With the introduction of enhanced depth imaging optical coherence tomography (EDI-OCT) technology for evaluating choroidal anatomy,1 its use in measuring choroidal thickness (CT) has been widely adopted for investigating the in vivo anatomy of normal choroids and choroids in ocular pathologies. As EDI-OCT is increasingly being used in evaluating choroidal structure, the question arose as to whether CT measurement could be affected by mydriatic use. Imaging of the choroid has been performed under different pupil conditions in either a dilated state,2 an undilated state,1,3 or a combination thereof,4 and there remains a lack of data on the potential influence of mydriatics on CT measurement. The effect of mydriatics has been explored in the fields of glaucoma and refractive surgeries with regard to the measurement of retinal nerve fiber layer thickness5 – 8 and the evaluation of the anterior segment using spectral-domain OCT.9 However, the potential influence of mydriatics on the measurement of choroidal parameters using EDI-OCT has not been examined.
Mydriatic application, depending on the specific chemical components and their concentrations, was reported to bring about changes in iris thickness, angle, and morphology of the iridociliary region.9 The choroid, being part of the uveal structure, could also be affected by the contraction and relaxation of ciliary muscles induced by the instillation of combined sympathomimetic and parasympatholytic agents that comprise commercially available mydriatics. Therefore, the issue of whether the cycloplegic effect of mydriatics need be taken into account is of clinical significance in the era of choroidal imaging, and it is important to identify clinically significant CT measurement variability that may potentially be induced by mydriatic use.
In view of the growing interest in the research of choroidal structures, we investigated the influence of mydriatics on CT measurement in healthy subjects using EDI-OCT.
SUBJECTS AND METHODS
Thirty-eight healthy subjects were initially recruited into this randomized, double-blind, paired-eye study between February 2011 and May 2011. Subjects with any history of ophthalmic disease were excluded. All subjects underwent a screening process involving a complete ophthalmologic examination, including fundus examination, to check for the presence of any ocular diseases, and their best-corrected visual acuity, intraocular pressures, and refractive errors were recorded. The spherical equivalent of refractive error was measured by manifest refraction, and the axial length was measured with the IOL Master (Carl Zeiss Meditec, Dublin, CA). Subjects were required to have a best corrected visual acuity of 20/40 or better, refractive error less than −8 diopters, and 3 diopters of cylinder. Subjects with pathologic myopia or whose acquired OCT images were of poor quality, including an indistinct chorioscleral interface, were subsequently excluded from the study. Fifty-eight eyes of 29 subjects satisfying the inclusion criteria were finally enrolled in the study. Both eyes were enrolled in the study, and one eye of each subject was designated randomly as experimental and the contralateral eye as control. Patients and examiners were masked as to which eye received mydriatics vs. saline, thus following a double-blinded protocol. A single drop of Mydrin-P (tropicamide 5 mg/mL and phenylephrine 5 mg/mL, Santen Pharmaceuticals, Japan) or a single drop of unpreserved saline was administered in the respective eyes of subjects three times at 5 min intervals. Immediately after the instillation of eye drops, patients were asked to keep their eyes closed for approximately 1 min. Pupil size >6 mm was considered as clinically effective pupillary dilation and deemed acceptable for examination.10
This study was performed in accordance with the tenets of the Declaration of Helsinki and with the approval of our institutional review/ethics boards. Informed consent was obtained from all participants before study enrollment at the Yonsei University Eye and ENT Hospital Vitreoretinal Service (Seoul, Korea).
Image Acquisition Method
The method of obtaining EDI-OCT has been described previously.1 Briefly, a spectral-domain OCT was placed close enough to the eye to obtain an image. The EDI option allowed the chorioretinal interface to be placed adjacent to the zero delay, and an upright image of the retina and choroid was obtained. All subjects were imaged by the same experienced retina specialist (M.K.). Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany) with software version 5.3 was used. It uses an 870-nm wavelength superluminescent diode and is capable of obtaining 40,000 A scans/second with an axial resolution of 7 μm and transversal resolution of 14 μm. Two high-quality horizontal and two vertical line scans passing through the fovea were obtained within a 5 × 30-degree area at the fovea, in which 100 scans were averaged for each section. The signal-to-noise ratio was maximized using the automatic real-time averaging mode to ensure high-quality images. EDI-OCT images of each subject were obtained before the drug instillation and 30 min after drug instillation in both experimental and control eyes. Among the two horizontal and two vertical line scans acquired, the higher quality image scan for each was chosen for analysis in cases with noise or an obscure chorioscleral interface, if any. If not, one horizontal and one vertical line scan were randomly chosen for analysis. Pupil size >6.0 mm diameter in eyes administered with mydriatics was checked in all subjects to ensure that subjects who might show a delayed response to mydriatics would undergo an OCT examination under similar conditions.
CT was defined as the distance from the outer border of the hyper-reflective line corresponding to the retinal pigment epithelium (RPE) perpendicular to the chorioscleral interface. Using the manual calipers provided by the software, CT was measured at the subfoveal region and at 0.5 mm intervals (up to 3.0 mm) from the fovea at nasal, temporal, superior, and inferior locations in OCT images obtained before and after eye drop instillation (Figure 2). A magnification of at least 100% was used to place the measurement line precisely at the outermost RPE layer and at the chorioscleral interface. Physicians measuring CT were masked as to which eyes received mydriatics. Scans were performed around the same time of the day (1:00 p.m.) to minimize the possibility of CT changes attributable to diurnal CT fluctuations.11 Two independent observers (M.K. and H.J.K.) measured CT, and these measurements were averaged for analysis. They were masked to each other's measurement and were allowed to adjust the contrast of the image to better delineate choroidal boundaries.
Results are presented as mean ± standard deviation. SAS software version 9.2 was used for statistical analysis (SAS Institute Inc., Cary, NC). For evaluation of intraobserver and interobserver agreement, intraclass correlation coefficient (ICC) was calculated. Because this study used a repeated measures design with each eye evaluated at two time points, the data were analyzed using a linear mixed model for repeated measures. Unstructured symmetry was assumed, and random components were assumed to be uncorrelated. CT at each location before and after the administration of eye drops, and CT changes between the two groups were compared for statistical significance using a linear mixed model, and p values <0.05 were considered statistically significant.
A total of 58 eyes of 29 subjects satisfying the inclusion criteria were included in the study, and patient demographics and ocular characteristics are summarized in Table 1. Ocular parameters between eyes in the mydriatics (n = 29) and placebo groups (n = 29) were similar and showed no significant differences (Table 1). In the mydriatics group, there was a statistically significant difference in the spherical equivalent before and after the administration of mydriatics (−2.16 ± 2.01 and −1.76 ± 2.06, respectively; p < 0.0001, paired t-test).
A summary of CTs measured at the fovea and at 0.5 mm intervals (up to 3.0 mm) from the fovea at nasal, temporal, superior, and inferior locations for both groups is shown in Table 2. CT measurements were slightly increased or decreased after the use of mydriatics, but these differences did not attain statistical significance (p > 0.05 for all comparisons, linear mixed model). In both the horizontal and vertical sections (Table 2) from OCT passing through the fovea, no significant CT differences were observed at any of the measurement points in either the mydriatics or placebo groups, before and after eye drop administration. Furthermore, when changes of CTs in the mydriatic group were compared with those in the placebo group, no statistically significant differences were observed (p > 0.05 for all comparisons, linear mixed model). Fig. 1 depicts CTs measured before and after eye drop instillation in both groups, neither of which showed clinically significant CT changes at any of the locations measured (Fig. 1A, mydriatics group; Fig. 1B, placebo group).
There was an excellent agreement in CT measured between the two independent observers with a mean ICC of 0.989. The difference in CT measurement between the observers ranged from 0 to 19 μm with a mean value of 2.2 ± 13.4 μm for all measured points. Intraobserver agreement with a mean ICC of 0.995 for every measured locations also showed good agreement within the observers.
Overall, these results indicate that CT measurements obtained before and after the use of mydriatics are not statistically different from each other.
We hypothesized that mydriatic use could influence CT measurements evaluated using EDI-OCT. The main finding of the present study indicates that mydriatics (Mydrin-P) do not significantly affect CT measurement by EDI-OCT. As expected, mydriatics induced cycloplegia as manifested by changes in refractive status and pupillary dilation. However, contrary to our hypothesis, no significant CT changes were observed.
The exact mechanisms behind CT changes currently remain unknown.12 However, possible mechanisms by which mydriatics could influence CT measurement include the direct influence of mydriatic agents on the uveal structures. Mydriatics regularly used in current ophthalmic practice frequently contain a combination of tropicamide and phenylephrine to achieve dilation for fundus examination. Phenylephrine is a sympathetic agonist which exerts its mydriatic effect by activating sympathetic receptors on the iris dilator muscle. Tropicamide, on the other hand, is an anticholinergic agent that enhances pupil dilation by inhibiting iris sphincter muscle action. Both of these mydriatic components also exert a cycloplegic effect on the ciliary muscle. The possible cause of hyperopic shift observed in our study after the instillation of Mydrin-P may be ascribed to the action of both phenylephrine and tropicamide on ciliary muscle. The sympathomimetic action of phenylephrine and the parasympatholytic action of tropicamide would both cause ciliary muscle relaxation, which could subsequently lead to an increase in the tension of the zonular fibers connected to the lens, thereby flattening the lens. This could cause hyperopic shift in refraction. Because mydriatics result in ciliary muscle relaxation, and the connection of non-vascular smooth muscle cells beneath the fovea to ciliary muscle fibers has also been suggested previously,12 one can speculate that mydriatic action can potentially cause CT changes. Furthermore, evidence for a muscular mechanism in modulating CT has been suggested in many studies previously; the choroid contains non-vascular smooth muscle, and these tissues are thought to be innervated by both sympathetic and parasympathetic inputs.13 – 15 Although topical mydriatic administration is thought to have a localized effect on the iris and ciliary body, the contiguity of these structures with the anterior and posterior choroid forming the uveal tract suggests a potential role of mydriatics in the contraction of non-vascular smooth muscle in the choroid. Contraction of these muscles might result in the efflux of fluid out of the choroid, thereby thinning it.12 Another possible mechanism for mydriatic-induced CT change involves the role in which choroidal blood flow and lymphatics play in the modulation of CT. Parasympathetic fibers terminating on perivascular plexuses control blood flow by vasodilation,12 and animal studies suggested a possible role of the fluid-filled lacunae in the choroidal stroma as a drainage reservoir.16 Choroidal lymphatics have been demonstrated to exist in humans, and histologic reports have shown that the choroid contains many large membrane-lined lacunae that can dramatically change their volume and thus alter CT by up to four-fold in primates.17,18
Surprisingly, the results from the present study were found to contrast our initial hypothesis; CTs did not significantly change with the administration of mydriatic agents. It is possible that CT changes caused by mydriatics (Mydrin-P) are opposed by other mechanisms that influence CT. Given that the alignment of the non-vascular smooth muscle is not perpendicular to the plane of the choroid, it is also possible that contraction of these muscles simultaneously modulates both the filling and the drainage of the lacunae.12 Currently, we can only speculate that there might be a very intricate interplay among choroidal non-vascular smooth muscle, choroidal blood flow, and choroidal lymphatics in modulating CT, and the proposed potential effect of mydriatics (Mydrin-P) or lack thereof on CT might not be sufficiently explained by any single proposed mechanism. Thus, any explanations for the lack of effects from mydriatic agents (Mydrin-P) on CT are currently speculative. Despite the lack of any statistically significant effect of mydriatics (Mydrin-P) on CT measurement with EDI-OCT, consistency in mydriatic use is recommended in the setting of clinical studies or longitudinal follow-up of patients with choroidal disorders that require CT evaluation.
There are some limitations in the study. First, only one type of commercially available mydriatic (Mydrin-P) was used, and the possible effects of other types of mydriatics still remain to be evaluated. Mydriatics containing different concentrations of sympathomimetic and parasympatholytic agents could have different effects on CT; i.e., the concentrations used in the study might not have been sufficient enough to induce choroidal structural changes that could be detected with EDI-OCT. We reduced this possibility by administering eye drops three times to compensate for any possible loss of medication through the punctum into the conjunctival sac, and by ensuring that pupils were uniformly dilated to >6 mm in diameter. Subjects enrolled in this study were mainly of Asian ethnicity, and because the effect of mydriatics might differ among diverse ethnic populations, this study should be expanded to include non-Asian participants. Currently, because there is no reliable automated segmentation software available to delineate choroidal boundaries yet, our measurements were taken manually. Beacuse only the posterior 7 mm section of the choroid was included in the analysis, the possibilities of changes in CT at other locations of the choroid remain to be evaluated. Examination of anterior choroidal structures might reveal thickness changes not detected in the current study. Also, our findings may not demonstrate the effect of mydriatics on patients with choroidal diseases in which these agents might display different degrees of mydriasis and cycloplegia, dependent on specific pathologies.
The strengths of the study are that eyes were randomized as to which eye received mydriatics and saline in the paired-eye study design, and that the subjects and the examiners followed a double-blind study protocol. Also, rather than just examining the CT at subfoveal area, to better assess any potential CT changes induced by mydriatics in the macular region, CTs were measured at a number of points distant from the fovea, in both horizontal and vertical planes.
In conclusion, this study shows that mydriatics (Mydrin-P) did not result in significant CT changes, suggesting that the use of Mydrin-P does not complicate or alter CT evaluation using Spectralis EDI-OCT technology; however, the limitations identified within this study should be considered when using EDI-OCT to diagnose and investigate retinal disorders involving the choroid. Further studies are warranted to investigate the influence of mydriatics in patients of different ethnicity, chorioretinal pathologies, and the comparative effects of mydriatics with different concentration and combination of drugs on CT measurement.
Sung Chul Lee
Institute of Vision Research
Department of Ophthalmology, Severance Eye and ENT Hospital,
Yonsei University College of Medicine
134 Shinchon-dong, Seodaemun-gu, Seoul
The authors would like to thank Kim Myo Jung in the Department of Biostatistics for assistance. The authors have no proprietary or commercial interest in any materials discussed in this article.
1. Spaide RF, Koizumi H, Pozzoni MC. Enhanced depth imaging spectral-domain optical coherence tomography. Am J Ophthalmol 2008;146:496–500.
2. Hirata M, Tsujikawa A, Matsumoto A, Hangai M, Ooto S, Yamashiro K, Akiba M, Yoshimura N. Macular choroidal thickness and volume in normal subjects measured by swept-source optical coherence tomography. Invest Ophthalmol Vis Sci 2011;52:4971–8.
3. Ikuno Y, Kawaguchi K, Nouchi T, Yasuno Y. Choroidal thickness in healthy Japanese subjects. Invest Ophthalmol Vis Sci 2010;51:2173–6.
4. Manjunath V, Taha M, Fujimoto JG, Duker JS. Choroidal thickness in normal eyes measured using Cirrus HD optical coherence tomography. Am J Ophthalmol 2010;150:325–9.
5. Hoh ST, Greenfield DS, Liebmann JM, Hillenkamp J, Ishikawa H, Mistlberger A, Lim AS, Ritch R. Effect of pupillary dilation on retinal nerve fiber layer thickness as measured by scanning laser polarimetry in eyes with and without cataract. J Glaucoma 1999;8:15907–63.
6. Massa GC, Vidotti VG, Cremasco F, Lupinacci AP, Costa VP. Influence of pupil dilation on retinal nerve fibre layer measurements with spectral domain OCT. Eye (Lond) 2010;24:1498–502.
7. Savini G, Carbonelli M, Parisi V, Barboni P. Effect of pupil dilation on retinal nerve fibre layer thickness measurements and their repeatability with Cirrus HD-OCT. Eye (Lond) 2010;24:1503–8.
8. Serbecic N, Beutelspacher SC, Aboul-Enein FC, Kircher K, Reitner A, Schmidt-Erfurth U. Reproducibility of high-resolution optical coherence tomography measurements of the nerve fibre layer with the new Heidelberg Spectralis optical coherence tomography. Br J Ophthalmol 2011;95:804–10.
9. Marchini G, Babighian S, Tosi R, Perfetti S, Bonomi L. Comparative study of the effects of 2% ibopamine, 10% phenylephrine, and 1% tropicamide on the anterior segment. Invest Ophthalmol Vis Sci 2003;44:281–9.
10. Bartlett JD, Jaanus SD. Clinical Ocular Pharmacology, 5th ed. St. Louis, MO: Butterworth-Heinemann/Elsevier; 2008.
11. Chakraborty R, Read SA, Collins MJ. Diurnal variations in axial length, choroidal thickness, intraocular pressure, and ocular biometrics. Invest Ophthalmol Vis Sci 2011;52:5121–9.
12. Nickla DL, Wallman J. The multifunctional choroid. Prog Retin Eye Res 2010;29:144–68.
13. Schrodl F, De Laet A, Tassignon MJ, Van Bogaert PP, Brehmer A, Neuhuber WL, Timmermans JP. Intrinsic choroidal neurons in the human eye: projections, targets, and basic electrophysiological data. Invest Ophthalmol Vis Sci 2003;44:3705–12.
14. Schrodl F, Tines R, Brehmer A, Neuhuber WL. Intrinsic choroidal neurons in the duck eye receive sympathetic input: anatomical evidence for adrenergic modulation of nitrergic functions in the choroid. Cell Tissue Res 2001;304:175–84.
15. May CA, Neuhuber W, Lutjen-Drecoll E. Immunohistochemical classification and functional morphology of human choroidal ganglion cells. Invest Ophthalmol Vis Sci 2004;45:361–7.
16. Meriney SD, Pilar G. Cholinergic innervation of the smooth muscle cells in the choroid coat of the chick eye and its development. J Neurosci 1987;7:3827–39.
17. Poukens V, Glasgow BJ, Demer JL. Nonvascular contractile cells in sclera and choroid of humans and monkeys. Invest Ophthalmol Vis Sci 1998;39:1765–74.
18. Yucel YH, Johnston MG, Ly T, Patel M, Drake B, Gumus E, Fraenkl SA, Moore S, Tobbia D, Armstrong D, Horvath E, Gupta N. Identification of lymphatics in the ciliary body of the human eye: a novel “uveolymphatic” outflow pathway. Exp Eye Res 2009;89:810–9.