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Dr.Chaitali Patel, 

Dr.Sayan Basu

Chaitali N Patel, MS1,2; Sayan Basu, MS1,2

1TejKohli Cornea Institute, L V Prasad Eye Institute, Hyderabad-500034, India

2Prof. Brien Holden Eye Research Center, L V Prasad Eye Institute, Hyderabad-500034, India

Short Title: Ocular Surface Angiography

Key Words: Optical Coherence Tomography (OCT); Angiography; Imaging; Ocular Surface; Ocular Surface Vasculature

Financial Disclosures: None of the authors have any commercial interests related to materials or techniques described in this study to disclose.

Conflicts of Interest: None of the authors have any conflicts of interest to disclose.

Author’s Contribution: All authors contributed equally to this manuscript.

Correspondence:

Sayan Basu, MBBS, MS

Room # 606A, Sixth Floor, KallamAnji Reddy Campus, L V Prasad Eye Institute, Hyderabad-500034, India

Email: sayanbasu@lvpei.org; Phone: +91-40-30612625; Fax: +91-40-23548271

ABSTRACT

Purpose:

To use optical coherence tomography angiography (OCTA) in determining the optimal timing and dosage of topical vasoconstrictor agents.

Design:

Prospectiveobservational study.

Participants:

This study included 370 angiograms of15 normal eyes of healthy volunteers and 10 eyes of 10 patients with nasal pterygiums.

Methods:

Standardized OCTA scans were performed using a commercially available split-spectrum amplitude-decorrelation angiography system.Superior, inferior, nasal and temporal quadrants of the bulbar surface were imaged with the patient looking in the opposite gaze, respectively.Post-processing of the images to quantify the area occupied by the blood vessels was performed using image-J software. The reliability of the algorithm was tested by evaluating the intra- and inter-observer variability amongst two technicians. Serial OCTA scans were then performed before and after instillation of vasoconstricting eye drops. The accuracy of the model was tested by comparing the temporal change in amount of vasoconstriction in healthy eyes as compared to eyes with primary nasal pterygiums.

Outcome Measures:

The main outcome measures were reliability of OSA and its accuracy in detecting relative change in the area occupied by the blood vessels after instillation of vasoconstrictor eye drops. To calculate the dosage and timing of the vasoconstrictor drops.

Results:

The intra-class correlation coefficient for intra and inter-observer agreement was noted to be 0.91 and 0.88 and respectively. Significant vasoconstriction was noted at 5 mins (13-15%) from baseline, which peaked at 10 minutes (14-17%) and was sustained until 20 minutes followed by gradual recovery. The degree of vasoconstriction was greatest with a combination of 5% phenylephrine and 0.15% brimonidine tartrate (BT) eye drops than either 1 or 2 drops of BT alone, both at 10 (p=0.0058) and 20-minute (p=0.0375) time-points. This temporal trend was replicated in eyes with primary nasal pterygium (p=0.31).

Conclusions:

The findings suggest that OSA using OCTA is a reliable and accurate technique of objectively quantifying the relative change in ocular surface vasculature. Optimal vasoconstriction during ocular surface surgery can be achieved by using a combination of 0.15% BT and 5% PE eye drops 10 minutes prior to the procedure.

Cornea being an avascular structure offers a bloodless field during various corneal procedures, however ocular surface procedures do not present the same scenario. Complete meticulous hemostasis is recommended to optimize surgical outcome. Traditionally it has been achieved by cauterization, suction drain or simple compression.

The conjunctival blanching effect of various topical alpha receptor agonists like brimonidine 0.15% and phenylephrine 5% has been well observed.1-2OCTA has recently been used as a tool for visualizing the presence of ocular blood flow in anterior segment and surface vessels without the use of dyes, such as fluorescein or indocyanine green.3In this study, we aimed to objectively test the reliability of OSA using serial OCTA imaging to detect a relative change in the caliber of the ocular surface vasculature after the use of topical vasoconstrictor eye drops in normal healthy eyes and eyes with pterygium.We also aim to document and quantify vasoconstriction in ocular surface vasculature using OCTA to effectively decide the optimal timing and dosage for the aforementioned drugs.

MATERIAL AND METHODS

Study design, duration and participants:This was a prospectivecomparative imaging study conducted at the TejKohli Cornea Institute, L V Prasad Eye Institute Hyderabad, India between 1st January 2018 and 30th April 2018. The study adhered to the tenets of the Declaration of Helsinki and local institutional review board approval was obtained prior to commencement of the study. Normal healthy volunteers and patients with primary nasal pterygium in at least one eye were eligible to be included in the study. Children, those unwilling to have drops put in their eyes and those who were not cooperative for imaging were excluded. All study participants underwent a complete slit-lamp biomicroscopic examination performed by a trained ophthalmologist for validation of their eligibility and patients with ocular co-morbidities were excluded.

Technique of OCTA imaging of the ocular surface:Ocular surface angiography imaging was performed by a technician using the AngioVue OCTA system (Optovue, Inc., Fremont, CA) in all four limbal quadrants (superior, inferior, nasal and temporal) with adjoining bulbar conjunctiva.The anterior segment optical adaptor lens was used to image the ocular surface vasculature using the split-spectrum amplitude-decorrelation angiography (SSADA) algorithm on the OCTA system originally intended for retinal imaging (HD AngioRetina 6mm mode, software version 2017 1.0). Since the auto scan selection system is for retinal OCTA, the autofocus along with follow up and tracking was turned off. Manual scan was chosen and the Z motor was set at 10.90, focus fixed at -15.00 and P motor left unchanged at 55 to optimize scan signal strength and image quality.

Image focus square was then manually centered while the subject was asked to look straight. All subjects looked at fixed targets and two images in each quadrant- Fast X and Fast Y were obtained by focusing on the limbus. Each scan was performed with axial resolution of 5 mm and a beam width of 22 mm with a light source emission at 840 nm. Scans were processed automatically to reduce motion artefacts like transverse saccadic and residual axial motion by the internal software (ReVue version 2017.1.0; Optovue, Inc.) The images were analyzed in en-face custom mode by setting the lower-RPE offset at 1200 and upper-RPE offset at -800. No Motion correction technology (MCT) was selected based on the scan quality obtained for each image. All images were exported, labeled and saved in the tagged image file format (TIFF).

Post-acquisition image processing:

Fiji (National Institutes of Health, Bethesda, MD) was used for post-acquisition image analysis. An area of interest (AOI) was identified in each quadrant around a landmarked vascular segment by selecting a 250×250 mm square centered on the vessel (Figure 1). The same area of interest was manually selected in subsequent time scans of that quadrant in each group. All selected AOIs were cropped for analysis. The percentage surface area occupied by the vessels was calculated after making each AOI a binary image and adjusting the threshold. The horizontal band pass filter and despeckle filter was used to minimize the line and dot artefacts.

Primary Outcome Measures:

The primary outcome measures were reliability and accuracy of OSA in objectively quantifying the ocular surface vasculature. Reliability was defined as acceptable intra- and inter-observer variability and accuracy was defined as being able to detect a relative change in ocular surface vasculature after the instillation of different doses and combinations of topical vasoconstrictor eye drops.

Secondary Outcome Measures:

To calculate the dosage and timing of the vasoconstrictor drops was the secondary outcome measure.

Reliability of OSA:

The intra and inter observer variability was tested using 5 normal healthy volunteers and 2 technicians (AA and HK). Each technician took two scans of each quadrant of the two eyes of the volunteers. The images were saved and randomly numbered using a computer-generated sequence. An observer (CNP) who was blinded to the identity of both the technician and the subjects, processed and quantified the percentage surface area occupied by the vessels in each image. Inter-class correlation (ICC) coefficient was calculated to estimate the agreement between two separate scans by the same technician (intra-observer variability) and between the two technicians for the same subjects (inter-observer variability). Conventionally, ICC values less than 0.5 are indicative of poor reliability, values between 0.5 and 0.75 indicate moderate reliability, values between 0.75 and 0.9 indicate good reliability, and values greater than 0.90 indicate excellent reliability. 4

Accuracy of OSA:

The accuracy of OSA in detecting the relative change in ocular surface vasculature was tested in 10 eyes each of normal volunteers and 10 eyes of patients with primary nasal pterygium. In both groups the subjects received the following eye drops on three separate days: a) one drop of topical Brimonidine tartrate 0.1% w/v (Allergan, Inc. Irvine, CA 92612, U.S.A.); b) two drops of topical brimonidine tartrate 0.1% w/v 5 minutes apart; c) one drop each of topical brimonidine tartrate 0.1%w/v and topical phenylephrine 5% (Cipla Ltd. Mumbai, Maharashtra, India)5minutes apart. The OCTA imaging was performed at baseline before application of the eye drops and repeated at 5, 10, 20, 60, 120 and 180 minutes after final eye drop instillation (Figure 2).

Sample Size Calculation:

To calculate theinter-class coefficients for estimating intra- and inter-observer variability a minimum sample size of 20 scans per technician was required.4Assuming analpha error of 5% and a power of 80% to detect a 5% difference in the percentage area occupied by vessels in the different groups, a sample size of minimum of 9 eyes with 4 scans each (total 36 scans) was needed in each group.

Statistical analysis:

MedCalc (version 11.4.3.0, MedCalc Software, Mariakerke, Belgium) statistical software was used for data analysis. Kruskal-Wallis test was used to compare the difference in percentage surface area occupied by the vessels in each group at different time-points. The Wilcoxonsigned-rank test was used to compare the difference in percentage surface area occupied by the vessels in different groups at each time point.  A 2-tailed P value less than 0.05 was considered statistically significant.

RESULTS

Baseline Demographics: The study included 20 patients, which included 10 normal healthy volunteers and 10 patients with primary nasal pterygium. The normal group included 6females and 4 male subjects whose average age was 26.4±3.1 years. The pterygium included 10 female subjects whose average age was 55.1±7.8 years.

Reliability of OSA:

The intra-class correlation coefficient for intra-observer (between two scans of the same quadrant by the same technician) agreement was noted to be 0.91, indicating excellent reliability.There was no statistically significant difference in the agreement for each individual quadrant (p=0.73). The intra-class correlation coefficient for inter-observer agreement (between two scans of the same quadrant by different technicians) was noted to be 0.88, indicating good reliability. There was no statistically significant difference in the agreement for each individual quadrant (p=0.63).

Accuracy of OSA:

In normal eyes a similar trend was noted in all quadrants (Figure 3). The area occupied by vessels at baseline was28.2%, 30.3%, 35%, 39.8% in the superior, inferior, nasal and temporal quadrants respectively. There was a dramatic reduction at 5 mins after the application of vasoconstrictor eye drops (13-15%) from baseline, which peaked at 10 minutes (14-17%) and was sustained until 20 minutes followed by gradual recovery. The degree of vasoconstriction was greatest with a combination of 5% phenylephrine and 0.15% brimonidine tartrate (BT) eye drops than either 1 or 2 drops of BT alone, both at 10 (p=0.0058) and 20-minute (p=0.0375) time-points. This temporal trend was replicated in eyes with primary nasal pterygium (Figure 4).

DISCUSSION

The normal ocular surface has an intricate network of conjunctival and episcleral blood vessels, while the cornea is completely avascular.4Dilatation of these blood vessels leads to the characteristic appearance of a red eye, which is symptomatic of scleral, episcleral, conjunctival or intra-ocular inflammation. Similarly, appearance of blood vessels in the cornea is also suggestive of chronic inflammatory pathologies.5 Imaging of the ocular surface vasculature, therefore has several potential clinical applications.6,7 However, unlike the posterior segment, angiography of the ocular surface is not commonly performed. Fluorescein leaks through the fenestrated capillaries in the uninflamed conjunctiva and obscures visualization of deeper vessels.5 Although indocyanine green (ICG) is primarily protein-bound and remains within the vessels for a longer time, both ICG and fluorescein angiography are invasive techniques that carry the risk of severe adverse events.5

With the advent of optical coherence tomography-based angiography (OCT-A) platforms it has now become possible to perform non-invasive ocular angiography which obviates the risks associated with ICG and fluorescein angiography.6 This technology detects phase variations or changes in reflectivity to vascular flow and split-spectrum amplitude-decorrelation angiography (SSDA) to improve the signal-to-noise ratio.8 While OCT-A was primarily developed for imaging of the posterior segment, techniques of using the same platform for limbal and corneal imaging have recently been described.9,10 The OCTA system when optimized for anterior segment imaging was found to be comparable with ICG angiography for corneal vascularization.11However,

The reliability of OSA in detecting and quantifying the relative change in ocular surface vasculature has not yet been validated. We created a model of dynamic change in ocular surface vasculature by using topical alpha receptor agonists as vasoconstrictors, which have been clinically used to reduce bleeding in strabismus surgery.12,13Our study proved that OCTA imaging for anterior segment is repeatable and reliable. Our results objectively document the previous non-quantitative observations that 0.15% brimonidine tartrate and 5% phenylephrine cause conjunctival blanching effect by inducing vasoconstriction. Brimonidine tartrate 0.15% is a partially selective alpha-2 receptor agonist and 5% phenylephrine being mainly an alpha-1 receptor agonist. Hence, maximum vasoconstriction was obtained when both the drugs were combined due the action on both alpha receptors by individual drugs.

One of the important potential applications of ocular surface angiography (OSA) will be in detecting limbal and episcleral ischemia in cases of ocular burns and graft vascularization after conjunctival or limbal transplantation.14

 

CONTRIBUTORSHIP STATEMENT

The corresponding author states that authorship credit of this manuscript was based on 1) substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published. All listed authors met conditions 1, 2, and 3. All persons designated as authors qualify for authorship, and all those who qualify are listed. Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content. The first and second authors contributed equally to this study.

ACKNOWLEDGMENTS AND DISCLOSURES

This work was funded by the Hyderabad Eye Research Foundation, Hyderabad, India. The sponsoring organizations had no role in the design or conduct of this research.

None of the authors have any conflicts of interest or financial interests to disclose.

The study followed the tenets of the Declaration of Helsinki and informed written consent was obtained from all patients enrolled in this study. The study was approved prospectively by the Institutional Review Board (IRB) and the Ethics Committee, L V Prasad Eye Institute, Hyderabad, India.

REFERENCES

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FIGURE LEGENDS

Figure 1

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