Trabecular meshwork (TM) migration into Schlemm’s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.
The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.
Glaucoma is the world’s second leading cause of irreversible blindness1. Elevated intraocular pressure (IOP) is a major causal risk factor for the presence and progression of glaucoma2-7. IOP is regulated by balance between the formation and outflow of aqueous humor8. The locations of greatest outflow resistance are the juxtacanicular tissue and the inner wall of Schlemm canal (SC), the interface between SC and the trabecular meshwork (TM) 9-11. While TM stiffness may contribute to the prevention of SC collapse in the face of IOP elevation, Overby et al. 12 recently demonstrated that gene expression in glaucoma is altered, resulting in increased SC endothelial stiffening, impeding formation of pores, leading to IOP elevation in glaucomatous eyes13. TM morphology and stiffness correlate with outflow facility14,15, emphasizing the need to measure its biomechanical characteristics.
Atomic force microscopy measurements of the TM show elevated stiffness in eyes donated by glaucoma patients (81 kPa) compared with eyes from donors without glaucoma (4.0 kPa) 16, but these measurements were made in dissected ex vivo tissues. The posterior TM is anchored into the ciliary muscle via anterior tendons of the longitudinal muscle cells which insert into the outer lamellated and cribiform TM17. Ciliary muscle (CM) activity may increase TM tautness, mimicking elevated TM stiffness17. The ability to observe alterations in resistance to SC collapse induced by perturbations of smooth muscle has been shown in an animal model18. We have demonstrated the ability to non-invasively image the primary aqueous humor outflow system in living human eyes distal to and including SC using spectral domain optical coherence tomography (OCT) 19-21. Using this technique, we have demonstrated the ability to quantify the morphometric response of the TM and SC to acute IOP elevation22.
The overall goal of the method described herein was to quantify the morphometric response of the living outflow tract to acute IOP elevation in living tissues in situ. This technique has the advantage of examining the TM under physiological conditions, which includes contributions of both contractile fiber activity within the TM and CM to TM stiffness, as compared to published measurements made in dissected tissues. The rationale behind applying this technique to observation of the mechanical TM response is that it provides us with otherwise unavailable insights into the mechanical behavior of the TM, which we now know to be linked directly to outflow resistance and IOP regulation13. To discern the contribution of contractile tissues to overall stiffness, a small cohort of subjects was examined without and with suppression of smooth muscle activity by administration of tropicamide.
Ethics Statement: Approval was obtained from the Institutional Review Board of the University of Pittsburgh School of Medicine before subject recruitment began. All subjects provided written informed consent before participation in the study.
1. Data Acquisition
2. Data Processing
Using these data acquisition and image analysis techniques, the effects of small and large changes in IOP on outflow tract morphological parameters such as SC cross-sectional area are obtained (Figure 1). We can see that high levels of IOP increase produce an observable collapse of SC, as represented by a large reduction in cross-sectional area. The eye appears to be able to accommodate small increases in IOP, as evidenced by the lack of change in SC-CSA (Figure 1). These results show that the technique is capable of quantifying the morphometric response of the outflow tract to an acute IOP challenge. No other family of technologies or techniques provides both visual and quantitative information about outflow tract biomechanics.
Throughout the study, no significant change in TM thickness was observed. In response to a 23 mmHg IOP increase, SC inner to outer wall distance was reduced by 5.03 µm. Without and with suppression of smooth muscle activity, a 6 mmHg increase in IOP caused SC inner to outer wall distance to decrease by 0.18 µm and 2.34 µm respectively. In addition, baseline SC-CSA dropped from 4,597 ± 2,503 µm2 to 3,588 ± 1,198 µm2 (mean ± standard deviation) with smooth muscle activity suppression. Together, with the insertion of anterior tendons from the ciliary muscle which insert into the outer lamellated and cribiform TM17, this implies a control system to maintain SC patency involving smooth muscle. Further study is merited.
Figure 1. Schlemm’s canal area versus intraocular pressure in living eyes. Schlemm’s canal (SC) cross-sectional areas from the two cohorts of subjects are provided. Error bars present 1 standard error in intraocular pressure (IOP) on the X-axis, and SC area on the Y-axis.
The present technique leverages non-invasive observation of the mechanical response of soft tissue to quantify SC collapse. Future work using human cadaver eyes is needed to calibrate tissue deflections actual tissue stiffness after dissection. But, such studies will suffer the same limitations of previous outflow models; specifically, that the contributions of living muscle to tissue tension will not be present. Further calibration in a living mammalian eye model may allow calibration of imaging and direct measurements of stiffness of the TM.
There are several limitations to the technique. It has yet to be demonstrated on other OCT platforms. The literature suggests that the same structures may be visualized on other OCT devices, however sensitivity to changes associated with acute IOP elevation on those devices has yet to be demonstrated in human eyes. The present device was used out of convenience, as no additional optics are required for anterior segment scanning. The greatest challenge to this work is identification of SC within the scans. It is impossible to definitely identify SC within a single slice. Interrogation of the volume is required to first locate the area of tissue containing SC. Its identity is then confirmed by observation of collector channel ostia, and interconnection of the various segments of SC that appear slice to slice. In our experience, SC will present as between 0 to 4 openings within the limbus that can merge into single large openings near a collector channel ostium, or collapse to a pinched section of complete closure.
The greatest significance of this break-through technique is that there is no other option for the assessment of TM stiffness in situ. The morphology and stiffness of the TM correlate with outflow facility14,15, emphasizing the need to measure the biomechanical characteristics of outflow pathway. In the future, such measurements may provide insights currently unavailable in the management of glaucoma.
The authors have nothing to disclose.
Supported in part by National Institute of Health contracts R01-EY13178, and P30-EY08098 (Bethesda, MD), the Eye and Ear Foundation (Pittsburgh, PA), and unrestricted grants from Research to Prevent Blindness (New York, NY).
Spectral Domain OCT | Zeiss | Cirrus | |
Imaging Workstation | Apple | iMac | |
Ophthalmodynamometer | (Baillairt Matalene Ophthalmodynamometer, Surgical instruments CO., Inc. New York, NY) | ||
Image Processing Program | rsb.info.nih.gov/ij | ImageJ, FIJI |