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Dr. Manpreet Brar, B16870, Dr. Grewal S P S, Dr. Ajay Aurora, Dr. Dilraj S Grewal

Abstract:

Title: Correlation of Ganglion Cell Layer Damage and Diabetic Macular Ischemia in Diabetic Retinopathy

Aim: To assess ganglion cell layer thickness and foveal avascular zone using OCT Angiography in eyes with diabetes but no diabetic retinopathy (DR) in comparison to age-matched healthy subjects

Settings and Design: Retrospective case series

Material and Methods: Spectral-domain optical coherence tomography (SD-OCT) scans of patients with diabetes but clinically no signs of diabetic retinopathy (DR) was performed. A control group of 45 eyes matched healthy was included for comparison. OCTA scans (3 × 3 mm, Zeiss AngioPlex OCT angiography) centred on the fovea were acquired and superficial capillary plexus (SCP) and deep capillary plexus (DCP) FAZ horizontal and vertical greatest linear diameter were calculated. Ganglion cell layer (GCL) thickness, defined as the distance from the internal limiting membrane to the boundary of the outer inner plexiform layer, was generated from the Macular Cube 512 × 218 protocol centred on the fovea.

Statistical analysis: Spearman’s correlation test was used to compare GCL thickness, horizontal and vertical SCP FAZ (SCP FAZH and SCP FAZV) and DCP FAZ (DCP FAZH and DCP FAZV) between the eyes with no diabetic retinopathy and control group.

Results: Compared to healthy normal eyes, patients with Diabetes had thinner GCL (74.27 vs 82.29 microns, p=0.00038), larger SCP FAZ H (747.1 vs 639.71 microns, p<0.0001) and SCP FAZ V (704.05 vs. 610.18 microns, p<0.05) and larger DCP FAZ H (1038.16 vs. 971.47 microns, p< 0.05) and DCP FAZ V (1016.40 vs. 939.71 microns, p<0.05)

Conclusions: Early thinning of the inner retina and microvascular ischemic changes happens in Type 2 diabetes, even before clinically visible vascular signs of Diabetic Retinopathyappear.

Introduction:Diabetic retinopathy (DR) is the leading cause of blindness and visual impairment in adults of working age with an increase in affected individuals predicted [1].

DR isclinicallycharacterizedbytheobservationofapparent microvascular lesions (e.g. micro aneurysms and hard exudates). However, apparent microvascular lesions do not timely reflect retinal microvascular damage because insidious vascular changes as reflected by vesselcalibrealterationswouldhavedevelopedpriorto the incidence and progression of DR. [2-4]. Furthermore, experimental studies have extensively shown that retinal ganglion cells (RGCs) are damaged in diabetes suggesting that DR also has a significant neuronal component underlying its pathogenesis. [5,6]

To evaluate early vascular damage,measurement of FAZ has been studies in the past using FFA [7]. However, FFA being an invasive imaging technique, it has been associated with rare but life-threatening complications such as anaphylaxis and cardiac arrest [8]. Spa ide et al. demonstrated in 2015 that OCTangiography could image all layers of the retinal vasculature in a non-invasive manner and its use to measure FAZ has been extensively studied now. [9,10]

Advancesintheanalysisofopticalcoherencetomography (OCT) derived images with newly developed algorithms have enabled objective quantification of structuralRGClossatspecificinnerlayersofthe retinal layers. [11].

With regard to the age of affected individuals and the predictedincreaseofDRinthepopulation,earlydetectionplaysa pivotal role in the treatment of DR. With theadvent ofnon-invasive imaging modalities currently used it has become possible to detect differences in foveal avascular zone amongst diabetics and also to study automated GCL loss using SD-OCTbefore clinical signs and symptoms appear.Thus,it is of great interest to determine whether FAZ measurements on OCT angiography correlate with neurological degeneration as measured by ganglion cell loss before clinically apparent changes of DR are seen and therefore might be helpful for the early recognition and grading of DR.

Methods:

A retrospective cross-sectional study was performed with enrolment of two groups: (A) control patients without diabetes; (B) patients with type 2 diabetes with no clinical or angiographically diagnosed DR. Patients were examined in our institute after that had signed an informed consent. IRB approval was taken for the project and the study was conducted according to Declaration of Helsinki.

Control subjects did not have a diagnosis of diabetes or any retinal disease. These subjects were randomly recruited from cataract unit or other eye of patient with unilateral retinal disease not linked to diabetes.

The inclusion criteria were individuals with type 2 DM and above the age of 40 years.No Diabetic retinopathy was considered as the absence of any micro aneurysm in the retina and no other diabetic lesions according to the classification of the international clinical diabetic retinopathy disease severity scale [12]. Patients were excluded from the study when OCT images were of inadequate quality (signal strength below 7), if DR equal or worse than moderate, lens opacity and other vision impairing diseases such as glaucoma, cataract, uveitis, or macular degeneration.

All patient’s medical history records were reviewed and the following variables were collected: Demographics(age,gender)time of diabetes and best corrected visual acuity.

Imaging:

After pupil dilation using tropicamide 1% and phenylephrine hydrochloride 2.5%, retinal scanning was performed using Cirrus HD-OCT (Carl Zeiss Meditec, Dublin, CA.) to obtain measurements of the ganglion cell-inner plexiform layer (GC-IPL) Cirrus HD-OCT is a commercially available spectral-domain OCT device with a scan speed of 27,000 axial scans per second and axial resolution of 5μm.[13] One macular scan was acquired using the Macular Cube 512X128 scan protocol where 6 x 6mm area centred on the fovea was scanned with 128 horizontal B-scans, each consisting of 512 A-scans per B-scan (total of 65536 sampled points) within a scan time of 2.4s in each eye.[14] The automated Ganglion Cell Analysis algorithm, incorporatedinCirrusHD-OCTsoftwareversion 6.0, was used to demarcate and measure thicknesses of  GC-IPL.Themeasurementswereobtained within an elliptical annulus centred on the fovea based on the three-dimensional data generated from the Macular Cube 512X128 scan protocol.  The Ganglion Cell Analysis algorithm measuredthicknessesoftheGCL-IPL of eight areas for each scan, determined by ETDRS grid: nasal, superior, temporal, inferior, nasal superior, nasal inferior, temporal superior and temporal inferior. Thicknesses were calculated as the distance between two segmented hyper reflectiveintraretinallayers; GC-IPL thickness, which is the distance between outer boundaries of the RNFL and the inner plexiform layer.

OCT angiography scans were captured using the same machine. The images were captured with the standard macula protocol with a resolution of 3 mm×3mm. For all measurements, the automated segmentation with the preset settings for the superficial vascular layer and deep vascular layer was utilized. Hereby, the upper border of the superficial vascular layer was defined as 3 μm below the internal limiting membrane(ILM)and the lower border as15μm below the inner plexiform layer (IPL). For the deep vascular layer, the borders were defined as 15 μm and 70 μm below the IPL, respectively.Images with motion artefact, centration error, algorithm segmentation error or signal strength of less than six were excluded from the analysis. Manual calipers inbuilt in the machine was used to measure greatest linear dimension in the horizontal and vertical axis. Measurements were manually done by single retina specialist.FAZ dimensions were measured on both superficial and deep retinal plexus.

Statistical analysis:

Analysis of variance test was used to compare age, duration of diabetes, GCL thickness, horizontal and vertical SCP FAZ (SCP FAZH and SCP FAZV) and DCP FAZ (DCP FAZH and DCP FAZV) between eyes with diabetes but no diabetic retinopathy and control group. Spearman’s correlation test (ρ) was used to study the correlation between FAZ size and GCL thickness.

Results:A total of 86 eyes of type 2 diabetic patients who did not have any signs of DR (no-DR group)were enrolled. There were 56 males, 30 females; mean age 64±8.3 years, range 31–83 years).A control group consisting of 45 subjects, homogenous for age and sex (35males, 10 females; mean age 56 ± 8.2 years, range 25–80 years), was selected for statistical comparisons.Patients affected by diabetes reported a mean duration of disease of 8.74±6.6 years.

The average and minimum GC-IPL thickness was 74.27±13.31 and 65.98±19.24 in diabetic patients.In control group,average and minimum GCL was82.29±8.72 78.89±9.82respectively. This difference in diabetic and control group was found to be statistically significant (p<0.05)(Table 1). In addition, in all quadrants, the mean GC-IPL thickness was lower in the diabetic patients as compared to controls but this difference was not statistically different across all regions. (Table 1).

Results of FAZ measurements: Mean value of Horizontal and Vertical Greatest linear dimension of Foveal avascular zone measured in the superficial capillary plexus(SCP) was found to be747.1 microns and 704.05microns respectively in the diabetic group. Similar measurement in control group; SCP-Horizontal was 639.71 microns and SCP vertical was 610.18microns. This difference in FAZ size was found to be statistically significant (p<0.0001, p<0.03) in the diabetic versus control group.

Furthermore, Foveal avascular zone measured in the deep capillary plexus(DCP) was alsofound to be significantlylarger in diabetics as compared to controls. Measurements of FAZ horizontal was 1038.16 vs. 971.47 micronsin diabetic and controls respectively and vertical measurement of FAZ in DCP was 1016.40 vs. 939.71 microns. This difference in FAZ size was found to be statistically significant.

The spearman’s correlation test showed that among all patients, there was a significant correlation of GCL thickness with SCP FAZ horizontal (ρ=-0.204, p=0.009) and vertical (ρ=-0.292, p<0.001), and DCP FAZ horizontal (ρ=-0.233, p=0.003) and vertical (ρ=-0.256, p=0.001).

Discussion:In this cross-sectional study using SD-OCT Angiography we investigated the GC-IPL thickness values and size of foveal avascular zone in asymptomatic type 2 diabetic patients with no NPDR, without diabetic macular oedema.

Overall, the current analysis revealed a significant reduction of the mean GC-IPL thickness in type 2 diabetic patients compared with normal healthy control group.Several other studies in the past had similar finding supporting the theory that early neuroretinal degeneration happens in diabetic patients. [15] Moreover, in type 2 diabetic patients, neuroretinal alterations are supported both by a retinal function test, electroretinogram or microperimetry and neuroretinal histological evaluation. [16-19]In contrast to our results, Van Dijk HW et al [20] showed no significant decrease in the GC-IPL and RNFL thickness values in patients affected by type 2 diabetes and without any signs of DR. However, this is probably secondary to the different SD-OCT types used (Cirrus and Topcon in ourand VanDijk HW’s study, respectively).It has been hypothesized that chronic hyperglycaemia, even without clinically detectable microvascular complications, can negatively affect RGCs, leading to the functional impairment and death of RGCs and, consequently, a reduction of GC-IPL thickness. These are suggested by Barber et al, [6] who showed increased apoptosis of retinal neural cells both in experimental diabetic rats and in diabetic patients. Increased apoptosis is probably due to the following: (a) neurofilament accumulation in RGC axons, related to changes in retrograde axonal transport [21] (b) elevated levels of glutamate; (c) increasing neurotoxic factors,[22] and (d) reactive changes in microglia. [1]

We also found significant correlation between GC thickness and FAZ size indicating that early neuroretinal degeneration is well correlated with earliest sign of microvascular damage as well. Enlargement of FAZ indicates that there are anatomical changes of the inner retinal microenvironment as demonstrated by OCT angiography that can be seen before clinically apparent DR changes develop and they are well correlated with the Changes in OCT parameters of the inner neuronal layers of the retina. In diabetic retinae of animal models, glial cells (prominently at the RNFL) and RGCs have shown an increased expression of vascular endothelial growth factor (VEGF).[23] As a result, the excessive level of VEGF promotes breakdown of the blood–retinal barrier, and thus allows entry of circulatory harmful agents into the neuronal retina.[24] Furthermore, the toxic effects of hyperglycemia, which arise from severalmetabolicpathways(e.g. ProteinkinaseCandformation of advanced glycation end products), not only causecirculatorydisturbancetotheretinalmicrovasculature but also enhance the production of reactive oxygen species leading to oxidative damage of retinal neurons.[25]Takentogether,metabolicfunctionsofboth retinal glia and neurons are altered early in DR progression. It is plausible that altered glial function affects the integrity of both the neuronal and vascular elements of the retina.[19]Consequently, this disturbs thecloseinteractionbetweenneuronalactivityandretinal blood flow, and hinders the homeostasis required for normal retinal function.[26] Therefore, maintaining retinal glia and neuronal functions may normalize retinal circulation to prevent or delay DR progression.[27]We hypothesize thatmechanisms of neuroretinal degeneration and  microvascular changes might be pathologically linked. Further studies are necessary to understand whether ganglion cell neuroretinal degeneration and microvascular damages are pathogenically linked and whether treatment targeted to prevent such damages can prevent development of DR.

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