Introduction {#sec1-1}
============

Diabetic retinopathy (DR) is a major cause of blindness in the working age group worldwide and remains one of the most serious complications of diabetes mellitus.\[[@ref1][@ref2]\] Visual impairment in DR can be due to macular edema,\[[@ref3]\] vitreous hemorrhage, tractional retinal detachment involving fovea, and macular ischemia.\[[@ref4]\] The early treatment DR study has clearly demonstrated that it is important to differentiate the severity of DR, as moderate nonproliferative DR (NPDR) (level 47) carries an 8.6% risk of developing high-risk proliferative DR (PDR) over a period of 1 year and severe NPDR (level 53e) carries a 45% risk of developing high-risk PDR.\[[@ref5]\] The varying severity is based on morphological features in each stage of retinopathy. The morphological features contribute to the tissue space and thus retinal thickness. These quantitative alterations in the thickness of retinal layers, overall and quadrant-wise, can be studied by spectral domain optical coherence tomography (SDOCT). These structural changes in various stages of DR have been poorly studied.\[[@ref6][@ref7][@ref8]\]

The aim of the present study is to elucidate the changes in the retinal thickness and individual layer thickness in subjects with no diabetes; subjects with diabetes but no DR; subjects with mild, moderate, and severe NPDR; and PDR.

Materials and Methods {#sec1-2}
=====================

Subjects with diabetes mellitus, with or without clinical evidence of DR, and normal healthy subjects were included in this study. All the subjects underwent a comprehensive eye examination including 45΀ digital fundus photography (Carl Zeiss fundus camera, Visucam Lite, Jena, Germany) and SDOCT (SDOCT Copernicus, Optopol Technologies, Zawierci, Poland). DR severity was graded based on the International Clinical DR Disease Severity Scale.\[[@ref9]\] The study was approved by the Institutional Review Board. Informed consent was obtained from all study subjects.

Subjects with history of systemic diseases other than diabetes mellitus and with vascular or pathological changes in eye other than DR were excluded from the study. Subjects with history of intravitreal injection or any retinal surgery and poor image quality on SDOCT due to media opacities were also excluded.

The subject\'s pupils were dilated with 5% phenylephrine and 1% tropicamide eye drops; if phenylephrine is contraindicated, 1% cyclopentolate eye drops are used. SDOCT scans were performed through a dilated pupil, and the reconstructed video image of the central retina was monitored. For the purpose of this study, we used a scan length of 7 mm with 6 B-scans and 3000 A-scans per B-scans through the center of the fovea for the asterisk scan protocol. Three-dimensional scan protocol was used with 7 mm scan length with 50 B-scans and 1000 A-scans per B-scan. All the measurements were calculated in microns. The foveal center was defined by a hyper-reflective dot echo at the innermost retinal layer. All the measurements were carried out at the same point for the uniformity of comparison using measurement software.

Definitions {#sec2-1}
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### Central foveal thickness {#sec3-1}

This was manually measured as the distance between the internal limiting membrane and the anterior surface of the retinal pigment epithelium (RPE).

### Photoreceptor layer thickness {#sec3-2}

It was defined as the distance between the external limiting membrane which appeared as a thin hyper reflective line on SDOCT and the anterior surface of the RPE.

### Retinal pigment epithelial thickness {#sec3-3}

It was defined as the distance between the inner and outer edge of the RPE layer.

### Retinal nerve fiber layer thickness {#sec3-4}

It was measured at 2.5 mm nasal to the central fovea by the automated software which provided the distance between the internal limiting membrane and the outer edge of the retinal nerve fiber layer (RNFL).

### Central subfield thickness {#sec3-5}

This was assessed as the retinal thickness in central 1 mm area of early treatment DR study (ETDRS) quadrants.

### Mean retinal thickness {#sec3-6}

Mean retinal thickness was given as the average retinal thickness of all ETDRS quadrants, i.e. central subfield thickness (CSFT), superior, inferior, nasal, and temporal quadrants, in the middle 3 mm and the outer 6 mm ring.

Central foveal thickness (CFT), photoreceptor layer (PRL), and RPE thickness were measured manually using the calipers available in the SD-OCT software. RNFL thickness was assessed using an automated measurement technique in the macular area (2.5 mm nasal to fovea); no glaucoma software was used for assessing the RNFL thickness. These measurement techniques can also be done using other OCT instrument, considering the segmentation landmarks used by different OCT instruments.

Intraobserver variability was seen by the same observer in a subset of the study sample 1 week after the initial measurements. For the interobserver variation in manual measurement of SDOCT, the thickness parameters were taken by a different observer in a subset of the study sample.

Statistical analysis {#sec2-2}
--------------------

Statistical analysis was performed using SPSS (Statistical Package for Social Sciences, version 15.0, Chicago, Illinois, USA). The mean comparison of the SDOCT parameters between groups and the difference in thickness between genders was done using t-test. The Pearson\'s correlation coefficients were used to find out the association between the parameters. Intraclass correlation was done to assess the intraobserver variability. Bland--Altman was plotted using MedCalc software to see the interobserver variation in measurement of SDOCT parameters. Type I error of 5% was considered statistically significant.

Results {#sec1-3}
=======

A total of 251 eyes from 170 subjects were included in this study. The mean age of the study population was 55.34 ± 9.02 years (range: 32--80 years), and 56.6% of these were males. The mean duration of diabetes mellitus from diagnosis was 110.9 ± 78.1 months (range: 1--444 months). [Table 1](#T1){ref-type="table"} shows the baseline parameters of the study population. We found a significant positive correlation between duration of diabetes and severity of DR (*r* = 0.21, *P* = 0.014).

###### 

Baseline characteristics of study population

![](OJO-9-32-g001)

[Table 2](#T2){ref-type="table"} shows gender difference in thickness parameters. Men had significantly greater CFT, CSFT, RNFL thickness and retinal thickness in all the quadrants of 3 mm and 6 mm zones compared to women (*P* \< 0.001). However, no significant difference was found in PRL (*P* = 0.91) and RPE (*P* = 0.11) thicknesses.

###### 

Retinal thickness measurements according to gender

![](OJO-9-32-g002)

[Figure 1](#F1){ref-type="fig"} depicts the quadrant-wise retinal thickness in the study population. When the retinal thickness in different quadrants was compared, there was no difference found between nondiabetic subjects and diabetes subjects with no DR. In DR group, all quadrants had a significantly increased retinal thickness compared to nondiabetic subjects (*P* = 0.004, *P* = 0.016, *P* = 0.024, and *P* = 0.005 in superior, inferior, nasal, and temporal quadrants, respectively). Only superior (*P* = 0.044) and temporal (*P* = 0.011) quadrants showed an increased retinal thickness in any DR group when compared with subjects with diabetes but no DR.

![Quadrant-wise retinal thickness](OJO-9-32-g003){#F1}

[Table 3](#T3){ref-type="table"} shows the thickness parameters in various DR groups. On comparing the thickness parameters between nondiabetic and diabetic subjects with no DR, PRL thickness was significantly reduced in diabetic subjects with no DR (nondiabetic - 65.04 ± 4.43 μm, no DR - 62.75 ± 4.82 μm; *P* = 0.014). On comparison of diabetic subjects with no DR and those with mild and moderated NPDR, CFT was significantly high, and the RPE thickness was significantly low in mild and moderate NPDR (CFT: No DR - 167.28 ± 14.56 μm, mild and moderate NPDR - 177.12 ± 20.99 μm; *P* = 0.003. RPE thickness: No DR - 43.25 ± 6.90 μm, mild and moderate NPDR - 40.51 ± 6.14 μm; *P* = 0.014). PRL thickness was significantly reduced in severe NPDR when compared with mild and moderate NPDR (mild and moderate NPDR - 61.98 ± 6.64 μm, severe NPDR - 57.53 ± 7.53 μm; *P* = 0.003). We did not find any significant difference in CSFT and RNFL thickness among the subgroups.

###### 

Comparison of thickness parameters in various groups

![](OJO-9-32-g004)

Intraobserver repeatability was found to be good in measuring the SDOCT outcomes with intraclass correlation of 0.99 for CFT, 0.635 for PRL thickness and 0.805 for RPE thickness. [Figure 2](#F2){ref-type="fig"} shows the Bland--Altman plot for various thickness parameters. The mean difference in the CFT measurements was 6.7 μm with limits of agreement ranging from −19.4 to 32.8 μm. The mean difference in the PRL thickness was 8 μm with 95% limits of agreement between −7.4 and 23.4 μm; the mean difference in RPE thickness was −2.2 μm with limits of agreement ranging from −9.1 to 4.7 μm.

![Bland--Altman plot for spectral domain optical coherence tomography parameters](OJO-9-32-g005){#F2}

Discussion {#sec1-4}
==========

The morphological changes in various stages of DR are reflected by the differences in the retinal thickness in total or thickness of individual layers. Yang *et al*. and Forooghian *et al*. have reported the changes in diabetic macular edema.\[[@ref10][@ref11]\] Significant difference in retinal thickness was reported in various stages of DR using stratus OCT.\[[@ref12][@ref13]\] Our group also

studied the neuronal changes that occur in diabetic subjects who did not have clinical evidence of DR.\[[@ref14]\] It was reported that SDOCT measured PRL thickness was 61.62 ± 4.48 μm in subjects with diabetes but no DR, and 68.79 ± 7.84 μm in nondiabetic subjects. CFT was 168.64 ± 16.46 μm in subjects with diabetes but no DR and 177.74 ± 14.58 μm in nondiabetic subjects.

Increased retinal thickness in men was reported in healthy individuals in the study done by Wong *et al*.\[[@ref15]\] Bressler *et al*.\[[@ref8]\] in their study reported that in type II diabetes subjects, CSFT was significantly greater in the retina of men with mean of 209 ± 18 μm and it was 194 ± 23 μm in women which was similar to our study results. However, they did not find any difference in the inner subfield and outer subfield retinal thickness which was noted in our study.

Significant difference in the retinal thickness between diabetic subjects with and without DR was only found in the superior and temporal quadrants. These findings make the superior and temporal quadrants the most likely regions where the earliest and clinically nondetectable changes in retinal thickness caused by DR can be detected. Our results were similar to the study done by Schaudig *et al*.,\[[@ref16]\] in which they found the superonasal quadrants to have the earliest DR changes in terms of changes in the retinal thickness values. This could happen in an area where the retinal thickness and density of ganglion cell bodies are higher, vascular damage resulting in edema will produce thickening, mainly in a posterio-anterior direction whereas in an area with lower density of cells it, will take a higher amount of edema to produce the same amount of thickening. We identified that in all cases, the nasal quadrant was thicker than the temporal. This was consistent with the anatomical relationship of the converging of nerve fibers with the optic disc.\[[@ref17]\]

An increase in retinal thickness was found in subjects with mild and moderate NPDR when compared to diabetic subjects with no DR. PRL thickness was significantly reduced in diabetic subjects with no DR compared to nondiabetic subjects and also in severe NPDR group compared to mild and moderate NPDR. RPE thickness was significantly reduced in diabetic subjects with mild and moderate NPDR compared to diabetic subjects with no retinopathy. Recent evidence suggests that the selective thinning of inner retinal layers supports the concept of early neurodegenerative component in patients with minimal DR.\[[@ref13][@ref14]\] Sufficient evidence also comes from the animal studies that thickness of the inner retinal layers decreases in diabetic condition, indicating early neurodegeneration.\[[@ref18][@ref19]\]

Earlier studies have assessed RNFL thickness in the peripapillary area using OCT, scanning laser polarimetry, etc., and reported that the RNFL thickness was significantly reduced with severity of DR;\[[@ref20][@ref21]\] however, we measured RNFL thickness in the macular area and did not find any difference in RNFL thickness between the groups of DR. This suggests that RNFL thickness did not vary among the subjects with diabetes and with DR in the macular area.

Conclusion {#sec1-5}
==========

In summary, this study demonstrates a loss of PRL thickness in diabetic subjects which support the concept of neurodegeneration. We also report that superior and temporal quadrants are the most likely regions where the earliest and clinically nondetectable changes in the retinal thickness can be detected. These results may help in close monitoring of diabetic subjects before developing DR.

Financial support and sponsorship {#sec2-3}
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Nil.

Conflicts of interest {#sec2-4}
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There are no conflicts of interest.
