A, The 15° × 5° rectangular area in the nasal limbus for 81 serial horizontal EDI OCT B-scans (box). B, Magnified horizontal EDI OCT B-scan with a clear visualization of the Schlemm canal (arrowhead). AC indicates anterior chamber.
A, The Schlemm canal (arrowhead) before administration of pilocarpine, 1%, to participant 2 (no glaucoma). B, The Schlemm canal (arrowhead) 1 hour after administration of pilocarpine, 1%, to participant 2. C, The Schlemm canal (arrowhead) before administration of pilocarpine, 1%, to participant 3 (no glaucoma). D, The Schlemm canal (arrowhead) 1 hour after administration of pilocarpine, 1%, to participant 3. AC indicates anterior chamber.
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Skaat A, Rosman MS, Chien JL, et al. Effect of Pilocarpine Hydrochloride on the Schlemm Canal in Healthy Eyes and Eyes With Open-Angle Glaucoma . JAMA Ophthalmol. 2016;134(9):976–981. doi:10.1001/jamaophthalmol.2016.1881
The in vivo effect of pilocarpine hydrochloride on the Schlemm canal may help explain its pharmacologic mechanism of action and better indicate its clinical use.
To investigate the effect of pilocarpine on the structure of the Schlemm canal in vivo in healthy eyes and eyes with glaucoma.
Design, Setting, and Participants
In this case-control study, healthy individuals and patients with open-angle glaucoma were prospectively enrolled between September 1, 2013, and June 30, 2014, after a complete ophthalmologic examination at a tertiary glaucoma referral practice. Eighty-one serial, horizontal, enhanced depth imaging optical coherence tomographic B-scans (interval between B-scans, approximately 35 µm) of the nasal corneoscleral limbus were performed before and 1 hour after topical administration of pilocarpine, 1%, in 1 eye of healthy volunteers and pilocarpine, 2%, in 1 eye of patients with glaucoma. Fifty B-scans in the overlapping area (circumferential length, approximately 1.7 mm) between the 2 sets of serial scans (before and after pilocarpine administration) were selected for analysis based on the structures of aqueous and blood vessels as landmarks. The cross-sectional area of the Schlemm canal was measured in each selected B-scan. Volume of the Schlemm canal was calculated using commercially available 3-dimensional reconstruction software.
Main Outcomes and Measures
Mean cross-sectional area of the Schlemm canal.
Enhanced depth imaging optical coherence tomographic scans of the Schlemm canal were performed successfully before and after administration of pilocarpine, 1%, in 9 healthy eyes (9 individuals) and pilocarpine, 2%, in 10 eyes with glaucoma (10 patients) (mean [SD] age, 31.9 [7.8] and 68.7 [13.2] years, respectively). Following pilocarpine administration, mean (SD) intraocular pressure decreased from 14.3 (1.3) to 13.7 (1.1) mm Hg in healthy eyes (P = .004) and from 17.5 (6.0) to 16.6 (6.1) mm Hg in eyes with glaucoma (P = .01). The mean (SD) cross-sectional area of the Schlemm canal increased by 21% (4667  to 5647  µm2) in healthy eyes (P < .001) and by 24% (3737  to 4619  µm2) in eyes with glaucoma (P < .001) (mean difference in percent increase, 2.2%; 95% CI, –8.5% to 12.9%). The mean (SD) volume of the Schlemm canal in the overlapping area increased from 8 004 000 (2 923 000) to 9 685 000 (3 277 000) µm3 in healthy eyes (P < .001) and from 6 468 000 (1 170 000) to 7 970 000 (1 199 000) µm3 in eyes with glaucoma (P < .001).
Conclusions and Relevance
These data suggest that pilocarpine expands the Schlemm canal in eyes with and without glaucoma. No differences in the effect were identified between the 2 groups. Enhanced depth imaging optical coherence tomography may be useful in investigating the effect of pharmacologic agents on the Schlemm canal.
Pilocarpine hydrochloride, a nonselective muscarinic receptor agonist, has been used in the treatment of glaucoma for more than 140 years1 owing to its ability to lower intraocular pressure by increasing facility of trabecular outflow.2 Pilocarpine binds and activates muscarinic M3 receptors on ciliary smooth muscle cells and stimulates contraction of the longitudinal ciliary muscle, which pulls on tendons terminating in the trabecular meshwork and inner wall of the Schlemm canal.3,4 Functional consequences of ciliary muscle contraction appear to include expansion of the juxtacanalicular portion of the trabecular meshwork and expansion of the Schlemm canal.5
Spectral-domain optical coherence tomography (OCT) provides high-resolution cross-sectional images of the trabecular outflow pathway in vivo and ex vivo, with details comparable to corrosion casting techniques.6-8 However, conventional spectral-domain OCT images of the Schlemm canal are often suboptimal, as this structure is located deep in the corneoscleral limbus and may often approach the limits of resolution of some anterior segment OCT devices. Enhanced depth imaging (EDI) OCT, a modification to the standard spectral-domain OCT technique, improves image quality of the deep-seated structures.9 Averaging of multiple OCT images to obtain a single image with the aid of an eye tracking function increases the signal to noise ratio. We applied the EDI method and averaging technology of spectral-domain OCT to image the Schlemm canal before and after administration of pilocarpine in healthy individuals and patients with glaucoma to assess the effect of pilocarpine on the anatomy of the Schlemm canal.
Question Does pilocarpine change the structure of the Schlemm canal in human eyes?
Findings In this case-control study, serial enhanced depth imaging optical coherence tomography B-scans of the corneoscleral limbus were performed before and after administration of pilocarpine, 1%, in healthy eyes and pilocarpine, 2%, in eyes with glaucoma. Following administration of pilocarpine, the mean cross-sectional area of the Schlemm canal increased by 21% in healthy eyes and by 24% in eyes with glaucoma.
Meaning These findings help in understanding pilocarpine’s pharmacologic mechanism of action and better indicate its clinical use.
We recruited healthy individuals and patients with various severities of open-angle glaucoma (primary open-angle glaucoma, exfoliation glaucoma, and pigmentary glaucoma) from the glaucoma referral practices at Glaucoma Associates of New York between September 1, 2013, and June 30, 2014. Patients with glaucoma who were using multiple topical antiglaucoma medications except for pilocarpine but who needed further reduction of intraocular pressure were recruited. All participants provided a detailed medical and ocular history and underwent slit lamp biomicroscopy, Goldmann applanation tonometry, gonioscopy, dilated fundus examination, stereo disc photography (Stereo Camera 3-DX; Nidek, Inc), and standard automated perimetry (Humphrey Visual Field Analyzer, 24–2 Swedish interactive threshold algorithm standard strategy; Carl Zeiss Meditec, Inc). This study was approved by the New York Eye and Ear Infirmary of Mount Sinai Institutional Review Board. Written informed consent was obtained from all participants, and the study adhered to the tenets of the Declaration of Helsinki.
Healthy individuals were recruited randomly from spouses and friends of patients with glaucoma at the glaucoma referral practices. They were required to have normal-appearing anterior segments, open iridocorneal angles, clinically normal optic discs and retinas, intraocular pressure between 10 and 21 mm Hg, normal circumpapillary retinal nerve fiber layer thickness and profile on spectral-domain OCT and normal visual fields. Glaucoma was defined by the presence of characteristic localized or diffuse neuroretinal rim thinning or retinal nerve fiber layer defect on stereo disc photography or spectral-domain OCT and of glaucomatous visual field defects. A glaucomatous visual field defect was defined as a glaucoma hemifield test result outside the normal limits on 2 consecutive visual field tests and the presence of at least 3 contiguous test points within the same hemifield on a pattern deviation plot at P < .01, with at least 1 point at P < .005. These tests required reliability indices better than 15%.
Exclusion criteria were best-corrected visual acuity of 20/40 or less, previous ocular trauma, previous intraocular surgery other than uncomplicated phacoemulsification of cataract more than 1 year before recruitment, any ocular or systemic conditions that may affect trabecular outflow pathway structures other than open-angle glaucoma (eg, anatomically narrow angles, angle-closure glaucoma, anterior segment anomaly, or ocular neovascularization), any ocular or systemic conditions that may reduce OCT image quality (eg, severe dry eye syndrome, pterygium, pinguecula, or nystagmus), and pregnancy or breastfeeding.
Serial, horizontal EDI OCT B-scans of the nasal corneoscleral limbal area were performed before and 1 hour after administration of pilocarpine, 1%, in 1 randomly selected eye of healthy individuals and pilocarpine, 2%, in 1 randomly selected eye of patients with open-angle glaucoma using the anterior segment module of Spectralis OCT (Heidelberg Engineering, GmbH) (Figure 1). Pilocarpine, 1%, was chosen for healthy individuals to minimize adverse effects. Pilocarpine, 2%, was chosen for patients with glaucoma owing to its widespread use. The OCT device was set to scan a 15° × 5° rectangle (81 EDI OCT B-scans; interval between scans, approximately 35 µm). Conjunctival vessels were used as landmarks to scan the same limbal area before and after pilocarpine administration. After acquisition of volumetric EDI OCT scans, the aqueous and blood vessels in each B-scan were carefully reviewed to identify the overlapping region between the 2 sets of volumetric scans (before and after pilocarpine administration).
Participants with an incomplete set of EDI OCT B-scans owing to poor patient cooperation (eg, excessive eye or head movement or excessive blinking) or with poor-quality EDI OCT B-scans in which the Schlemm canal could not be delineated reliably were excluded from analysis.
The hyporeflective Schlemm canal lumen was identified at 1 side of the triangular trabecular meshwork. The cross-sectional area of the Schlemm canal was measured in each EDI OCT B-scan in the overlapping region by manually delineating the Schlemm canal lumen using commercial software (Amira, version 5.4.5; Visage Imaging, Inc). The Schlemm canal in the overlapping region was reconstructed 3-dimensionally after manually aligning the EDI OCT B-scans using the same software, and the volume of the Schlemm canal in this region was calculated. The Schlemm canal in each EDI OCT image was delineated by an investigator (A.S.), and images with Schlemm canal delineations were reviewed and adjudicated by a second investigator (S.C.P.). The 2 investigators were masked to the clinical status of participants, including the timing of EDI OCT in regard to pilocarpine administration (before or after administration of pilocarpine). When the investigators disagreed on the delineation, a mutual conclusion was reached after discussion.
Statistical analysis was performed using a paired t test to compare intraocular pressure and cross-sectional area and volume of the Schlemm canal between the EDI OCT B-scans performed before and after administration of pilocarpine. Data were analyzed using Microsoft Excel 2010 (Microsoft Corp) and SPSS, version 13.0 (SPSS Inc).
Twelve healthy individuals (12 eyes) and 13 patients with glaucoma (13 eyes) were enrolled. Three healthy individuals (3 eyes) and 3 patients with glaucoma (3 eyes) were excluded owing to poor patient cooperation or poor quality of the EDI OCT image. The remaining 9 healthy eyes and 10 eyes with glaucoma were included for measurement and analysis of the Schlemm canal. Demographic and clinical characteristics of the participants are described in Table 1. Mean (SD) age was 31.9 (7.8) years (range, 24-42 years) in healthy individuals and 68.7 (13.2) years (range, 48-87 years) in patients with glaucoma. Mean intraocular pressure was reduced after administration of pilocarpine in healthy individuals (14.3 [1.3] to 13.7 [1.1] mm Hg; P = .004) and patients with glaucoma (17.5 [6.0] to 16.6 [6.1] mm Hg; P = .01). Miosis was noted in all eyes 1 hour after administration of pilocarpine.
The number of overlapping EDI OCT B-scans varied between participants, ranging from 50 to 79 B-scans in healthy individuals and 50 to 81 B-scans in patients with glaucoma. To maintain consistency among participants, 50 matched EDI OCT B-scans from before and after pilocarpine administration were selected for measurement of the cross-sectional area of the Schlemm canal. Following pilocarpine administration, the mean cross-sectional area of the Schlemm canal increased in all participants (Table 2). The mean cross-sectional area of the Schlemm canal increased by 21% (4667  to 5647  µm2; P < .001) in healthy individuals and by 24% (3737  to 4619  µm2; P < .001) in patients with glaucoma (mean difference in percent increase, 2.2%; 95% CI, –8.5% to 12.9%). Representative EDI OCT B-scans before and after pilocarpine administration are shown in Figure 2. Mean (SD) volume of the analyzed region of the Schlemm canal (approximately 1.7 mm of circumferential length in the nasal limbus) increased from 8 004 000 (2 923 000) µm3 to 9 685 000 (3 277 000) µm3 in healthy individuals (P < .001) and from 6 468 000 (1 170 000) µm3 to 7 970 000 (1 199 000) µm3 in patients with glaucoma (P < .001). The percent increase of the cross-sectional area of the Schlemm canal was similar between healthy individuals and patients with glaucoma (P = .66 in a univariable analysis; P = .58 in a multivariable analysis controlling for age, refractive error, and intraocular pressure before the administration of pilocarpine).
The conventional outflow pathway consists of drainage through the trabecular meshwork into the Schlemm canal and then to aqueous veins, which eventually empty into scleral veins.10 Although the effects of pilocarpine on ciliary muscle contraction and lens rounding have been well demonstrated,11,12 to our knowledge, its in vivo effects on human conventional outflow pathway structures, especially the Schlemm canal, have not been studied. Electron microscopic studies by Grierson et al on baboons (Papio cynocephalus)13 and human eyes14 showed prominent cellular bulges in the Schlemm canal lining endothelial monolayer in eyes treated with pilocarpine compared with controls. Endothelial pores were larger and more frequent in eyes treated with pilocarpine. The authors speculated that this increase in vacuolization was produced by the direct action of pilocarpine on the Schlemm canal endothelium or was a consequence of increased fluid passage through the drainage system. The authors did not remark on cross-sectional area or volume of the Schlemm canal. More recently, Li et al5 demonstrated that pilocarpine increases lumen area and outflow facility of the Schlemm canal in mouse eyes in vivo. Imaging of the Schlemm canal in mice was simplified by relatively thin sclera and large Schlemm canal lumen.
We demonstrated an increase in cross-sectional area and volume of the Schlemm canal after administration of pilocarpine in eyes with and without glaucoma. A study on conventional outflow pathway architecture in mice15 demonstrated the structural association between the ciliary muscle, trabecular meshwork, and inner wall of the Schlemm canal. Trabecular meshwork contains a net of elastic fibers connecting the inner wall of the Schlemm canal to the cornea anteriorly and to the ciliary muscle posteriorly. Nets of elastic fibers were also demonstrated in nonhuman primate,16 bovine,17 and porcine18 trabecular meshwork. The ciliary muscle bifurcates into external and internal branches near the posterior trabecular meshwork. Tendons of the external branch of the ciliary muscle and the elastic fiber net of the choroid bend to insert into the juxtacanalicular elastic net of trabecular meshwork or connect directly to the inner wall endothelium of the Schlemm canal, while the internal branch of the ciliary muscle connects to the lamellated trabecular meshwork that is continuous with the ciliary body stroma. Therefore, it seems reasonable that pilocarpine, which parasympathomimetically stimulates ciliary muscle contraction and pulls on the tendons and the elastic fiber net terminating in the inner wall of the Schlemm canal,3,4,15 will cause an increase in the cross-sectional area and volume of the Schlemm canal, as demonstrated in our study. Such an increase in the cross-sectional area and volume of the Schlemm canal seems to explain the increase in outflow facility, as demonstrated in anesthetized owl monkeys19 and enucleated mouse eyes perfused with pilocarpine,15 leading to reduction of intraocular pressure. Our study demonstrated for the first time, to our knowledge, the in vivo effect of pilocarpine on the human Schlemm canal in both healthy eyes and eyes with glaucoma, which may help explain its pharmacologic mechanism of action and better indicate its clinical use.
In addition, our study suggests the potential role of EDI OCT in clinical assessment of the Schlemm canal with regard to medical or surgical treatment of glaucoma. Patients’ response to pharmacologic agents that are supposed to increase aqueous outflow through trabecular meshwork can be monitored using EDI OCT for tailored treatment. Regarding newer glaucoma surgeries targeting iridocorneal angles and the Schlemm canal, preoperative EDI OCT may be helpful in selecting good surgical candidates and better targeting areas in such candidates. Postoperative EDI OCT may be useful in evaluating surgical success and causes for surgical failure.
Several studies have pointed out the problem of imaging the Schlemm canal in vivo in human eyes.5,7 The causes include thick sclera, artifacts owing to the coexistence of different tissues (cornea, sclera, iris, Schlemm canal, and trabecular meshwork) at different layers, different light reflection properties and polarization characteristics of the tissues, shadowing effect from superficial blood or aqueous vessels, changes in anatomical orientation, the large distance of the location of the Schlemm canal from the zero reference position in most current tomographic systems, and the fact that axial resolution is inversely proportional to the center wavelength (if longer wavelengths are used for imaging deeper tissue, the axial resolution becomes poorer). Enhanced depth imaging OCT provides superior imaging of the deeper ocular tissues compared with that provided by conventional spectral-domain OCT.9 In EDI OCT, the OCT apparatus is placed closer to the eye, creating an inverted view of the ocular structures, which increases the image quality of the deeper tissue layers.9 Enhanced depth imaging OCT has been used mainly for imaging the posterior segment deep structures and their pathologic conditions including age-related macular degeneration, central serous chorioretinopathy, optic nerve head drusen, birdshot chorioretinopathy, retinitis pigmentosa, choroidal melanocytosis, and lamina cribrosa changes in glaucoma.20-24 Using this advantage provided by EDI OCT, our study evaluated the pharmacologic effect of pilocarpine on the structure of the Schlemm canal.
In our study, the effect of pilocarpine was measured 1 hour after administration because its maximal effect on anterior chamber depth, lens thickness, and pupil miosis is between approximately 30 to 90 minutes following administration.12,25 The nasal corneoscleral limbal area was chosen because the Schlemm canal has been found to be larger in the nasal than in the temporal side in healthy eyes, suggesting preferential nasal aqueous drainage.7
Although the relatively small number of participants resulted in relatively wide confidence intervals around the findings reported, an increase in the cross-sectional area and volume of the Schlemm canal was observed consistently in all participants. A small part (5° [approximately 2.8 mm] in the nasal quadrant) of the Schlemm canal circumference was evaluated to minimize any adverse effect of patient fatigue on image quality and acquisition. Therefore, it is possible that other parts of the Schlemm canal may respond differently to pilocarpine. Although we attempted to scan the same nasal limbal area before and after administration of pilocarpine based on conjunctival vessels, not all 81 EDI OCT B-scans were matched between scans performed before and after administration of pilocarpine. To maintain consistency among participants, the minimum number of matched EDI OCT B-scans (50 scans representing approximately 1.7 mm of Schlemm canal circumference) were included for analysis in all eyes. The mean (SD) age was different between the healthy individuals (31.9 [7.8] years) and patients with glaucoma (68.7 [13.2] years) (P < .001), so direct comparison of the cross-sectional area of the Schlemm canal between the 2 groups is inappropriate. However, this finding suggests that pilocarpine expands the Schlemm canal in both young and older people. Correlation between changes in cross-sectional area of the Schlemm canal and changes in intraocular pressure is an interesting subject to explore, but we were unable to conduct reliable correlation analysis because the mean intraocular pressure decrease after administration of pilocarpine was less than 1 mm Hg in healthy eyes (0.7 mm Hg) and eyes with glaucoma (0.9 mm Hg).
We have demonstrated that pilocarpine expands the Schlemm canal in vivo in healthy individuals and patients with glaucoma, which provides additional insights into pilocarpine’s mechanism of action. No differences in the effect of pilocarpine were identified in eyes with glaucoma compared with eyes without glaucoma. Whether this finding results from a direct effect of pilocarpine on the Schlemm canal or secondarily from its effect on the ciliary musculature and/or trabecular aqueous outflow remains to be determined. Enhanced depth imaging OCT of the Schlemm canal may prove to be a useful and valuable tool for evaluating the mechanisms of action of pharmacologic agents and surgical techniques and devices for glaucoma.
Submitted for Publication: January 1, 2016; final revision received April 23, 2016; accepted May 1, 2016.
Corresponding Author: Sung Chul Park, MD, Department of Ophthalmology, Manhattan Eye, Ear and Throat Hospital, 210 E 64th St, New York, NY 10065 (email@example.com).
Published Online: June 23, 2016. doi:10.1001/jamaophthalmol.2016.1881.
Author Contributions: Dr Park had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Skaat, Rosman, Ren, Park.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Skaat, Rosman, Chien, Ren, Liebmann, Park.
Critical revision of the manuscript for important intellectual content: Skaat, Rosman, Mogil, Liebmann, Ritch, Park.
Statistical analysis: Skaat, Park.
Obtained funding: Liebmann, Park.
Administrative, technical, or material support: Skaat, Ren, Liebmann, Ritch, Park.
Study supervision: Liebmann, Ritch, Park.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Liebmann reported receiving instrument support from Heidelberg Engineering, GmbH, and Topcon Medical Systems and serving as a consultant for Carl Zeiss Meditec, Inc. Dr Ritch reported serving as a consultant for Aeon Astron, Sensimed AG, iSonic Medical, Allergan, Santen, Ministry of Health of Kuwait, and Ocular Instruments, Inc, and receiving stock options from Diopsys. Dr Park reported receiving an honorarium from Heidelberg Engineering, GmbH. No other disclosures were reported.
Funding/Support: This study was supported by the Mentoring for Advancement of Physician-Scientists (MAPS) Award of the American Glaucoma Society (Dr Park), the James Cox Chambers Research Fund (Dr Ren), and the Joseph and Deena La Motta Research Fund of the New York Glaucoma Research Institute.
Role of the Funder/Sponsor: The funding sources had no role in the study design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Previous Presentations: This study was presented in part at the Association for Research in Vision and Ophthalmology Annual Meeting; May 5, 2014; Orlando, Florida; and at the American Glaucoma Society 2015 Annual Meeting; February 26, 2015; Coronado, California.