Dynamic Changes of Scleral Spur Length In Different Accommodation States

This study aimed to evaluate the scleral spur length in response to different accommodation stimuli states, as well as the correlation with Schlemm’s canal and trabecular meshwork. 74 children were recruited for this study. The 0D, -4D, and − 8.0 D accommodation state was stimulated by looking at a variable distance optotype. The ciliary muscle, scleral spur, Schlemm’s canal, and trabecular meshwork were imaged by swept-source optical coherence tomography. The scleral spur length increased with accommodation stimulation (p < 0.05). Method I was 127.97 ± 20.32 µm, 138.29 ± 20.11 µm, and 150.73 ± 21.75 µm at 0D, -4D and − 8D, respectively. Method II was 158.15 ± 19.97 µm, 168.47 ± 18.72 µm, and 179.79 ± 20.83 µm at 0D, -4D and − 8D, respectively. Method III was 221.56 ± 30.74 µm, 234.99 ± 30.11 µm, and 250.09 ± 29.87 µm at 0D, -4D and − 8D, respectively. Method III had the largest areas under ROC curves (0.798, 95% CI 0.721–0.875). Moreover, ciliary muscle 1, Schlemm’s canal, and trabecular meshwork length were signicantly correlated with SSL (Method III) (p < 0.05). These ndings suggest that the contractile ability and compliance of the scleral spur play an important role in maintaining the morphology of the SC. Moreover, the force of accommodation regulates the SC size by increasing the length of SS.


Introduction
Primary open-angle glaucoma (POAG) is a common form of glaucoma and one of the most common causes of irreversible blindness in the world 1 . Elevated intraocular pressure (IOP) is a primary risk factor for POAG and is caused by increased aqueous humor out ow resistance somewhere in the trabecular meshwork (TM) and out ow pathway of Schlemm's canal (SC) [2][3][4] . Many previous studies have shown that the principal sites of out ow resistance are located in the inner wall of the endothelium of SC, the basement membrane of SC, the underlying extracellular matrix of juxtacanalicular tissue, and the TM [5][6][7] .
Thus, in the clinic, the SC and TM have been considered the most important therapeutic targets for the treatment of POAG.
The scleral spur (SS) has been considered to play an important role in maintaining the structure of the SC and TM, which are key components in the pathways of aqueous humor ow. The SS is a projection of the sclera that is located in the ciliary muscle bers and at the base of the TM. It has a rigid nature because it contains elastic and collagenous bers as well as myo broblast cells 8-9 . Swain DL et al. provided different methods for measuring the scleral spur length (SSL) in pathological sections; they found that compared with healthy subjects, in POAG subjects, the SSL was signi cantly shorter, suggesting that a shorter SS may be a risk factor for the progression of POAG because short SS would be insu cient for maintaining the structure of the SC and TM 10 . In real-time and in vivo, Li M et al. also found the SSL was signi cantly shorter in POAG eyes compared with healthy eyes by swept-source optical coherence tomography (SS-OCT). Moreover, the SC area (SCA) was signi cantly associated with the SSL in both POAG and healthy groups. They suggested that the SS plays an important role in the maintenance of SCA and the SSL could be a novel biomarker for POAG evaluation clinically 11 .
Accommodation is the ability of the visual system to alter its optical power to see objects clearly at a nite distance from the eye, it is done by contraction of the ciliary muscles that are innervated by the parasympathetic nervous system 12 . Previous studies have reported that accommodation stimulation could promote aqueous humour out ow and decreases IOP by expanding the SC size, but the mechanisms underlying these changes are unclear [13][14] . Most researchers suggest that the mechanical effects of ciliary muscles under accommodation states mediate structural changes of TM and SC by the SS. But, changes in SS morphology with accommodation stimuli have not yet been reported in humans. Besides that, pilocarpine can also promote aqueous humor out ow by expanding TM and the inner wall of SC, thus keeping SC lumen open [15][16] . However, the drug's effectiveness is lost upon severing the anterior attachment of the ciliary muscle from the SS 17 . These results suggested that the SS is an indispensable part of regulating the SC and TM under accommodation states. Moreover, the accommodative ability is at its peak in childhood and gradually declines with age [18][19] . Therefore, in this study, we aimed to evaluate the SSL in children in response to different accommodation stimuli states, as well as the correlation with SC and TM, in an exploration of the mechanisms underlying the regulation of SC.

Results
A total of 74 children were enrolled in this study. 7 children were excluded because of low quality images caused by poor patient cooperation. Thus, 67 children (  chose the SSL measurement of Method III, which had the largest AUC, to investigate the associations of scleral spur length with chamber parameters (Fig. 4B-4F). The result shows that SSL was signi cantly correlated with CM1, SC, and TM.

Discussion
Ciliary muscle changes provide direct evidence of accommodation stimulation. Past research has reported that the ciliary muscle changes with accommodation using UBM, and OCT [25][26] . The ciliary muscle showed a contractile shortening and a thickening of the anterior portion with accommodation 27 . In this study, the CM1 increased signi cantly with accommodation state and CM3 decreased with accommodation state, consistent with past results.
In addition, we observed that the SSL increased with increasing accommodation force. We speculate that the changes in the SSL under different accommodation states may be due to several causes. On the one hand, the SS is a wedge structure formed by the projection of the inner sclera, the posterior border of the corneoscleral portion of the TM and the anterior border of the longitudinal bers of the ciliary muscle. The ciliary muscle was thicker and showed a greater contractile response on the anterior portion 27 . Thus, the force of accommodation derived from the ciliary muscle mainly acts on the base of the SS rather than simply pulling on the tip of the SS. Moreover, elastic ber tendons from the longitudinal bers of the ciliary muscle are continuous anteriorly into the SS and attach to the posterior elastic bers of the SS 8 . The wedge-shaped structure results in more longitudinal bers of the ciliary muscle attached to the base of the SS, which results in more accommodation force derived from the ciliary muscles. On the other hand, the SS may possess contractile ability and compliance. The SS contains circumferentially oriented collagenous and elastic bers that provide rigidity. Within the aggregated bers of the SS, a population of circularly oriented and spindle-shaped cells (scleral spur cells) stain intensely for α-smooth muscle actin and smooth muscle myosin 28 . In addition, scleral spur cells are innervated by nerve endings, which contain granular and agranular vesicles that are regarded as typical for adrenergic terminals 29 . This evidence indicates that, in the state of accommodation stimulation, the contraction force of the ciliary muscle can posteriorly and internally pull the SS, which increases the SSL.
The TM and SC are other important structures in mediating aqueous humor out ow [3][4] . In this study, the SCA and SCL increased signi cantly with accommodation stimulation, and these changes showed a signi cantly positive association with the SSL. The previous study has reported the SC size correlated with out ow facility and had an effect on out ow resistance 2 . The SC inner wall and the juxtacanalicular tissue (JCT), which are the majority of out ow resistance, also respond to changes in mechanical tension 30 . Although IOP or aqueous out ow could not be simultaneously monitored during the OCT scans in this study. However, earlier research reported that accommodation stimulation could promote aqueous humour out ow and decreases IOP [13][14] . In addition, the TML increased signi cantly with accommodation stimulation. We speculate that the contractile force of the ciliary muscle with the accommodation stimulation is transmitted to the TM via pulling SS, causing the TM to be stretched, which increases the TML, thus leading to an expansion of the spaces of SC. Clinically, pilocarpine is one treatment for lowering IOP in POAG, and it is a drug that increases aqueous out ow by inducing contraction of the ciliary muscle, leading to an expansion of the spaces between the beams of the TM 31 .
Furthermore, the posterior part of the SC where the SS exerts the most force is wider than the anterior part, consistent with the function of the SS in maintaining the opening of the SC 23 . These evidences suggest that the SS can regulate the morphology of the TM and SC.
The previous histological sections study by Swain DL et al. found that the signi cantly shorter SS in POAG eyes than in age-matched normal eye, which could not provide su cient support for the SC and TM 10 . In addition, Li M et al. also found the scleral spur length was signi cantly shorter in POAG eyes compared with healthy eyes by SS-OCT in real-time and in vivo 11 . These results suggest that the shorter SS than in normal eyes contains fewer ciliary muscle bers and TM attachments, circumferentially oriented collagenous and elastic bers, and circularly oriented and spindle-shaped cells. The shorter SS may compromise its contractile ability and compliance. When the ciliary muscle contracts and pulls the SS of POAG eyes, it moves only a short distance posteriorly, opening few layers of meshwork beams and failing to support the SC lumen; however, this hypothesis needs further con rmation. Additionally, high myopia is an independent risk factor for open-angle glaucoma, but the reason why myopic eyes appear to be more susceptible to glaucomatous damage is unclear 32 . High myopia is characterized by a marked thinning of the sclera, choroid and retina as well as elongation of the axial length 33 . High myopia patients have a series of collagen ber changes, including a predominantly laminar collagen ber bundle arrangement, loss of ber cross-links, and reductions in collagen and glycosaminoglycan synthesis 34 .
Whether these changes will affect the collagen ber structure, biochemistry or biomechanical properties of the SS in high myopia, resulting in changes in the contractile ability and compliance of SS, needs further investigation. Thus, further revealing the effect of SS contractile ability on the SC and TM may help to elucidate the underlying pathophysiological mechanisms involved in open-angle glaucoma.
There were several limitations in this study. First, we only observed SSL, SC, and TM changes in normal subjects. It is unclear whether similar changes would be observed in high myopic subjects or POAG patients; thus, further veri cation is needed. Second, in a previous study, it was found that age had an impact on structural compliance. However, in this study, the subjects' ages ranged from 7 to 14 years (10.91 ± 1.99 years), so whether smaller changes would occur in adults and older people requires further con rmation. Third, in the current study, when provided with accommodation stimulation, IOP or aqueous out ow could not be simultaneously monitored during the OCT scans. Future studies and additional advanced and convenient methods to evaluate out ow function may provide more insight into the mechanisms underlying the pathology of glaucoma.
In conclusion, by SS-OCT, we found that the SSL increased with different accommodation stimulation, and SSL was signi cantly correlated with SC size. These ndings suggest that the contractile ability and compliance of the scleral spur play an important role in maintaining the morphology of the SC. Moreover, the force of accommodation regulates the SC size by changing the length of SS.

Methods
The research was approved by the ethics committee (Renmin Hospital of Wuhan University). The study protocol was registered with chictr.org.cn (ChiCTR-ROC-1900026923). Written informed consent was obtained from the children's parents. The study followed the tenets of the Declaration of Helsinki. In total, 74 children were recruited from Renmin Hospital of Wuhan University, Hubei Province, China, from June 2020 to September 2020. The inclusion criteria were as follows: (1)  All participants underwent a serial ophthalmologic examination, including slit-lamp biomicroscopy (Haagstreit, Bern, Swiss), fundus examination, IOP (NIDEK noncontact tonometer RT-2100), subjective refraction, amplitude of accommodation and spherical equivalent refraction (SER, spherical error plus one-half of the cylindrical error) (RT-2100, NIDEK CO. LTD, Gamagori, Japan). The SS-OCT scan used a 1310-nm wavelength with a scan speed of 30, 000 A-scans/s and an axial resolution of less than 10 µm. Participants were imaged with the 3D-angle high-de nition scans (dimension, a raster of 64 B-scans each with 512 A-scans over 8 mm). All participants' left eyes underwent examinations with swept-source OCT (CASIA SS-1000; Tomey Corporation, Nagoya, Japan) in different states (0D, -4D, -8D). To make sure the same direction of visual axial in different states. A tilted mirror with a frame carrying a rotation axis was xed on the external xation lights. According to the individual interpupillary distance and testing items adjusted the tilting angle of the mirror. The participants were instructed to stare at the optotypes through the mirror with their left eye under the 0D, -4D and the − 8.0 D accommodation (distance based on the formula: 100/−(− M + X) cm, where X was the refractory error value in diopters, M is the accommodative response) 20 stimulation state, and right eye was covered with a gauze. According to the recommendations of Gabriel, the mean of each 5 s was determined for both accommodative response and pupil size 21 . Thus, the participants must accurately identify the optotypes through the mirror with their left eye after 5 s for each accommodation, then the left eye was scanned by the SS-OCT, each measurement took approximately 30 s to complete (Fig. 1). Conjunctival vessels and iris features were used as landmarks to scan the same cross-section under different states to ensure accurate measurements of the CM, SC, TM, and SS. All the eyes were imaged at nasal positions. The scans of each cross-section were repeated three times, and the best quality image was chosen for analysis. All images were obtained under darkroom conditions by the same examiner.
Thickness of the ciliary muscle width at 1, 2, and 3 mm at posterior to the scleral spur was assessed (CM-1, CM-2, CM-3) ( Fig. 2A). Schlemm's canal area (SCA) was drawn and de ned as the black area surrounded by the white outline. Schlemm's canal length (SCL) was measured from the posterior to anterior SC end point by the a-b dotted line. Trabecular meshwork length (TML) was de ned as the dotted arrow line from the tip of the SS to Schwalbe's line. Trabecular meshwork width (TMW) was de ned as the average of the solid arrow line measurements obtained from the anterior and middle points of the SC 22 (Fig. 2B). The SSL was measured by three different methods from previous studies. Method 20 images from 20 eyes in the participants were randomly chosen. To measure the intraobserver repeatability, a single masked observer (HX) measured the images at two different times with an interval of 3 days, and agreement between the two observations was analyzed. To measure the interobserver reproducibility, the same images were independently evaluated by two observers (HX, LX) blinded to the treatments, and the agreement between them was determined. Intraclass correlation coe cients were calculated using a two-way mixed-effects model. The measurements were performed using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).
Data are presented as the mean ± standard deviation. Repeated measures analysis of variance (rANOVA) was performed to analyze the differences in different accommodation states. Sphericity was tested with Mauchly's test, and if not statistically signi cant, Greenhouse-Geisser corrected p-values for F-tests were reported. After a signi cant F-test, post hoc analysis with Bonferroni correction was used for comparisons between groups. Statistical signi cance was de ned as p < 0.05. Figure 1 Simulated diagram of the setup used to stimulate different accommodation during optical coherence tomography (OCT) imaging. A tilted mirror with a frame carrying a rotation axis was xed on the OCT machine and used to place the stimuli at different vergences. The participants were instructed to stare at the optotypes through the mirror with their left eye, and right eye was covered with a gauze. Then, the left eye was imaged by OCT.  A. CM1 increased signi cantly with accommodation state. CM2 did not change signi cantly. Compared to the base state, CM3 decreased at -8D accommodation state. B. SSL that was measured by three different methods increased with accommodation stimulation. C. SCA and SCL increased signi cantly with accommodation stimulation. D. TML increased signi cantly with accommodation stimulation, but TMW did not.