within the limits of acceptable medical control. Their glycosylated hemoglobin A 1c (HbA 1c ) values at baseline ranged from 5.4% to 9.4% (the percentage given for the total hemoglobin level), and the duration of type 2 DM ranged from 2 to 15 years (Table 1). The tenets of the Declaration of Helsinki were followed and approval of the institutional review board was obtained for this study. Informed consent was obtained from the patients before they were enrolled in the study. PROCEDURES Stereoscopic Fundus Photography Stereoscopic fundus photography was performed according to the Early Treatment Diabetic Retinopathy Study protocol. Stereoscopic pairs of fields were obtained using a 30 fundus camera to be classified as level 20 or level 35 according to the Wisconsin Card-Sorting Test grading, although only field 2 was used to examine and correlate with other data (retinal leakage and retinal thickness). Fundus Fluorescein Angiography Fluorescein angiography was performed using a fundus camera with a 45 -field after patients received an injection of fluorescein sodium of 14 mg/kg of body weight. Retinal Leakage Analyzer The retinal leakage analyzer (RLA) is a new method that quantifies localized fluorescein leakage from the retina into the vitreous across the BRB in humans. The instrumentation and the entire image processing procedures were described previously. 2,4 The RLA obtains images of the fundus with real 3-dimensional information. Two types of information are obtained simultaneously, one for optical imaging and the other for fluorescence measurements being scanned. Axial graphics of the fluorescein concentrations obtained from the vitreous, representing a volume of µm, were converted into RLA maps. Multiple measurements of retinal leakage can be graphically assembled in a false-color RLA map that represents the distribution of alterations in the BRB in any chosen area of the total area of the posterior pole under examination. Intravisit and intervisit reproducibility values of the method are ±10.2% and ±13%, respectively. 2 Retinal Thickness Analyzer The retinal thickness analyzer (RTA) is a quantitative and reproducible method for evaluating retinal thickness. 5 The system acquires optical images of the retina in sections by projecting the laser into the retina at an angle, allowing the reflection or scattering of the laser light from the vitreoretinal and chorioretinal interfaces to be viewed. The separation between the reflections (and scatter) from these 2 interfaces is a measure of retinal thickness. These data are analyzed and the retinal thickness is given as a numerical value and as a color-coded map. 212

3 Table 2. Retinal Leakage Analyzer (RLA) and Retinal Thickness Analyzer (RTA) Data Area of Increase Maximum Increase Area of Increase Maximum Increase Patient No./ Eye/Age, y V0 V6 V12 V18 V24 V30 V36 V0 V6 V12 V18 V24 V30 V36 V0 V6 V12 V18 V24 V30 V36 V0 V6 V12 V18 V24 V30 V36 1/L/ /R/ /L/ /R/ /L/ /L/ * * * * 7/L/ * NA * NA * NA * NA 8/R/ /L/ /L/ /L/ /L/ /R/ /R/ Abbreviations: NA, not applicable; V, visit. *Patient had clinically signficant macular edema. RLA, % RTA, % DATA ANALYSIS Data from the group of individuals with type 2 DM were compared with those from a healthy control population (n=14; mean age, 48 years; age range, years) to establish reference maps for the RLA and RTA. 4 Because of the differences in resolution between the 2 instruments (RLA and RTA), a value of retinal leakage is obtained for each µm area, while a single value of retinal thickness covers a µm area. To compare information from both the RLA and RTA, the maps are composed of 63 (9 7) values, each representing a µm area, for a total of µm. 4 By comparing values from the patients with type 2 DM with those of a healthy control population, we can compute maps of increased retinal leakage or increased retinal thickness as percentages. All of the increases are computed considering the reference mean +2 SDs of the healthy control population. An extended description of the data analysis method is available elsewhere. 2 Correlations between the percentage of increases or decreases in RLA and RTA values occurring between examinations and HbA 1c values were tested using the 2 test to compare proportions and the nonparametric Spearman rank correlation coefficient (Base 11.0; SPSS Inc, Chicago, Ill). CHARACTERIZATION OF THE FUNDUS LESIONS Combined information is obtained only from field 2, using color stereoscopic fundus photography, fundus fluorescein angiography, RLA, and RTA. Lesions were characterized using Early Treatment Diabetic Retinopathy Study definitions. The findings on fundus photographs and fluorescein angiograms were registered independently by 3 different observers (C.L.L., J.R.F.A., and J.G.C.-V.). Disagreements were resolved after joint examinations. The alterations recorded in field 2 with the different methods used were as follows. Stereoscopic Fundus Photography Red dots considered to be microaneurysms were counted in field 2 at each examination. Their location was identified and each time a new microaneurysm was identified in a new location it was counted and added, giving the cumulative incidence of microaneurysms throughout the study. Fundus Fluorescein Angiography During the early phase the foveal avascular zone (FAZ) was outlined. A normal FAZ occurred when a smooth symmetrical outline of the FAZ was observed, with defects no larger than 10% of the entire outline. Retinal Leakage Analyzer Areas of increased fluorescein leakage ( µm) are areas in which fluorescein leakage is above the reference made equal to the mean+2 SDs of a healthy control population. The increase is expressed as a percentage over the reference. Retinal leakage analyzer leaking sites are identified as the central sites ( µm) of the maximum percentage of increased fluorescein leakage occurring in each area. 3,4 Retinal Thickness Analyzer Localized measurements of retinal thickness are obtained from a µm area. RESULTS The results are summarized in Table 1 and Table 2. During the 3-year follow-up period, 12 patients completed the entire set of visits, with the study eye being examined 7 times at 6-month intervals. The other 2 patients (patients 6 and 7) developed clinically significant macular edema and received laser photocoagulation therapy, one during the period before the sixth visit and the other before the final visit. In total there were 95 visits performed on the 14 patients included in the study. RETINAL LEAKAGE ANALYZER The RLA leaking sites, that is, sites of maximal fluorescein leakage for each area of abnormally increased leakage, reached values as high as 483% above normal levels, but in 20 of the 95 examinations performed, the 213

4 amount of fluorescein leakage recorded was within normal levels. Reversibility of the abnormally increased fluorescein leakage was statistically associated with documented improvement in HbA 1c levels. There was a good correlation between stabilization or a decrease in HbA 1c levels between successive visits and the occurrence of normal values of fluorescein leakage. In all 13 visits in which a return to normal values was registered, there was an associated decrease or stabilization in HbA 1c levels. There was also good correlation between increases or decreases in HbA 1c levels between successive visits and increases or decreases in the percentage of maximum increases in fluorescein leakage into the vitreous (P=.004). When comparing the RLA-leaking sites among the 7 examinations in the different eyes examined, they remained, in general, in the same location, but there was a clear fluctuation in the percentage of increase. Eyes showing a higher percentage of increase of abnormal fluorescein leakage showed higher leakage values (patients 3, 7, 8, and 9) throughout the 3-year follow-up period. FUNDUS PHOTOGRAPHY On successive visits special attention was given to counting microaneurysms in a cumulative way. On the first visit, there were a total of 39 red dots and at the end of the 3-year follow-up period, there was a cumulative count of 122 red dots. The rate of increase in microaneurysms varied between eyes, but increased significantly in the eyes of patients 3, 4, 7, 8, 9, and 13. FLUORESCEIN ANGIOGRAPHY Special attention was given to changes in the contour of the FAZ. Changes in the contour of the FAZ were detected in the eyes of patient 6 on the fourth and subsequent visits, in patient 12 on the second and subsequent visits, and in patient 13 on the fifth and subsequent visits. RETINAL THICKNESS ANALYZER Increases in retinal thickness were detected in all eyes examined at some time during the 3-year follow-up period. The sites of maximum percentage of increase in retinal thickness were generally lower than 35% above normal values. Values higher than 35% were observed on visit 4 of patient 1, visits 3 and 4 of patient 14, and on the last visit of the 2 eyes that were later diagnosed as having clinical significant macular edema. Changes were observed between the location of sites of maximum percentage of increase in retinal thickness when comparing the results of examinations performed on different visits in the same eye. No correlation could be established between changes in retinal thickness and changes in HbA 1c values. Similarly, no correlation could be found between the percentage of increase in fluorescein leakage and the percentage of increase in retinal thickness. MULTIMODAL MACULA MAPPING ANALYSIS When looking for associations between the main alterations found in this group of eyes with mild nonproliferative diabetic retinopathy from patients with type 2 DM, followed up for a 3-year period and examined at 6-month intervals, the following 3 major evolutionary patterns emerged (Figure). Pattern A Pattern A is defined as eyes showing reversible retinal leakage and a relatively low degree of abnormal fluorescein leakage in the RLA, a slow rate of microaneurysm accumulation, and a normal FAZ contour. The duration of the type 2 DM reported in these 7 eyes (patients 1, 2, 4, 5, 10, 11, and 14) was 10, 2, 15, 9, 4, 5, and 2 years, respectively. The eye of patient 4 has a relatively higher rate of microaneurysm accumulation, possibly associated with having a longer duration of type 2 DM. Pattern B Pattern B is defined as eyes showing persistent retinal leakage, with high fluorescein leakage values at some time during the 3-year follow-up period, relatively high rates of microaneurysm accumulation, and a normal FAZ contour. The duration of the type 2 DM registered in these 4 eyes (patients 3, 7, 8, and 9) was 10, 6, 8, and 2 years, respectively. Pattern C Pattern C is defined as eyes showing reversible retinal leakage and a low degree of abnormal fluorescein leakage during most of the 3-year follow-up period, variable degrees of progression of microaneurysm accumulation, and the development of abnormalities in the capillary net surrounding the FAZ. The duration of type 2 DM registered in the 3 eyes fulfilling the requirements (patients 6, 12, and 13) was 10, 15, and 3 years, respectively. COMMENT Diabetic retinopathy is fundamentally a posterior pole disease, preferentially involving the macular area, since its initial stages. Multimodal imaging of the macular area was used in this study by combining maps of fluorescein leakage into the vitreous, maps of changes in retinal thickness, and fundus images. The combination of these methods allows for an integrated view of the different changes occurring in the macular area. This prospective 3-year follow-up study of the macular region, in patients with type 2 DM and mild nonproliferative diabetic retinopathy, using these combined imaging methods, opens new perspectives for our understanding of diabetic retinopathy by showing the progression of the initial alterations of the retinal microcirculation. In a span of 3 years, eyes with minimal changes at the start of the study (level 20 or level 35 of Early Treat- 214

5 A 100 B C Multimodel images from patients 2, 9, and 13 of visits 0, 12, 24, and 36 showing for each visit the foveal avascular zone contour (FAZ), retinal leakage analyzer results, and retinal thickness analyzer results. The retinal leakage analyzer color-coded maps of the blood-retinal barrier permeability indexes are shown; retinal thickness analyzer views show white dot density maps of the percentage increases in retinal thickness. A, Pattern A, patient 2. Note the little amount of retinal leakage over the 4 represented visits and the normal FAZ contour. This patient showed a slow rate of microaneurysm formation. B, Pattern B, patient 9. Note the high retinal leakage showing a certain degree of reversibility and the normal FAZ contour. This patient showed a high rate of microaneurysm accumulation over the 3-year follow-up period. C, Pattern C, patient 13. Note the reversible retinal leakage and the development of an abnormal FAZ contour. This patient showed a high rate of micoraneurysm formation. ment Diabetic Retinopathy Study Wisconsin Card- Sorting Test grading for fundus lesions) were followed up at 6-month intervals to monitor the progression of the retinal changes. The most frequent alterations observed were, by decreasing order of frequency, RLA-leaking sites, areas of increased retinal thickness, and the occurrence of microaneurysms or hemorrhages. The RLA-leaking sites were a most frequent finding and reached high BRB permeability values in some eyes (patients 3, 6, 7, 8, and 9). These sites of alteration of the BRB, well identified in RLA-maps, maintained, in most cases, the same location on successive examinations, but their BRB permeability values fluctuated greatly between examinations, indicating reversibility of this alteration. There was, in general, a correlation between the BRB permeability values and the changes in HbA 1c levels occurring in each patient. This correlation was particularly clear when looking at eyes that showed, at some time during the 3-year follow-up period, BRB permeability values within the normal range. A return to normal levels of BRB permeability was, in this study and in each patient, always associated with a stabilization or a decrease in HbA 1c values. The frequent finding of RLA-leaking sites in these 14 eyes confirms previous reports using fluorescein angiography, 6 vitreous fluorometry, 7,8 and retinal leakage analysis, 4 which show that alteration of vascular permeability is one of the most frequent alterations occurring in the initial stages of diabetic retinal disease. Areas of increased retinal thickness were another frequent finding in these study eyes. They were present in every eye at some time during the 3-year follow-up period and were absent, at baseline, in only 2 eyes (patients 2 and 10). This confirms previous observations by our group 4 and by others. 9 However, the areas of increased retinal thickness varied in their location over subsequent examinations, did not correlate directly with the location of the RLAleaking sites, and did not correlate in any clear way with changes in HbA 1c levels. They may represent a delayed temporary response in time to other changes occurring in the retina, such as increased leakage, as we have suggested previously. 4 The areas of increased retinal thickness may, indeed, represent in most cases zones of extracellular edema, an interpretation supported by the frequent shift observed in their location. However, there were eyes showing generalized changes in retinal thickness with little retinal leakage (patients 1, 4, 5, and 6). Association with capillary closure was well demonstrated in 1 eye, with clear alterations of the FAZ contour (patient 6), which may indicate that in some instances intracellular edema associated with ischemia may be dominant. Microaneurysms were counted in field 2 and their rate of accumulation over the 3-year follow-up period may indicate progression of retinopathy and remodeling of the retinal microvasculature. Increased rates of micro- 215

6 aneurysm accumulation were found in eyes that had more microaneurysms at baseline (patients 3, 7, and 13) and higher values of BRB permeability during the 3-year study (patients 3, 7, 8, and 9). The study eye of patient 13 showed a clearly increased rate of microaneurysm accumulation with little retinal leakage, but had a high number of microaneurysms at baseline and developed an abnormal FAZ contour, demonstrating capillary closure. By combining different imaging techniques, multimodal imaging of the macula made apparent 3 major evolutionary patterns occurring during the 3-year follow-up period. One group included eyes with reversible and little abnormal fluorescein leakage, a slow rate of microaneurysm formation, and a normal FAZ contour. Patients included in this group had a longer duration of type 2 DM. This group, designated as having pattern A, seems to consist of eyes that will progress slowly and may never develop clinically significant macular edema or proliferative diabetic retinopathy. A second group included eyes with persistently high leakage values, indicating an important alteration of the BRB, high rates of microaneurysm accumulation, and a normal FAZ contour. The duration of type 2 DM in these patients was short. All these features suggest a rapid and progressive form of the retinal disease. This group, designated as having pattern B, could also identify a wet form of diabetic retinopathy. The third group included eyes with reversible retinal leakage and an abnormal FAZ contour. This group is less well characterized considering the few eyes that showed an abnormal FAZ contour. It may be that abnormalities of the FAZ may occur as a late development of those having patterns A or B or progress rapidly as a specific ischemic form (pattern C). It must be realized that levels of hyperglycemia and duration of type 2 DM, that is, exposure to hyperglycemia, will certainly influence the evolution and rate of progression tentatively classified in these 3 major patterns. If diabetic retinopathy is a multifactorial disease in the sense that different factors or different pathways may predominate in different groups of cases with diabetic retinopathy then it is crucial that these differences and the possible different phenotypes be identified. 10 Diabetes mellitus is a familial metabolic disorder with equally strong genetic and environmental causes. Familial aggregation is more common in type 2 DM than in type 1 DM. Rema et al 11 reported that familial clustering of diabetic retinopathy was 3 times higher in siblings of patients with type 2 DM who have diabetic retinopathy. Polymorphisms involved in differences in gene regulation of the receptor for advanced glycation endproducts have been linked to endothelial dysfunction in diabetes mellitus. 12 Intercelullar adhesion molecule 1 polymorphism has also been proposed as a genetic risk factor for retinopathy in type 2 DM. 13 Presence or absence of genetic factors may play a fundamental role in determining specific pathways of vascular disease and, as a consequence, different progression patterns of diabetic retinal disease. It could be that certain polymorphisms would make the retinal circulation more susceptible to an early breakdown of the BRB (pattern B) or microthrombosis and capillary closure (pattern C). The absence of these specific genetic polymorphisms would lead to an evolutionary pattern of pattern A. It is clear from this study and from previous large studies such as the Diabetes Control and Complications Trial Epidemiology of Diabetes Interventions and Complications Research Group, 14 and the United Kingdom Prospective Diabetes Study Group 15 that hyperglycemia plays a determinant role in the progression of retinopathy. It is interesting to note that HbA 1c levels are also largely genetically determined. 16 An interesting perspective of our observations, analyzed in the light of the available literature, depicts diabetic retinopathy as a microvascular complication of DM conditioned in its progression and prognosis by a variety of different genetic polymorphisms, and modulated in its evolution by HbA 1c levels, partly genetically determined and partly dependent on individual management of DM. The interplay of these multiple factors and the duration of this interplay would finally characterize different clinical pictures or phenotypes of diabetic retinopathy. The ultimate goal, therefore, should be the characterization of relationships between genetic factors (represented by distinct genotypes) and their medically significant expression (distinct diabetic retinopathy phenotypes). Characterization of different phenotypes of diabetic retinopathy has been suggested by previous descriptions of extreme situations that may occur in diabetic retinal disease. 10 We present herein, based on our observations of a 3-year prospective study of eyes with mild nonproliferative diabetic retinopathy of patients with type 2 DM, an attempt to identify phenotypes of diabetic retinopathy: a wet or leaky type, an ischemic type, and finally, an apparently more common, slow progression type. It must be realized, however, that our observations were made using new technologies, still in the research domain, and the sample size of the population examined was small. A classification of diabetic retinopathy, based on both relevant genotypes and disease phenotypes, is an ambitious goal. We believe that this route may help identify the particular form that threatens an individual patient and, consequently, offer an opportunity for specific and more effective therapies. Submitted for publication April 1, 2003; final revision received August 17, 2003; accepted September 10, Corresponding author and reprints: Conceição L. Lobo, MD, PhD, Centre of Ophthalmology, University Hospital and Institute of Biomedical Research on Light and Image, Faculty of Medicine, University of Coimbra, 3000 Coimbra, Portugal ( clobo@aibili.pt). REFERENCES 1. Aiello LP, Gardner TW, King GL, et al. Diabetic retinopathy [technical review]. Diabetes Care. 1998;21: Lobo C, Bernardes R, Santos F, Cunha-Vaz JG. Mapping retinal fluorescein leakage with confocal scanning laser fluorometry of the human vitreous. Arch Ophthalmol. 1999;117:

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