Optical coherence tomography biomarkers DROL, PROS, SND, hyperreflective walls of foveal cystoid spaces as predictors of central macular thickness and visual acuity in diabetic macular edema treated with intravitreal ranibizumab

Abstract

Purpose:

This study aims to establish DROL (disruption of retinal outer layers), PROS (photoreceptor outer segment length), SND (subfoveal neuroretinal detachment), and hyperreflective walls of foveal cystoid spaces (HRW) as optical coherence tomography (OCT) biomarkers and predictors of central macular thickness (CMT) and visual acuity in diabetic macular edema (DME) treated with intravitreal ranibizumab (IVR).

Methods:

In this prospective, interventional study performed at a tertiary care center over a span of 1 year from December 2021 to December 2022, 50 eyes of 46 patients of DME were included. Visual acuity and spectral domain OCT imaging were performed at baseline. Using inbuilt calipers on SD-OCT, the horizontal extent of DROL and the vertical extent of PROS were measured manually. SND and HRW were assessed qualitatively. IVR was administered and patients were followed up at 4, 8, and 12 weeks.

Results:

The eyes without DROL had statistically significant (P < 0.05) lesser CMT and better BCVA (best-corrected visual acuity) (P < 0.05) after pro re nata injection of IVR. There was a positive correlation between the extent of baseline DROL with final CMT (P < 0.05) and final logMAR BCVA (P > 0.05), whereas negative correlation with the extent of baseline PROS with final CMT (P < 0.05) and final logMAR BCVA (P > 0.05). The presence of HRW and SND predicted non-resolution of CMT and worse visual acuity after treatment with IVR in DME

Conclusion:

DROL, PROS, SND, and hyperreflective walls of foveal cystoid spaces may be utilized as qualitative as well as quantitative biomarkers to predict the post-treatment CMT and visual acuity in DME.

Diabetes is a chronic, metabolic disease that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces.[1] When ophthalmic manifestations are considered, diabetic retinopathy (DR) is taking center stage today. DR is one of the leading causes of blindness worldwide in working adult age groups.[2] DR naturally progresses from non-proliferative abnormalities to proliferative diabetic retinopathy (PDR), characterized by neovascularization involving disc (NVD) or neovascularization elsewhere (NVE). The leading cause of vision loss in DR patients is diabetic macular edema (DME). DME is characterized by retinal thickening and edema, which can develop in all stages of retinopathy. Macular edema results from abnormal accumulation of fluid in the central retina and indicates a compromised function in inner, outer or both blood–retinal barriers. Any abnormal pooling of extracellular fluid may result in the displacement of spatial relationships between retinal neuronal components. Small amounts of fluid may lead to an increase in the overall retinal thickness, whereas larger amounts may give rise to cell-free spaces as observed in cystoid macular edema (CME).[3] Basic research has postulated the molecular mechanisms including vascular endothelial growth factor in DME and proliferative diabetic retinopathy (PDR).

Optical coherence tomography (OCT) provides retinal sectional images as in histology studies and is useful for qualitative and quantitative evaluation of pathological retinal changes. The purpose of this study is to establish OCT biomarker DROL (disruption of retinal outer layers), PROS (photoreceptor outer segment length), SND (subfoveal neuroretinal detachment), and hyperreflective walls of foveal cystoid spaces as predictors of visual acuity and central macular thickness in diabetic macular edema treated with intravitreal ranibizumab. The diabetic retinopathy clinical research network (DRCRnet), especially defined an increase in the mean central thickness within 1 mm of fovea as center-involved DME, which is the new standard for applying treatments and a surrogate marker for evaluating treatment efficacy. In addition, the DRCRnet reported that “paradoxical visual acuity” (VA) changes are observed after intervention, that is, VA reduction despite resolution of ME or VA improvement with increased retinal thickening.” Spectral-domain OCT with higher resolution and reduced speckle noises has enabled the segmentation of individual retinal layers, and several kinds of OCT instruments provide automated segmentation.[4] This feature has encouraged the quantification of the thickness of retinal layers and qualitative evaluation of lesions in individual layers, which helps us to establish various biomarkers and explain the paradoxical changes in visual acuity in relation to macular edema.

Methods

Study Design-A prospective, single center, interventional study was conducted at a tertiary care center over a span of 1 year from December 2021 to December 2022. A total of 50 eyes of 46 patients were included in the study. After ethical approval and informed consent, the study enrolled only individuals of (1) age above the age of 18 years of either gender diagnosed with type 1 or type 2 diabetes. (2) Non-proliferative diabetic retinopathy patients having diabetic macular edema with central macular thickness ≥275 μm. Exclusion criteria were (1) history of ocular trauma and surgery, (2) advanced media opacities (cataract > grade 2 LOCS III classification[5]), (3) corneal scarring, (4) proliferative diabetic retinopathy at baseline, (5) foveal avascular zone (FAZ) distortion on optical coherence tomography (OCTA), (6) other ocular conditions (retinal detachment, macular hole, retinal vein occlusion, age-related macular degeneration, abnormality of vitreoretinal interface, glaucoma), and (7) patients on anticoagulants or contraceptive pills.

Study Protocol-All patients underwent a thorough ophthalmic examination that included best-corrected visual acuity (BCVA) (Snellen’s chart), slit-lamp examination, intraocular pressure measurement, dilated fundus examination by slit-lamp biomicroscopy, following which detailed fundus examination was performed with an indirect ophthalmoscope. For statistical analysis, BCVA was transformed into the logarithm of the resolution angle minimum (logMAR). Spectral-domain OCT (SD-OCT) imaging was performed using OCT Macula, 3D Wide 12 × 9 mm scans (TOPCON 3D OCT-1 Maestro2, Topcon Healthcare, Japan). Inbuilt IMAGEnet 6 capture software was used for imaging data. Only images with signal strength intensity (SSI) ≥0.5 were used for analysis. Routine blood tests such as Complete Blood Count, Fasting Blood Sugar, Post prandial blood sugar, Hemoglobin A1C, Liver Function Test, Kidney Function Test, Lipid profile, Bleeding Time, and Clotting Time were performed before intravitreal ranibizumab injection.

Measurement of biomarkers on OCT

• Quantitative biomarkers

  • DROL was taken as the horizontal extent in microns, for which any boundaries between the external limiting membrane (ELM), ellipsoid zone (EZ), and interdigitation zone (IZ) could not be identified in the 1 mm foveal area.[6] Using the inbuilt caliper of the IMAGEnet 6 capture software, a vertical line was drawn from the center of the fovea to the outermost retinal hyperreflective layer, that is, RPE. One horizontal line measuring 500 µm was drawn on either side of the vertical line to delineate the central 1 mm foveal area. Within this 1 mm zone, the horizontal extent of disrupted outer retinal layers was measured and taken as DROL.

  • PROS-Using the inbuilt caliper of the IMAGEnet 6 capture software, a vertical line was drawn from the center of the fovea to the outermost retinal hyperreflective layer, that is, retinal pigment epithelium (RPE). One horizontal line measuring 500 µm was drawn on either side of the vertical line to delineate the central 1 mm foveal area. Within this 1 mm zone, the vertical extent of PROS was measured from the inner surface of the IS/OS band to the inner surface of RPE.[7]

• Qualitative Biomarkers-

  • Hyperreflective walls of foveal cystoid spaces were present when there was hyperreflectivity along the walls of cystoid spaces in the 1 mm foveal area.[8]

  • SND was present when there was a detachment of the neurosensory retina from the underlying RPE layer in the 1 mm foveal area on OCT.[9]

Outcome Measures–Patients were followed up at 4, 8, and 12 weeks post-injection and all baseline investigations were repeated. Repeat doses of pro re nata intravitreal ranibizumab injections were administered at 1-month intervals in patients whose central macular thickness remained ≥275 μm.[10]

Statistical Analysis-The data were collected with a predesigned proforma and entered in Microsoft Excel 2010 (Microsoft Corporate Excel version 2010). The data were analyzed with Epi info version 7.1.4.0. Continuous data (age, log MAR BCVA, CMT, extent of DROL, etc.) are presented with mean and standard deviation, whereas categorical data (gender, eyes with presence of DROL, etc.) are presented with frequency and percentage. Spearman’s r correlation coefficient and receiver operating characteristic (ROC) curves for the development and validation of the data were performed. For all analyses, P < 0.05 was considered statistically significant.

Results

Fifty eyes of 46 patients of NPDR with DME were analyzed. The mean age of patients was 57.76 years (SD ± 8.76). Also, 56% were male and 44% were female.

DROL as a qualitative biomarker

At the first visit pre-injection, DROL was present in 36 eyes (72%) (taken as baseline DROL). Eyes that did not have DROL had statistically significant (P < 0.05) lesser CMT and better BCVA at 12 weeks than those that had DROL before intervention [Table 1].

Table 1.

Comparison of OCT biomarkers (DROL, HRW, and SND) at baseline and 12 weeks for mean CMT and logMAR BCVA

		Mean CMT at 12 weeks (μ)		Mean logMAR BCVA at 12 weeks
DROL
+		276.78±61.96		0.69±0.10
-		253.88±11.32		0.32±0.08
P		<0.05		<0.05
HRW
+		241.40±47.49		0.467±0.08
-		289.42±84.09		0.553±0.26
P		<0.05		0.08
SND
+		284.15±83.24		0.571±0.23
-		255.75±59.22		0.325±0.18
P		0.36		<0.05

DROL as a quantitative biomarker

We found a statistically significant (P < 0.05) positive correlation of the extent of pre-injection DROL with CMT achieved at 12 weeks. There was also a weak positive correlation between the extent of pre-injection DROL with logMAR BCVA achieved at 12 weeks; however, this result was not statistically significant (P > 0.05) [Table 2]. We also found a statistically significant (P < 0.05) decrease in the horizontal extent of DROL from the baseline up to 12 weeks post-intravitreal injection of ranibizumab [Table 3, Fig. 1a and b].

Table 2.

Correlation of pre-inj DROL length and pre-inj PROS length with CMT and with logMAR BCVA at 12 weeks

		Pearson Correlation		P		n
Pre-inj DROL length
CMT at 12 weeks		0.5		<0.05		36
logMAR BCVA at 12 weeks		0.23		1.6		36
Pre-inj PROS length
CMT at 12 weeks		-0.028		<0.05		50
logMAR BCVA at 12 weeks		-0.13		0.36		50

Table 3.

Effect of intravitreal ranibizumab on OCT biomarkers at 4, 8, and 12 weeks

		Pre-inj		4 weeks		8 weeks		12 weeks
Mean DROL (μ)		712.93±233.92		577.93±241.92		433.67±217.72		260.87±161.05
Mean PROS (μ)		31.04±8.17		32.48±8.03		33.04±7.33		33.36±5.90
HRW+ eyes (%)		76%		64%		32%		16%
SND+ eyes (%)		16%		16%		16%		16%

Figure 1.

(a) Patient 1- DROL at pre-injection = 999 μ (yellow arrow). (b) Patient 1- DROL at 12 weeks post-injection = 679 μ (yellow arrow). (c) Patient 2 - PROS at pre-injection = 16 μ (red arrow head). (d) Patient 2- PROS at 12 weeks post-injection = 30μ (red arrow head)

Photoreceptor outer segment length (PROS)

The extent of baseline PROS correlated negatively with CMT, and this result was statistically significant (P < 0.05). That is, the longer the baseline PROS length, the lesser was CMT achieved at 12 weeks. While evaluating its correlation with visual acuity, we found a weak negative correlation between baseline PROS and logMAR BCVA. That is, the more was the baseline PROS length, the lesser was logMAR BCVA and the better the BCVA achieved at 12 weeks; however, this result was not statistically significant (P > 0.05) [Table 2]. There was an increase in the PROS length from pre-injection up to 12 weeks post-injection; however, its increase was not statistically significant (P > 0.05). [Table 3, Fig. 1c and d]

Hyperreflective walls of foveal cystoid spaces (HRW)

We found that the eyes in which they were present at the baseline, that is, 38 eyes (76%) had statistically significant (P < 0.05) more CMT at 12 weeks post-intravitreal ranibizumab injection than in those they were absent. Also, the latter had better visual acuity at 12 weeks; however, the difference in visual acuity was not statistically significant (P > 0.05) between the two groups [Table 1]. Also, at 12 weeks post-intravitreal injection, there was a progressive decrease in the number of eyes in which hyperreflective walls of foveal cystoid spaces were present before injection [Table 3, Fig. 2a and b].

Figure 2.

(a) Patient 3-Presence of HRW at pre-injection (yellow arrow). (b) Patient 3-Reduction in HRW at 12 weeks post-injection (yellow arrow). (c) Patient 4-Presence of SND at pre-injection (red arrowhead). (d) Patient 4- Persistence of SND at 12 weeks post-injection (red arrowhead)

Subfoveal neuroretinal detachment (SND)

On evaluating SND as a qualitative biomarker in DME treated with as-needed intravitreal ranibizumab, we found that the eyes in which SND was not present at pre-injection visit, had lesser CMT at 12 weeks; however, this result was not statistically significant (P > 0.05). As far as BCVA is concerned, we found that eyes in which SND was not present at the pre-injection visit had statistically significant (P < 0.05) better visual acuity at 12 weeks than in those in which it was present [Table 1]. Also, SND persisted in all eight eyes (16%), in which it was previously present, and did not disappear completely in any eye up to the follow-up period of 12 weeks [Table 3, Fig. 2c and d].

Discussion

The pathophysiology of DME is complex and is presumed to be dependent on biochemical changes such as oxidative stress, accumulation of intracellular sorbitol, increased activity of protein kinase C, and advanced glycosylation end products that lead to endothelial damage with altered leucocyte function and a consecutive breakdown of the blood–retinal barrier with dilated capillaries, microaneurysms, and loss of pericytes. Vascular leakage of fluid and serum proteins, plasma constituents, and lipids into the intraretinal space leads to increased retinal thickness and DME. A number of growth factors including the vascular endothelial growth factor (VEGF) appear to be major mediators of signal pathways, resulting in microangiopathy, angiogenesis, and neovascularization in patients with diabetic retinopathy and appear to play a key role in the therapy of DME. In the current study, we found a statistically significant (P < 0.05) improvement in BCVA and central macular thickness 12 weeks after intervention with intravitreal ranibizumab injection.

To the best of our knowledge, this is the first study to investigate DROL, that is, DROL, as a combined qualitative and quantitative biomarker for functional and anatomical outcomes in DME eyes treated with as-needed intravitreal ranibizumab injections. DROL is a combined biomarker that includes DROL including external limiting membrane, ellipsoid zone, and interdigitation zone.

While evaluating DROL as a qualitative marker, we found a statistically significant (P < 0.05) difference in CMT as well as visual acuity achieved at 12 weeks post-injection, when eyes with baseline DROL were compared with those without it. That is, eyes that did not have DROL had statistically significant (P < 0.05) better visual acuity and a lesser CMT at 12 weeks than those that had DROL before intervention.

While evaluating DROL as a quantitative biomarker, we found a statistically significant (P < 0.05) positive correlation of the extent of pre-injection DROL with CMT achieved at 12 weeks. That is, the more the baseline DROL, the greater the CMT at 12 weeks post-injection. We also found a weak positive correlation between the extent of pre-injection DROL with logMAR BCVA achieved at 12 weeks. That is, the longer the baseline DROL length, the worse the visual acuity at 12 weeks post-injection; however, this result was not statistically significant (P > 0.05). Eraslan et al.,[11] in their study, found that the presence of ERM, EZ irregularity, and DRIL were associated with significantly lower final visual acuity after intravitreal ranibizumab treatment for DME. Similar results were observed when we divided the extent of baseline DROL into four categories in the order of increasing extent.

We also found a statistically significant (P < 0.05) improvement in DROL as shown by a significant decrease in its horizontal extent from pre-injection up to 12 weeks post-intervention with intravitreal ranibizumab, which conformed with the findings by Somnath De et al.[12] and Otani et al.[13] who proposed the mechanism of ELM and EZ restoration after anti-VEGF therapy in DME and that VA improvement was positively correlated with the shortening of the disrupted EZ at 12 months. In the current study, because statistically significant (P < 0.05) improvement in DROL corresponded to improvement in visual acuity after treatment with intravitreal ranibizumab, we hypothesize that this might be due to its anti-inflammatory effect on Müller cells. Those have been shown to be the first to be affected in DME by intracellular edema. With the progression of the disease, Müller cells become apoptotic (Romero-Aroca et al.)[14] Preclinical studies show that gliosis of Müller cells in the diseased retina promotes as well as inhibit the process of neuronal degeneration (Reichenbach and Bringmann)[15] Activated Müller cells release cytotoxic substances that are responsible for the recruitment of leucocytes, blood–retinal barrier breakdown, direct glial dysfunction, and neuronal cell death. Vujosevic et al.,[16] in their review, suggested that the same mechanisms are responsible for the disorganization of the inner retina and the disruption of the outer retina.

In this study, we also evaluated PROS length to look for its correlation with visual acuity and CMT in DME eyes treated with as needed intarvitreal ranibizumab injections. PROS length was measured on OCT as the vertical extent between the photoreceptor inner and outer segment junctions and RPE. We found an increase in the PROS length from pre-injection up to 12 weeks post-injection; however, its increase was not statistically significant (P < 0.05). The extent of baseline PROS correlated negatively with CMT, and this result was statistically significant (P < 0.05). That is, the longer the baseline PROS length, the lesser was CMT achieved at 12 weeks. While evaluating its correlation with visual acuity, we found a weak negative correlation between baseline PROS and logMAR BCVA. That is, the longer the baseline PROS length, the lesser was logMAR BCVA and the better the BCVA achieved at 12 weeks; however, this result was not statistically significant (P > 0.05). Our findings are consistent with those reported by Ozkaya et al.[17] and Suciu et al.[18] who also found that shorter PROS was significantly associated with the presence of DR or DME and with worse visual acuity in patients with DME.

On evaluation of hyperreflective walls of foveal cystoid spaces as a qualitative biomarker in DME treated with as-needed intravitreal ranibizumab, we observed that eyes in which they were present had statistically significant (P < 0.05) more CMT at 12 weeks post-intravitreal ranibizumab injection than in those they were absent. Also, the latter had better visual acuity at 12 weeks; however, the difference in visual acuity was not statistically significant (P > 0.05) between the two groups. We also observed that there was a progressive decrease in the number of eyes in which hyperreflective walls of foveal cystoid spaces were present at baseline up to 12 weeks post-injection. Our findings are consistent with those of Tomoaki Murakami et al.[8] who found that the eyes with hyperreflective walls in foveal cystoid spaces had poorer visual acuity and more severe photoreceptor disruption than those without such findings and also designated them as a predictor of no DME remission under ranibizumab injections. Thus, hyperreflective walls of foveal cystoid spaces can serve as a qualitative OCT biomarker, whose presence can predict worse visual acuity and non-resolution of CMT after intervention with intravitreal ranibizumab injection for DME.

We evaluated SND as a qualitative biomarker in DME treated with as-needed intravitreal ranibizumab and found that eyes in which SND was not present at pre-injection visit had statistically significant (P < 0.05) better visual acuity at 12 weeks than in those in which it was present. As far as CMT is concerned, eyes in which SND was not present at pre-injection visit had lesser CMT at 12 weeks; however, this result was not statistically significant (P > 0.05). Thus, the presence of SND is a predictor of worse visual acuity after intravitreal ranibizumab injection in DME, although CMT decreased in both SND+ and SND− eyes with no statistically significant difference between the two groups (P > 0.05). These findings are consistent with those of studies performed by Eraslan et al.[11] Also, when followed up to 12 weeks post-intravitreal injection and observing only qualitatively and not quantifying the extent of SND, we found that SND persisted in all eyes in which it was previously present, and did not disappear completely up to the pre-determined follow-up period. Our findings are consistent with those of Maggio et al.,[19] who found a higher prevalence of SND in those DME eyes in whom CMT did not resolve or resolved inconsistently.

One of the major strengths of this study is that this is the first-ever study to evaluate DROL as a prognostic biomarker to evaluate changes in outer retinal layers in DME. The advanced OCT machine (TOPCON 3D OCT-1 Maestro2, Topcon Healthcare, Japan) and inbuilt IMAGEnet 6 capture software allowed very fine manual measurements using inbuilt calipers.

The presence of subfoveal neuroretinal detachment and hard exudate plaque at the fovea, hampering measurements is a limiting factor in the measurement of OCT biomarkers. Also, manual measurements on OCT may have resulted in subjective errors in data collection.

Conclusion

DROL, PROS, SND, and hyperreflective walls of foveal cystoid spaces may be utilized as qualitative as well as quantitative biomarkers to prognosticate and predict the post-treatment visual acuity and CMT in DME, even before any intervention.

Financial support and sponsorship:

Nil.

Conflicts of interest:

There are no conflicts of interest.