- Email: [email protected]
Impaired photoreceptor inner segment ellipsoid layer reflectivity in mild diabetic retinopathy
ORIGINAL ARTICLE
Impaired photoreceptor inner segment ellipsoid layer reflectivity in mild diabetic retinopathy Ibrahim Toprak, MD,* Cem Yildirim, MD,† Volkan Yaylali, MD† ABSTRACT ● RÉSUMÉ Objective: To determine alterations in retinal pigment epithelium (RPE), photoreceptor inner segment ellipsoid layer (ISel), and external limiting membrane (ELM) reflectivities using optical coherence tomography (OCT) image analysis in patients with mild nonproliferative diabetic retinopathy (NPDR). Design: Retrospective, single-centre, controlled clinical study. Participants: The study cohort included 42 eyes of 42 patients with mild NPDR (NPDR group) and 40 eyes of 40 healthy subjects (control group). Eyes with moderate and severe NPDR, proliferative DR, macular edema, and other macular pathologies were excluded. Methods: The reflectivities of RPE, ISel, and ELM were calculated using a medical image-processing software based on greyscale OCT images. The differences in the reflectivity values between the NPDR and control groups were analyzed. Results: The NPDR group comprised 22 males and 20 females (with a mean age of 61.3 ⫾ 6.5 years), and the control group consisted of 14 males and 26 females (with a mean age of 63.0 ⫾ 4.1 years) (p 4 0.05). The ISel had significantly lower reflectivity (both absolute and relative) in eyes with mild NPDR compared with that of the control eyes (p o 0.001), whereas the reflectivities of RPE and ELM did not differ between the 2 groups (p ¼ 0.126, p ¼ 0.053 respectively). Conclusions: Although previous ex vivo studies reported photoreceptor degeneration in diabetic retinopathy, this is the first clinical study to investigate retinal layer reflectivities in NPDR using OCT. In eyes with mild NPDR, ISel seems to have lower reflectivity, and this finding might indicate early photoreceptor degeneration in diabetic retinopathy pathogenesis. Objet : Mesurer des altérations de la réflectivité de l'épithélium pigmentaire rétinien (EPR), de la couche ellipsoïde du segment interne des photorécepteurs et de la membrane limitante externe (MLE) grâce à l'analyse d'imagerie de tomographie de cohérence optique (TCO) chez des patients atteints de rétinopathie diabétique non proliférante (RDNP) légère. Nature : Étude clinique unicentrique rétrospective avec cas témoins. Participants : L'étude a porté sur 42 yeux de 42 patients présentant une RDNP légère (groupe RDNP) et sur 40 yeux de 40 sujets sains (groupe témoin). Les cas de RDNP modérée et sévère, de rétinopathie diabétique proliférante, d'œdème maculaire et d'autres pathologies maculaires étaient exclus. Méthodes : On a calculé la réflectivité de l'EPR, de la couche ellipsoïde du segment interne des photorécepteurs et de la MLE au moyen d'un logiciel de traitement d'imagerie médicale, à partir d'imagerie TCO en échelle de gris. On a analysé l'écart entre les valeurs de réflectivité du groupe RDNP et du groupe témoin. Résultats : Le groupe RDNP était composé de 22 hommes et de 20 femmes (âge moyen : 61,3 ⫾ 6,5 ans), tandis que le groupe témoin était formé de 14 hommes et de 26 femmes (âge moyen : 63,0 ⫾ 4,1 ans) (p 4 0,05). Dans le groupe RDNP, la réflectivité (tant absolue que relative) de la couche ellipsoïde du segment interne des photorécepteurs était significativement moindre que dans le groupe témoin (p o 0,001). Par contre, il n'y avait pas d'écart entre les deux groupes en ce qui concerne la réflectivité de l'EPR et de la MLE (p ¼ 0,126, p ¼ 0,053, respectivement). Conclusions : Des études ex vivo antérieures avaient fait état de la détérioration des photorécepteurs chez les patients atteints de rétinopathie diabétique, mais cette étude était la première étude clinique à explorer la réflectivité de couches rétiniennes à l'aide d'imagerie TCO chez des patients atteints de RDNP. Dans les cas de RDNP légère, la réflectivité de la couche ellipsoïde du segment interne des photorécepteurs semble plus faible, et cette conclusion pourrait être indicatrice d'une dégénérescence précoce des photorécepteurs dans la pathogénèse de la rétinopathie diabétique.
Early characteristics of nonproliferative diabetic retinopathy (NPDR) include microcapillary abnormalities such as microaneurysm formation, basement membrane thickening, and loss of pericytes.1,2 In the later stages of the retinopathy, capillary nonperfusion, microvascular abnormalities, retinal ischemia, and disruption of the blood– retina barrier manifest in fundus fluorescein angiography (FFA).1,3 Furthermore, ex vivo studies suggested that microcirculatory alterations lead neuronal degeneration
before the retinal neovascularization (proliferative diabetic retinopathy [PDR]).4–6 From a clinical perspective, advances in optical coherence tomography (OCT) devices allow visualization of retinal layers very close to histologic sections, whereas OCT does not provide functional data. In contrast, decrease in retinal layer reflectivity was found to be related with functional deterioration, and OCT reflectivities were evaluated previously in patients with
From the *Department of Ophthalmology, Servergazi State Hospital; and †Department of Ophthalmology, Faculty of Medicine, Pamukkale University, Denizli, Turkey
Can J Ophthalmol 2015;50:438–441 0008-4182/15/$-see front matter & 2015 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcjo.2015.07.009
Originally received Mar. 12, 2015. Final revision Jul. 4, 2015. Accepted Jul. 12, 2015 Correspondence to Ibrahim Toprak, MD, Department of Ophthalmology, Servergazi State Hospital, Tedas Mevki, Bereketli Beldesi, Denizli 20070, Turkey; [email protected]
438
CAN J OPHTHALMOL — VOL. 50, NO. 6, DECEMBER 2015
Photoreceptor reflectivity in diabetic retinopathy— Toprak et al. cystoid diabetic macular edema and idiopathic epimacular membrane.7–10 In this study, we firstly analyzed OCT reflectivities of foveal retinal pigment epithelium (RPE), photoreceptor inner segment ellipsoid layer (ISel, previously named photoreceptor inner segment/outer segment junction), and external limiting membrane (ELM) using a medical image-processing software (ImageJ v 1.47) in eyes with mild NPDR to reveal retinal neurodegeneration in vivo.
METHODS In this retrospective, single-centre, controlled clinical study, 42 eyes of 42 patients with mild NPDR (NPDR group) and 40 eyes of 40 age- and sex-matched healthy subjects (control group) were enrolled. The reliability of the sample size was tested at 95% power and 0.05 significance level (95% CI) using statistical software (PASS version 11.0.1; NSCC, LLC, Kaysville, Utah). The study followed the tenets of the Declaration of Helsinki, and permission was obtained from the local ethics committee. All participants underwent a complete ophthalmologic examination, which included corrected distance visual acuity measurement with Snellen charts, slit-lamp biomicroscopic examination, Goldmann applanation tonometry, dilated funduscopy (with þ90 D lens), and FFA. Inclusion and exclusion criteria
Patients with a diagnosis of mild NPDR (microaneurysms only) based on dilated fundus examination and FFA were included in the study. Patients with moderate and severe NPDR (severe retinal hemorrhages or venous beading or intraretinal microvascular abnormalities), disrupted or absent RPE, ISel and ELM bands on OCT, PDR, macular edema, or any other macular pathology and poor quality of OCT image were excluded.
Age- and sex-matched healthy subjects with a normal ophthalmologic examination comprised the control group. All patients and control subjects had a corrected distance visual acuity of 20/20 Snellen equivalent, and right eyes were used for the statistical analysis. Assessment of optical coherence tomographic reflectivities
The 6 mm 6 mm macular cube 512 128 scanning protocol of the Zeiss Cirrus HD-OCT 400 (Carl Zeiss Meditec, Dublin, Calif.) was used to acquire macular OCT images. A single experienced technician (MA) performed the OCT imaging. All participants had reliable (signal strength Z 7/10) macular OCT images. The greyscale OCT images were transferred to the ImageJ v1.47, a Java-based imageprocessing computer software developed under the National Institutes of Health, to perform reflectivity analyses.11 The ImageJ calculates distances, angles, areas, intensity, and pixel value statistics on an image with high reliability and reproducibility.9,10,12,13 A single masked ophthalmologist (I.T.) analyzed the randomly selected standardized greyscale OCT images for 2 times at different days, and the mean values were used for statistical analysis. The intraobserver intraclass correlation coefficients (ICC) were calculated for the repeated measurements to determine intraobserver reliability (a value 40.80 indicated good reliability). The reflectivities of the RPE, ISel, and ELM in the fovea centre were calculated using plot profile function of the software as previously described.9–11 The ImageJ provided reflectivity values along a vertical straight line passing through the centre of the fovea, and a reflectivity graph was created (Fig. 1). In a healthy eye, the RPE layer has the highest reflectivity on OCT followed by ISel and ELM. Hence the highest value on the reflectivity graph was accepted as the reflectivity of the RPE layer, and localizations of the RPE layer, ISel, and ELM on OCT
Fig. 1 — Vertical white line passing through the fovea centre indicates the reference line, which was used for reflectivity calculation. Reflectivity graph obtained from the ImageJ (right) and corresponding retinal layers on the greyscale macular optical coherence tomography scan (left) of a patient with mild nonproliferative diabetic retinopathy. ELM, external limiting membrane; ISel, inner segment ellipsoid layer; RPE, retinal pigment epithelium. CAN J OPHTHALMOL — VOL. 50, NO. 6, DECEMBER 2015
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Photoreceptor reflectivity in diabetic retinopathy— Toprak et al.
Fig. 2 — Comparison of the retinal pigment epithelium (RPE), photoreceptor inner segment ellipsoid layer (ISel) and external limiting membrane (ELM) reflectivities between the mild nonproliferative diabetic retinopathy (NPDR) and control groups.
image were confirmed simultaneously on the reflectivity graph using live plot profile function of the software.9,10 The reflectivity values on the graph were noted (as absolute reflectivity) and converted to the relative reflectivity according to the following formula9,10: Relative reflectivity (arbitrary unit) ¼ (reflectivity of a retinal layer [ISel or ELM]) / (reflectivity of the fixed reference retinal layer [RPE]) 100. The reflectivity values of RPE, ISel, and ELM were compared between the NPDR and control groups.
groups regarding the RPE (206.3 ⫾ 17.7 vs 211.5 ⫾ 12.2 arbitrary units, respectively; p ¼ 0.126) and ELM (89.5 ⫾ 14.7 vs 95.5 ⫾ 12.6 arbitrary units, respectively, p ¼ 0.053) reflectivities. Moreover, relative ISel reflectivity was also significantly lower in the patients with NPDR (71.7 ⫾ 7.3 arbitrary units) than in the healthy subjects (85.4 ⫾ 10.1 arbitrary units) (p o 0.001), whereas relative ELM reflectivity did not differ between the NPDR (43.4 ⫾ 6.3 arbitrary units) and control groups (45.2 ⫾ 6 arbitrary units) (p ¼ 0.192). Figure 2 represents comparison of the reflectivity values between the NPDR and control groups.
Statistical analysis
The Statistical Package for Social Sciences software version 16.0 (SPSS Inc, Chicago, Ill.) was used for the statistical analysis. Results were expressed as the mean ⫾ SD. The χ2 was used to compare 2 groups in terms of sex. The ICCs were calculated to test the intraobserver reliability of the repeated measurements (ICC 4 0.80 indicated good reliability). Comparison of the quantitative data (age and reflectivity values) between the 2 groups was performed using the independent samples t test. A p value less than 0.05 was considered statistically significant at 95% CI.
RESULTS Forty-two patients with mild NPDR and 40 healthy subjects were included in the study. The mean age was 61.3 ⫾ 6.5 years in the NPDR group and 63.0 ⫾ 4.1 years in the control group (p ¼ 0.182). The NPDR group consisted of 22 males and 20 females, and the control group comprised 14 males and 26 females (p ¼ 0.126). All patients and control subjects had normal macular thickness, and the repeated OCT measurements showed excellent intraobserver reliability (with an ICC 4 0.90). In the NPDR group, the absolute ISel reflectivity was significantly lower (148.2 ⫾ 21.3 arbitrary units) when compared with that of the control group (180.2 ⫾ 21.1 arbitrary units) (p o 0.001). However, there were no significant differences between the NPDR and control
440
DISCUSSION Microvascular insufficiency is known as the major hallmark of diabetic retinopathy (DR). However, previous studies demonstrated evidences of neurodegeneration in DR, such as increased apoptosis and glial activation, decreased retinal nerve fiber layer thickness, electroretinographic abnormalities, and impaired retinal function tests.2–6,14–16 In eyes with DR, researchers focused on alterations in the photoreceptor layer (particularly in the ISel).6,17 An experimental study by Alvarez et al.6 showed photoreceptor dysfunction in a hyperglycemic model of NPDR, and the authors suggested that cone photoreceptor dysfunction was a clinical hallmark of DR. Although previous ex vivo studies presented photoreceptor degeneration in patients with DR, a few clinical studies have investigated functional status of photoreceptor layer in DR. In a clinical study, Unoki et al.18 demonstrated decreased retinal sensitivity (using microperimetry) in eyes with severe NPDR and PDR. They also detected hyperreflective depositions between the photoreceptor layer and the RPE in OCT imaging. Similarly, Jackson et al.14 showed photoreceptor dysfunction based on dark-adapted visual sensitivity tests in patients with NPDR. In this study, we firstly evaluated reflectivities of RPE, photoreceptor ISel, and ELM in patients with mild NPDR using OCT. Previous neurophysiologic studies showed that decreased retinal layer reflectivity on OCT was
CAN J OPHTHALMOL — VOL. 50, NO. 6, DECEMBER 2015
Photoreceptor reflectivity in diabetic retinopathy— Toprak et al. associated with structural and/or functional damage.7,8 This study demonstrated a significant decrease in the ISel reflectivity (both absolute and relative) in eyes with mild NPDR. This finding appears to be parallel with the previous ex vivo studies. Furthermore, it might be important for showing photoreceptor degeneration (structural and/or functional) in early NPDR. In conclusion, DR is a complex pathologic process that includes both vascular and neuronal damage. This study is the first to evaluate retinal layer reflectivities using OCT image analysis in eyes with mild NPDR, and it showed the impaired ISel reflectivity, which might indicate contribution of the photoreceptor degeneration to DR pathogenesis from the early stages of the disease.
Disclosure: The authors have no proprietary or commercial interest in any materials discussed in this article. REFERENCES 1. Burns SA, Elsner AE, Chui TY, et al. In vivo adaptive optics microvascular imaging in diabetic patients without clinically severe diabetic retinopathy. Biomed Opt Express. 2014;5:961-74. 2. Antonetti DA, Barber AJ, Bronson SK, et al. Diabetic retinopathy: seeing beyond glucose-induced microvascular disease. Diabetes. 2006;55:2401-11. 3. Lorenzi M, Gerhardinger C. Early cellular and molecular changes induced by diabetes in the retina. Diabetologia. 2001;44:791-804. 4. Gardner TW, Antonetti DA, Barber AJ, LaNoue KF, Levison SW. Diabetic retinopathy, more than meets the eye. Surv Ophthalmol. 2002;47:253-62. 5. Garcia-Ramírez M, Hernández C, Villarroel M, et al. Interphotoreceptor retinoid-binding protein (IRBP) is downregulated at early stages of diabetic retinopathy. Diabetologia. 2009;52:2633-41.
6. Alvarez Y, Chen K, Reynolds AL, et al. Predominant cone photoreceptor dysfunction in a hyperglycaemic model of non-proliferative diabetic retinopathy. Dis Model Mech. 2010;3:236-45. 7. Bizheva K, Pflug R, Hermann B, Povazay B, Sattmann H, Qiu P. Optophysiology: depth-resolved probing of retinal physiology with functional ultrahigh-resolution optical coherence tomography. Proc Natl Acad Sci USA. 2006;103:5066-71. 8. Hood DC, Zhang X, Ramachandran R, Talamini CL, Raza A, Greenberg JP. The inner segment/outer segment border seen on optical coherence tomography is less intense in patients with diminished cone function. Invest Ophthalmol Vis Sci. 2011;52:9703-9. 9. Murakami T, Nishijima K, Akagi T, Uji A, Horii T, Ueda-Arakawa N. Optical coherence tomographic reflectivity of photoreceptors beneath cystoid spaces in diabetic macular edema. Invest Ophthalmol Vis Sci. 2012;53:1506-11. 10. Toprak I, Yaylalı V, Yildirim C. Decreased photoreceptor inner segment/outer segment junction reflectivity in patients with idiopathic epimacular membrane. Eye (Lond). 2014;28:1126-30. 11. Rasband W. Image J medical imaging software. Bethesda, MD: National Institutes of Health. Available at: http://rsb.info.nih.gov/ij/. Accessed September 26, 2014. 12. Collins TJ. ImageJ for microscopy. Biotechniques. 2007;43:25-30. 13. Zhao WJ, Duan F, Li ZT, Yang HJ, Huang Q, Wu KL. Evaluation of regional bulbar redness using an image-based objective method. Int J Ophthalmol. 2014;7:71-6. 14. Jackson GR, Scott IU, Quillen DA, Walter LE, Gardner TW. Inner retinal visual dysfunction is a sensitive marker of non-proliferative diabetic retinopathy. Br J Ophthalmol. 2012;96:699-703. 15. Carrasco E, Hernandez C, de Torres I, Farres J, Simo R. Lowered cortistatin expression is an early event in the human diabetic retina and is associated with apoptosis and glial activation. Mol Vis. 2008;14:1496-502. 16. Toprak I, Yildirim C, Yaylali V. Optic disc topographic analysis in diabetic patients. Int Ophthalmol. 2012;32:559-64. 17. Jain A, Saxena S, Khanna VK, Shukla RK, Meyer CH. Status of serum VEGF and ICAM-1 and its association with external limiting membrane and inner segment-outer segment junction disruption in type 2 diabetes mellitus. Mol Vis. 2013;19:1760-8. 18. Unoki N, Nishijima K, Sakamoto A, et al. Retinal sensitivity loss and structural disturbance in areas of capillary nonperfusion of eyes with diabetic retinopathy. Am J Ophthalmol. 2007;144:755-60.
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Impaired photoreceptor inner segment ellipsoid layer reflectivity in mild diabetic retinopathy Ibrahim Toprak, MD,* Cem Yildirim, MD,† Volkan Yaylali, MD† ABSTRACT ● RÉSUMÉ Objective: To determine alterations in retinal pigment epithelium (RPE), photoreceptor inner segment ellipsoid layer (ISel), and external limiting membrane (ELM) reflectivities using optical coherence tomography (OCT) image analysis in patients with mild nonproliferative diabetic retinopathy (NPDR). Design: Retrospective, single-centre, controlled clinical study. Participants: The study cohort included 42 eyes of 42 patients with mild NPDR (NPDR group) and 40 eyes of 40 healthy subjects (control group). Eyes with moderate and severe NPDR, proliferative DR, macular edema, and other macular pathologies were excluded. Methods: The reflectivities of RPE, ISel, and ELM were calculated using a medical image-processing software based on greyscale OCT images. The differences in the reflectivity values between the NPDR and control groups were analyzed. Results: The NPDR group comprised 22 males and 20 females (with a mean age of 61.3 ⫾ 6.5 years), and the control group consisted of 14 males and 26 females (with a mean age of 63.0 ⫾ 4.1 years) (p 4 0.05). The ISel had significantly lower reflectivity (both absolute and relative) in eyes with mild NPDR compared with that of the control eyes (p o 0.001), whereas the reflectivities of RPE and ELM did not differ between the 2 groups (p ¼ 0.126, p ¼ 0.053 respectively). Conclusions: Although previous ex vivo studies reported photoreceptor degeneration in diabetic retinopathy, this is the first clinical study to investigate retinal layer reflectivities in NPDR using OCT. In eyes with mild NPDR, ISel seems to have lower reflectivity, and this finding might indicate early photoreceptor degeneration in diabetic retinopathy pathogenesis. Objet : Mesurer des altérations de la réflectivité de l'épithélium pigmentaire rétinien (EPR), de la couche ellipsoïde du segment interne des photorécepteurs et de la membrane limitante externe (MLE) grâce à l'analyse d'imagerie de tomographie de cohérence optique (TCO) chez des patients atteints de rétinopathie diabétique non proliférante (RDNP) légère. Nature : Étude clinique unicentrique rétrospective avec cas témoins. Participants : L'étude a porté sur 42 yeux de 42 patients présentant une RDNP légère (groupe RDNP) et sur 40 yeux de 40 sujets sains (groupe témoin). Les cas de RDNP modérée et sévère, de rétinopathie diabétique proliférante, d'œdème maculaire et d'autres pathologies maculaires étaient exclus. Méthodes : On a calculé la réflectivité de l'EPR, de la couche ellipsoïde du segment interne des photorécepteurs et de la MLE au moyen d'un logiciel de traitement d'imagerie médicale, à partir d'imagerie TCO en échelle de gris. On a analysé l'écart entre les valeurs de réflectivité du groupe RDNP et du groupe témoin. Résultats : Le groupe RDNP était composé de 22 hommes et de 20 femmes (âge moyen : 61,3 ⫾ 6,5 ans), tandis que le groupe témoin était formé de 14 hommes et de 26 femmes (âge moyen : 63,0 ⫾ 4,1 ans) (p 4 0,05). Dans le groupe RDNP, la réflectivité (tant absolue que relative) de la couche ellipsoïde du segment interne des photorécepteurs était significativement moindre que dans le groupe témoin (p o 0,001). Par contre, il n'y avait pas d'écart entre les deux groupes en ce qui concerne la réflectivité de l'EPR et de la MLE (p ¼ 0,126, p ¼ 0,053, respectivement). Conclusions : Des études ex vivo antérieures avaient fait état de la détérioration des photorécepteurs chez les patients atteints de rétinopathie diabétique, mais cette étude était la première étude clinique à explorer la réflectivité de couches rétiniennes à l'aide d'imagerie TCO chez des patients atteints de RDNP. Dans les cas de RDNP légère, la réflectivité de la couche ellipsoïde du segment interne des photorécepteurs semble plus faible, et cette conclusion pourrait être indicatrice d'une dégénérescence précoce des photorécepteurs dans la pathogénèse de la rétinopathie diabétique.
Early characteristics of nonproliferative diabetic retinopathy (NPDR) include microcapillary abnormalities such as microaneurysm formation, basement membrane thickening, and loss of pericytes.1,2 In the later stages of the retinopathy, capillary nonperfusion, microvascular abnormalities, retinal ischemia, and disruption of the blood– retina barrier manifest in fundus fluorescein angiography (FFA).1,3 Furthermore, ex vivo studies suggested that microcirculatory alterations lead neuronal degeneration
before the retinal neovascularization (proliferative diabetic retinopathy [PDR]).4–6 From a clinical perspective, advances in optical coherence tomography (OCT) devices allow visualization of retinal layers very close to histologic sections, whereas OCT does not provide functional data. In contrast, decrease in retinal layer reflectivity was found to be related with functional deterioration, and OCT reflectivities were evaluated previously in patients with
From the *Department of Ophthalmology, Servergazi State Hospital; and †Department of Ophthalmology, Faculty of Medicine, Pamukkale University, Denizli, Turkey
Can J Ophthalmol 2015;50:438–441 0008-4182/15/$-see front matter & 2015 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcjo.2015.07.009
Originally received Mar. 12, 2015. Final revision Jul. 4, 2015. Accepted Jul. 12, 2015 Correspondence to Ibrahim Toprak, MD, Department of Ophthalmology, Servergazi State Hospital, Tedas Mevki, Bereketli Beldesi, Denizli 20070, Turkey; [email protected]
438
CAN J OPHTHALMOL — VOL. 50, NO. 6, DECEMBER 2015
Photoreceptor reflectivity in diabetic retinopathy— Toprak et al. cystoid diabetic macular edema and idiopathic epimacular membrane.7–10 In this study, we firstly analyzed OCT reflectivities of foveal retinal pigment epithelium (RPE), photoreceptor inner segment ellipsoid layer (ISel, previously named photoreceptor inner segment/outer segment junction), and external limiting membrane (ELM) using a medical image-processing software (ImageJ v 1.47) in eyes with mild NPDR to reveal retinal neurodegeneration in vivo.
METHODS In this retrospective, single-centre, controlled clinical study, 42 eyes of 42 patients with mild NPDR (NPDR group) and 40 eyes of 40 age- and sex-matched healthy subjects (control group) were enrolled. The reliability of the sample size was tested at 95% power and 0.05 significance level (95% CI) using statistical software (PASS version 11.0.1; NSCC, LLC, Kaysville, Utah). The study followed the tenets of the Declaration of Helsinki, and permission was obtained from the local ethics committee. All participants underwent a complete ophthalmologic examination, which included corrected distance visual acuity measurement with Snellen charts, slit-lamp biomicroscopic examination, Goldmann applanation tonometry, dilated funduscopy (with þ90 D lens), and FFA. Inclusion and exclusion criteria
Patients with a diagnosis of mild NPDR (microaneurysms only) based on dilated fundus examination and FFA were included in the study. Patients with moderate and severe NPDR (severe retinal hemorrhages or venous beading or intraretinal microvascular abnormalities), disrupted or absent RPE, ISel and ELM bands on OCT, PDR, macular edema, or any other macular pathology and poor quality of OCT image were excluded.
Age- and sex-matched healthy subjects with a normal ophthalmologic examination comprised the control group. All patients and control subjects had a corrected distance visual acuity of 20/20 Snellen equivalent, and right eyes were used for the statistical analysis. Assessment of optical coherence tomographic reflectivities
The 6 mm 6 mm macular cube 512 128 scanning protocol of the Zeiss Cirrus HD-OCT 400 (Carl Zeiss Meditec, Dublin, Calif.) was used to acquire macular OCT images. A single experienced technician (MA) performed the OCT imaging. All participants had reliable (signal strength Z 7/10) macular OCT images. The greyscale OCT images were transferred to the ImageJ v1.47, a Java-based imageprocessing computer software developed under the National Institutes of Health, to perform reflectivity analyses.11 The ImageJ calculates distances, angles, areas, intensity, and pixel value statistics on an image with high reliability and reproducibility.9,10,12,13 A single masked ophthalmologist (I.T.) analyzed the randomly selected standardized greyscale OCT images for 2 times at different days, and the mean values were used for statistical analysis. The intraobserver intraclass correlation coefficients (ICC) were calculated for the repeated measurements to determine intraobserver reliability (a value 40.80 indicated good reliability). The reflectivities of the RPE, ISel, and ELM in the fovea centre were calculated using plot profile function of the software as previously described.9–11 The ImageJ provided reflectivity values along a vertical straight line passing through the centre of the fovea, and a reflectivity graph was created (Fig. 1). In a healthy eye, the RPE layer has the highest reflectivity on OCT followed by ISel and ELM. Hence the highest value on the reflectivity graph was accepted as the reflectivity of the RPE layer, and localizations of the RPE layer, ISel, and ELM on OCT
Fig. 1 — Vertical white line passing through the fovea centre indicates the reference line, which was used for reflectivity calculation. Reflectivity graph obtained from the ImageJ (right) and corresponding retinal layers on the greyscale macular optical coherence tomography scan (left) of a patient with mild nonproliferative diabetic retinopathy. ELM, external limiting membrane; ISel, inner segment ellipsoid layer; RPE, retinal pigment epithelium. CAN J OPHTHALMOL — VOL. 50, NO. 6, DECEMBER 2015
439
Photoreceptor reflectivity in diabetic retinopathy— Toprak et al.
Fig. 2 — Comparison of the retinal pigment epithelium (RPE), photoreceptor inner segment ellipsoid layer (ISel) and external limiting membrane (ELM) reflectivities between the mild nonproliferative diabetic retinopathy (NPDR) and control groups.
image were confirmed simultaneously on the reflectivity graph using live plot profile function of the software.9,10 The reflectivity values on the graph were noted (as absolute reflectivity) and converted to the relative reflectivity according to the following formula9,10: Relative reflectivity (arbitrary unit) ¼ (reflectivity of a retinal layer [ISel or ELM]) / (reflectivity of the fixed reference retinal layer [RPE]) 100. The reflectivity values of RPE, ISel, and ELM were compared between the NPDR and control groups.
groups regarding the RPE (206.3 ⫾ 17.7 vs 211.5 ⫾ 12.2 arbitrary units, respectively; p ¼ 0.126) and ELM (89.5 ⫾ 14.7 vs 95.5 ⫾ 12.6 arbitrary units, respectively, p ¼ 0.053) reflectivities. Moreover, relative ISel reflectivity was also significantly lower in the patients with NPDR (71.7 ⫾ 7.3 arbitrary units) than in the healthy subjects (85.4 ⫾ 10.1 arbitrary units) (p o 0.001), whereas relative ELM reflectivity did not differ between the NPDR (43.4 ⫾ 6.3 arbitrary units) and control groups (45.2 ⫾ 6 arbitrary units) (p ¼ 0.192). Figure 2 represents comparison of the reflectivity values between the NPDR and control groups.
Statistical analysis
The Statistical Package for Social Sciences software version 16.0 (SPSS Inc, Chicago, Ill.) was used for the statistical analysis. Results were expressed as the mean ⫾ SD. The χ2 was used to compare 2 groups in terms of sex. The ICCs were calculated to test the intraobserver reliability of the repeated measurements (ICC 4 0.80 indicated good reliability). Comparison of the quantitative data (age and reflectivity values) between the 2 groups was performed using the independent samples t test. A p value less than 0.05 was considered statistically significant at 95% CI.
RESULTS Forty-two patients with mild NPDR and 40 healthy subjects were included in the study. The mean age was 61.3 ⫾ 6.5 years in the NPDR group and 63.0 ⫾ 4.1 years in the control group (p ¼ 0.182). The NPDR group consisted of 22 males and 20 females, and the control group comprised 14 males and 26 females (p ¼ 0.126). All patients and control subjects had normal macular thickness, and the repeated OCT measurements showed excellent intraobserver reliability (with an ICC 4 0.90). In the NPDR group, the absolute ISel reflectivity was significantly lower (148.2 ⫾ 21.3 arbitrary units) when compared with that of the control group (180.2 ⫾ 21.1 arbitrary units) (p o 0.001). However, there were no significant differences between the NPDR and control
440
DISCUSSION Microvascular insufficiency is known as the major hallmark of diabetic retinopathy (DR). However, previous studies demonstrated evidences of neurodegeneration in DR, such as increased apoptosis and glial activation, decreased retinal nerve fiber layer thickness, electroretinographic abnormalities, and impaired retinal function tests.2–6,14–16 In eyes with DR, researchers focused on alterations in the photoreceptor layer (particularly in the ISel).6,17 An experimental study by Alvarez et al.6 showed photoreceptor dysfunction in a hyperglycemic model of NPDR, and the authors suggested that cone photoreceptor dysfunction was a clinical hallmark of DR. Although previous ex vivo studies presented photoreceptor degeneration in patients with DR, a few clinical studies have investigated functional status of photoreceptor layer in DR. In a clinical study, Unoki et al.18 demonstrated decreased retinal sensitivity (using microperimetry) in eyes with severe NPDR and PDR. They also detected hyperreflective depositions between the photoreceptor layer and the RPE in OCT imaging. Similarly, Jackson et al.14 showed photoreceptor dysfunction based on dark-adapted visual sensitivity tests in patients with NPDR. In this study, we firstly evaluated reflectivities of RPE, photoreceptor ISel, and ELM in patients with mild NPDR using OCT. Previous neurophysiologic studies showed that decreased retinal layer reflectivity on OCT was
CAN J OPHTHALMOL — VOL. 50, NO. 6, DECEMBER 2015
Photoreceptor reflectivity in diabetic retinopathy— Toprak et al. associated with structural and/or functional damage.7,8 This study demonstrated a significant decrease in the ISel reflectivity (both absolute and relative) in eyes with mild NPDR. This finding appears to be parallel with the previous ex vivo studies. Furthermore, it might be important for showing photoreceptor degeneration (structural and/or functional) in early NPDR. In conclusion, DR is a complex pathologic process that includes both vascular and neuronal damage. This study is the first to evaluate retinal layer reflectivities using OCT image analysis in eyes with mild NPDR, and it showed the impaired ISel reflectivity, which might indicate contribution of the photoreceptor degeneration to DR pathogenesis from the early stages of the disease.
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