Segmental Unconventional Outflow in Mouse Eyes

Abstract

Purpose: To investigate the flow pattern in unconventional outflow and its correlation with conventional outflow in mouse eyes.

Methods: Fluorescent microspheres were injected into the anterior chamber of one eye of anesthetized C57BL/6J mice (n = 4), followed by perfused fixation with 4% paraformaldehyde in situ after 45 minutes. Post-euthanasia, the injected eyes were enucleated, further immersion fixed, and dissected into 12 equal radial segments. Both sides of each segment were imaged using a confocal microscope after nuclear counterstaining. Both unconventional and conventional outflow patterns of each eye were analyzed by ImageJ and ZEN 2.3 imaging software.

Results: Segmental outflow patterns were observed in both the ciliary body (CB) and the supraciliary space and suprachoroidal space (SCS). In the CB, the tracer intensity was the lowest at 12 o'clock and highest at 9 o'clock, whereas in the SCS it was the lowest at 2 o'clock and the highest at 10 o'clock. Consequently, a segmental unconventional outflow was observed, with the lowest and highest flow regions in the superior and temporal quadrants, respectively. The overall segmental uveoscleral outflow has no correlation with trabecular outflow (P > 0.05). Four different outflow patterns were observed: (1) low-flow regions in both outflows, (2) primarily a high-flow region in conventional outflow, (3) primarily a high-flow region in unconventional outflow, and (4) high-flow regions in both outflows.

Conclusions: Uveoscleral outflow is segmental and unrelated to the trabecular segmental outflow. These findings will lead to future studies to identify the best location for the placement of drainage devices and drug delivery.

Aqueous humor (AH) exits the eyes through both conventional (trabecular) and unconventional (uveoscleral) outflow pathways.^1,2 Conventional outflow is pressure dependent, such that as the intraocular pressure (IOP) increases the outflow rate increases.³ Specifically, AH flows sequentially from the trabecular meshwork (TM), Schlemm's canal (SC), and collector channels (CCs) and eventually exits the eyes via the episcleral veins (ESVs).^2,4 In contrast, unconventional outflow is relatively pressure independent,³ and its route remains less comprehensively understood. AH infiltrates the interstitial spaces among the ciliary muscle bundles and the supraciliary space and suprachoroidal space (SCS), and it either leaves the eyes through the vortex veins^5,6 or becomes absorbed by the orbital vasculature.^7,8 Both outflow systems can be modulated by different pharmaceutical agents or surgical procedures, which in turn can modulate IOP. For example, Rho kinase inhibitors^9–12 and excimer laser trabeculostomy^13,14 enhance conventional outflow, whereas prostaglandin analogs such as bimatoprost and latanoprost improve unconventional outflow.^15,16 Techniques enhancing the SCS have demonstrated a remarkable IOP reduction up to 32% 1 year after surgery.¹⁷ Both outflow systems play an irreplaceably important role in regulating IOP, albeit through different mechanisms. The induced IOP reduction significantly delays the progression of visual field defect in glaucoma patients.^18–20 

Tracer intracameral injection or fluorescein aqueous angiography studies have revealed the segmental or non-uniform nature of conventional outflow circumferentially around the eye in both human^21–24 and non-human^25–28 subjects. In human eyes, the temporal and nasal quadrants demonstrate the lowest and highest tracer intensities, respectively, in both the TM and ESVs and are considered as the low-flow (LF) and high-flow (HF) regions in conventional outflow.²³ However, despite the equal importance of conventional and unconventional outflows, no previous study, to the best of our knowledge, has investigated the pattern of uveoscleral outflow. Additionally, there has been a lack of quantitative comparison regarding tracer distribution circumferentially around the eye between these two outflow pathways, and the interrelation between these two systems at different locations remains unclear. In this study, we sought to characterize unconventional outflow. The study aimed to determine whether uveoscleral outflow is segmental and, if so, whether there is a quantitative correlation between unconventional and conventional outflows at various circumferential eye locations. 

Materials and Methods

All animal procedures were approved by Boston University Institutional Animal Care and Use Committee and conducted and complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Four male C57BL/6J mice, 2 months old, were purchased from Charles River Laboratories (Wilmington, MA, USA) and accommodated in the Animal Science Center of Boston University with a 12-hour light/12-hour dark cycle and unrestricted access to food and water for a minimum of 3 days. All mice were confirmed to have no signs of abnormality. 

Tracer Injection

To label the outflow pattern, one eye (left or right eye) of each mouse was injected with the fluorescent tracers following established protocols.^25,28,29 The mice (n = 4) were anesthetized with 87.5 mg/kg ketamine and 12.5 mg/kg xylazine (Covetrus, Dublin, OH, USA) by intraperitoneal injection and positioned on a mounting stage (Stoelting Mouse and Neonatal Rat Adaptor; Stoelting Co., Wood Dale, IL, USA). The AH volume in mouse eyes was approximately 5.9 ± 0.5 µL.³⁰ A fixed volume of 1 µL (equivalent to 17% of the AH volume) of 20-nm fluorescent microspheres (excitation/emission: 505/515, 2%; Invitrogen, Carlsbad, CA, USA), diluted 1:50 in Dulbecco's phosphate-buffered saline solution (v/v), was injected into the anterior chamber of one eye of each mouse using a 35-gauge beveled needle (NanoFil; World Precision Instruments, Sarasota, FL, USA) in a bevel-up position. The fluorescent tracer solution was delivered at a rate of 4 nL/s by a microprocessor-based microsyringe pump controller (Micro4; World Precision Instruments). After tracer injection, the needle remained in the eyes for 45 minutes to allow the tracers to migrate throughout the anterior chamber and enter both conventional and unconventional outflow systems. Artificial tears (Henry Schein, Melville, NY, USA) were topically administered to prevent dehydration of the eye. To fix the eyes in situ, 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA, USA) was subsequently introduced intracamerally (2 µL) and applied topically to the injected eye at the same time for 30 minutes. After euthanasia, both eyes were enucleated and immersion fixed in 4% PFA at 4°C for 48 hours. All eyes were transferred in PBS and stored at 4°C for further processing. Eyes without tracer injection were used as controls, and the tracer distributions in both conventional and unconventional outflow pathways in injected eyes were examined. 

Confocal Microscopy

All of the tracer-injected eyes were radically dissected into 12 equal segments according to clock-hour positions. After nuclear counterstaining through immersion in an antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Newark, CA, USA), cross-sectional images of both sides of each segment were captured using a confocal microscope (Zeiss LSM 700; Carl Zeiss Microscopy, White Plains, NY, USA) at a 20× objective magnification with a 16-bit color depth (darkest = 0; brightest = 65,536). The excitation wavelengths for the nucleus (blue) and tracer (green) were 405 nm (channel 0) and 488 nm (channel 1), respectively. Tracer intensities for all experimental samples were imaged with the same setting of gain, digital offset, and pinhole size, with no observable tracer signal in control eyes (Fig. 1A). To ensure the capture of fluorescent signals and all structures across different focus planes, Z-stack images were acquired with a step size of 1.5 µm. 

Figure 1.

 

Methods for the segmentation of tracer intensity in conventional and unconventional outflow pathways. Representative images of a negative control without tracer injection (A) and segmentation for image analysis (B). Blue represents nuclear counterstaining with DAPI, and green represents the tracers. The green tracer intensity above and below the horizontal line represents the tracer intensity in the conventional and unconventional outflows, respectively. In the area below the horizontal line, the tracer intensity on the right (anterior) and left (posterior) sides of the vertical line represents the tracer intensity in the CB and other locations such as supraciliary space and suprachoroidal space (SCS).

Analysis of Tracer Intensity in Confocal Images

Two-dimensional images for both nucleus and tracer channels were generated for each radical segment using the Z Project function in ImageJ (National Institutes of Health, Bethesda, MD, USA), employing maximum intensity as the projection method. Tracer intensity within the range of 5952 to 65,536 was analyzed by ZEN 2.3 imaging software (Carl Zeiss Microscopy), and no background noise was observed in the negative control image (Fig. 1A). In the pilot study, immunostaining of the CD31 for endothelial cells and α-smooth muscle actin for the ciliary muscle was conducted in cross-sections of normal C57BL/6J mouse eyes. Phase-contrast images were also captured. Based on these observations, the images were segmented according to anatomic positions identified by the nucleus. To ensure the repeatability of measurement, a horizontal straight line was drawn from the root of the iris to the end of the ciliary body. A perpendicular vertical line was then added, originating from the posterior end of Schlemm's canal and intersecting the horizontal line (Fig. 1B). Tracer intensities above and below the horizontal line were attributed to conventional and unconventional outflow, respectively. For the uveoscleral outflow, the areas anterior and posterior to the vertical line were defined as (1) ciliary body (CB) and (2) other locations such as the supraciliary or suprachoroidal spaces, respectively. 

Statistical Analysis

For each eye, the superior and nasal sides were designated as 12 o'clock and 3 o'clock, respectively. All data are presented as mean ± SEM. The image from each side of the radial segment was considered an independent image, resulting in a sample size of n = 8 for each clock-hour location. The total tracer intensity for the four quadrants was calculated by summing the tracer intensity at three clock-hour locations. Specifically, for each eye, positions from 2 to 4 o'clock represented the nasal (N) region; 5 to 7 o'clock represented the inferior (I) region; 8 to 10 o'clock the represented temporal (T) region; and 11 to 1 o'clock represented the superior (S) region. Subsequently, the percentage of tracer intensity in each quadrant relative to the total tracer intensity of each eye was calculated. One-way ANOVA followed by Fisher's least significant difference post hoc tests was used for the analysis of tracer intensity at different locations compared to the lowest intensity region, and two-way ANOVA was used for comparisons among outflow pathways at different locations. A paired t-test was used for the comparison of total intensity between the SCS and CB, as well as between the conventional and unconventional outflow pathways. Analysis involved summing up tracer intensities in images from different clock-hour locations for each eye, resulting in a sample size of n = 4. A Spearman correlation test was performed to investigate correlations. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; *** P < 0.001). 

Results

Upon analyzing all 96 images (16-bit color depth images), the mean tracer intensity measured from all clock-hour locations of both conventional and unconventional outflow pathways was 956 million. Clock-hour locations with tracer intensities higher than 956 million in either the conventional or unconventional outflow pathways were categorized as HF regions in their respective pathways, whereas those with tracer intensities lower than 956 million were categorized as LF regions. 

Segmental Uveoscleral Outflow in Mouse Eyes

A segmental tracer distribution was observed in the uveoscleral outflow pathway at the two distinct locations: (1) CB (Fig. 2A) and (2) SCS (Fig. 2B). The mean tracer intensity varied circumferentially around the eye in the CB, with the lowest intensity at 12 o'clock (Figs. 2A, 2C) and the highest at 9 o'clock (Figs. 2A, 2E). The tracer intensity at 12 o'clock was significantly lower than that at 9 o'clock by 1226 ± 68 million (P < 0.01; 87% ± 5%). Similarly, the tracer intensity displayed variation around the eye in the SCS, with the lowest intensity at 2 o'clock (Figs. 2B, 2D) and highest at 10 o'clock (Figs. 2B, 2F). The tracer intensity at 2 o'clock was significantly lower than that at 10 o'clock by 126 ± 6 million (P < 0.05; 91% ± 4%). When comparing the results between the CB and SCS, no correlation was found in tracer patterns between these two locations (P > 0.05; Spearman’s r = 0.54). Additionally, the total tracer intensity (sum of all tracer intensity at each clock-hour position in each eye) in the SCS was significantly lower than that in the CB by 17,973 ± 6410 million (P < 0.05; 91% ± 4%) 45 minutes after injection. 

Figure 2.

 

Tracer distribution along the uveoscleral outflow pathway around the eyes. (A, B) Mean tracer intensities in the CB (A) and other locations (B), such as the SCS, are presented. The solid line represents the mean tracer intensity, and the blue zone is the SEM of the intensity (*P < 0.05, **P < 0.01, compared to the lowest intensity location). (C–F) Representative images of the lowest (12 o'clock) (C) and the highest (9 o'clock) (E) intensity regions in the CB, as well as the lowest (2 o'clock) (D) and the highest (10 o'clock) (F) intensity regions in the SCS, respectively.

The tracer intensity in the uveoscleral outflow pathway was calculated by combining the tracer intensities in the CB and SCS of each sample. The tracer distribution was segmental circumferentially around the eye, with the HF region on the temporal side (Figs. 3A, 3B). The percentage of tracer intensity on the temporal side was significantly higher than on the superior side by 13% ± 3% (P < 0.001). Notably, the tracer intensity was greatest at 9 o'clock and lowest at 12 o'clock. Tracer intensity at 12 o'clock was significantly lower than at 9 o'clock by 1283 ± 71 million (P < 0.01, 86% ± 5%). The tracer distribution pattern in uveoscleral outflow exhibited a significant correlation with that in the CB (P < 0.001, Spearman’s r = 1) but not with the SCS (P > 0.05; Spearman’s r = 0.54), possibly due to the higher tracer intensity in the CB compared to the SCS. 

Figure 3.

 

Tracer distribution in both unconventional and conventional outflows around eye. Mean tracer intensities at different clock-hour positions are presented for unconventional (A) and conventional (C) outflow pathways. The solid line represents the mean intensity of the tracer, and the gray zone represents the SEM of the intensity. The percentages of tracer intensity at each quadrant of the total tracer intensity are presented for unconventional (B) and conventional (D) outflow pathways. S, superior quadrant; N, nasal quadrant; I, inferior quadrant; T, temporal quadrant. Data are presented as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, compared to the lowest intensity location).

Segmental Conventional Outflow in Mouse Eyes

A segmental nature was also observed in the conventional outflow pathway by the mean tracer intensity circumferentially around the eyes, with the HF region on the nasal side (Figs. 3C, 3D). The lowest flow region was identified at 11 o'clock (Figs. 3C, 4A), and the highest flow region was observed at 8 o'clock (Figs. 3C, 4B). Tracer intensity at 11 o'clock was significantly lower than at 8 o'clock by 793 ± 167 million (P < 0.05; 58% ± 12%). Furthermore, HF regions were observed from 1 to 5 o'clock, displaying tracer intensities comparable to those at 8 o'clock, with differences of less than 16% (P > 0.05). In quadrant results, a higher tracer intensity was observed in the nasal quadrant (Fig. 3D). Although this difference did not reach statistical significance, potentially due to the limited sample size in the quadrant analysis, combining the findings at clock-hour locations (Fig. 3C) indicated that the HF regions in the conventional outflow pathway were primarily located on the nasal side. 

Figure 4.

 

Tracer distribution in the conventional outflow. Representative images of the LF region in conventional outflow at 11 o'clock (A) and the HF region in conventional outflow at 8 o'clock (B).

Relationship Between Uveoscleral and Conventional Outflow in Normal Eyes

To visually elucidate the correlation between unconventional and conventional outflow pathways, mean tracer intensities from both outflow pathways are combined in Figure 5. The overall tracer distribution pattern circumferentially around the eyes did not exhibit correlation between the two pathways (P > 0.05; Spearman’s r = 0.24). Consequently, various statistical relationships were evident between these pathways when comparing tracer intensities at individual clock-hour positions. For example, a significant difference in the tracer intensity between the two outflow pathways was revealed at 1 o'clock and 2 o'clock, such that the maximum significant difference of tracer intensity was 875 ± 268 million (P < 0.05; 60% ± 12%) at 1 o'clock. Conversely, at certain clock-hour positions, the tracer intensity in both outflow pathways displayed a significant positive correlation. For example, at 8 o'clock (P < 0.005; Spearman’s r = 0.90), which was the HF region in both pathways, and at 11 o'clock (P < 0.05; Spearman’s r = 0.79), the LF region was observed in both pathways. Analyzing all 96 images, four distinct outflow patterns were identified (Figs. 5, 6): (1) LF regions in both unconventional and conventional outflows, (2) primarily a HF region in conventional outflow, (3) primarily a HF region in unconventional outflow, and (4) HF regions in both unconventional and conventional outflows. Based on the total tracer retained in the eyes 45 minutes after injection (sum of all tracer intensity in both outflow pathways for each eye), the ratios of tracer intensities for conventional and uveoscleral outflow ranged from 55% ± 3% to 45% ± 3%, and no significant difference was demonstrated between the total tracer intensities of these two pathways (P > 0.05, paired t-test). 

Figure 5.

 

Tracer distribution in both unconventional and conventional outflows around the eye. The solid red and blue lines represent the mean intensities of the tracers in the conventional and unconventional outflow pathways, respectively (*P < 0.05, for comparison of conventional and unconventional outflows).

Figure 6.

 

Four different patterns of outflow. (A) LF region in both unconventional and conventional outflows, (B) primarily HF region in conventional outflow, (C) primarily HF region in unconventional outflow, and (D) HF regions in both unconventional and conventional outflows.

Discussion

This study investigated the segmental nature of unconventional outflow and its interaction with conventional outflow in mice. By introducing fluorescent tracers into live mouse eyes for 45 minutes, we analyzed tracer intensities at all 12 clock-hour locations around the eye. We discovered that both conventional and unconventional outflows displayed a segmental pattern. This pattern was evident in both the CB and SCS, with higher tracer intensity in the CB compared to the SCS. Furthermore, although the overall segmental outflow patterns in conventional and unconventional pathways were independent of (or not correlated with) each other, the total retained tracers in both pathways were comparable at 45 minutes after injection. 

Previous studies have demonstrated the segmental nature of conventional outflow,^{21–24,26,27} but none, to the best of our knowledge, has explored the flow pattern of unconventional outflow or quantitatively compared the flow patterns between the two pathways at different locations. Currently, unconventional outflow rates are studied through indirect estimations and direct measurements using tracers.^31,32 Indirect estimation involves computing unconventional outflow via parameters such as AH inflow rate, outflow facility, and episcleral venous pressure, using the modified Goldmann equation. Direct measurement entails tracking unconventional outflow rates by monitoring tracer accumulation within diverse ocular tissue, vortex veins, lymphatic vessels, and lymphatic nodes. However, none of these methods provides a comprehensive flow pattern along the entire unconventional outflow pathways. Therefore, our study introduced a quantitative method that measures tracer intensity at 24 locations around the circumference of the eye along both outflow pathways. Our results revealed a segmental conventional outflow pattern in mouse eyes, similar to the HF region observed in the nasal quadrant of human eyes.²³ This similarity suggests that studying uveoscleral outflow pattern and its relationship with trabecular outflow in mouse eyes could offer insights into human eyes. 

Our results showed that unconventional outflow was also segmental, including the CB and SCS, with varying resistance circumferentially. We observed, in unconventional outflow, a HF region (lower resistance) in the temporal quadrant and a LF region (higher resistance) in the superior quadrant. A nearly 90% difference in tracer intensity was found between the lowest (12 o'clock; superior) and the highest (9 o'clock; temporal) flow region. Although we did not find a correlation between the overall flow patterns in uveoscleral and trabecular outflows, we identified significant differences or positive correlations in tracer intensity between these outflow pathways at specific clock-hour locations, resulting in four distinct outflow patterns (Fig. 6): (1) LF regions in both outflows, (2) primarily a HF region in conventional outflow, (3) primarily a HF region in unconventional outflow, and (4) HF regions in both outflows. 

To better understand segmental conventional outflow, previous studies have reported a higher expression of fibronectin and laminin³³ and lower matrix metalloproteinase (MMP) levels³⁴ in LF than HF regions. Similarly, to enrich our understanding of segmental flow and the relationship between the two pathways, further studies are warranted to determine whether there are segmental differences in protein or gene expression in unconventional outflow and shared regulators reported in both outflow pathways, such as MMPs.^35–37 Identifying the segmental nature of both outflow pathways could have implications for future glaucoma surgery or drug delivery strategies. For example, trabecular bypass surgery performed in the LF region of the conventional outflow pathway yielded a greater increase of outflow facility and IOP reduction.³⁸ Tracers consistently flow toward regions with lower outflow resistance. Our previous studies have shown distinct morphological differences in high-flow (or high-tracer) and low-flow (or low-tracer) regions, including thickening (expansion) of the TM and the juxtacanalicular region, an increased number of giant vacuoles with basal openings and I-pores, and a greater number of collector channels in high-flow regions.^4,23,39 Regarding unconventional outflow, our findings suggest that uveoscleral outflow and its resistance vary circumferentially. Although the morphological correlations have not yet been thoroughly investigated, the segmental flow may be attributed to differences in anatomical arrangements, such as interstitial spaces between ciliary muscle bundles and the spaces between the choroid and sclera. Consequently, the expansion of these spaces through procedures such as cyclodialysis,⁵ supraciliary microstenting implantation,^17,40 or injection of hyaluronic acid hydrogel in the SCS⁴¹ significantly lowers the outflow resistance and IOP. However, no studies, to the best of our knowledge, have explored the correlation among procedure location, IOP-lowering effects, and the occurrence of ocular adverse effects, such as corneal endothelial cell loss and IOP spikes.⁴⁰ Given the segmental nature of unconventional outflow and its resistance, future studies could investigate how the location of surgery and implant placements influences the extent of resistance and IOP reduction differently. This research will help to identify optimal locations for procedures targeting uveoscleral outflow, ultimately achieving better reduction in outflow resistance and IOP. 

The segmental outflow nature also applied to the CB and SCS, with the tracer intensity in CB about 90% higher than that in the SCS, indicating that most tracers reached the CB rather than the SCS 45 minutes after injection, or a lower outflow resistance was found in the CB than the SCS. Given the branching nature of the unconventional outflow pathway, tracers entering the CB are likely to leave the eyes through different routes^32,42 depending on the resistance, such as lymphatic drainage (“uveolymphatic” outflow pathway),^43–45 but the detailed routes and the existence of this drainage in normal eyes remain controversial and unclear.⁴⁶ Another example, in monkey⁶ and mouse⁴⁷ eyes, is that it has been suggested that small molecules are reabsorbed by uveal vessels. Capillaries in the CB are lined with fenestrated endothelial cells⁴⁸ and the posterior ciliary epithelium, which responds differently to the perturbation of epithelial transport, potentially serving as a reabsorptive structure allowing AH to move back into stroma.⁴⁹ Therefore, tracers that reach the CB may enter fenestrated capillaries in the ciliary stroma and leave through the uveal vessels. Our study provides better insight into the resistance allocation in uveoscleral outflow, with the tracer distribution varying around the eyes in the SCS and CB, and higher tracer intensity was observed in the CB than in the SCS at all locations. This suggests that the CB potentially consists of a lower resistance outflow route compared to the SCS. 

In addition to identifying the segmental outflow pattern and correlation in both outflow pathways at different positions, our analysis indicated that unconventional outflow constitutes approximately 45% of the total outflow in mouse eyes, emphasizing its significance in the overall outflow dynamics. Our result (45%) closely aligns with the percentage of unconventional outflow to total outflow (42%) reported in mice of the same age range (2.5–4.5 months) and strain (C57BL/6J), albeit with differences in perfusion duration (10 minutes) and size of tracers (70-kDa FITC–dextran with 13-nm hydrodynamic diameter⁵⁰), using direct measurement.⁵¹ Our findings are also comparable to the unconventional flow proportions relative to total outflow calculated from the indirect estimation in other studies in mice (53%),⁵¹ monkeys (45%–70%),⁵² and young adults (54%).⁵³ However, it is essential to acknowledge the variability in uveoscleral outflow rates across the literature (e.g., 20%–35% in monkeys,^3,54 3% in cats⁵⁵) due to differences in measurement methods and factors such as species, age, tracers, and perfusion and post-injection duration, which are also limitations of the current study. 

Previous studies have demonstrated a time-dependent increase of fluorescence intensity across different anterior segment tissues in mouse eyes^42,47 and vortex veins in monkey eyes⁶ after intracameral injection of fluorescent tracers. Tracers were observed to traverse from the anterior (iris root and ciliary process) to the posterior (stromal of adjacent equatorial sclera) along the SCS.⁴² This underscores the impact of the perfusion and post-injection duration on the amount of tracer reaching the SCS. Moreover, different tracer sizes and diffusion coefficients can affect the penetration and flow rate within the outflow system.^6,47,56 In this study, we adapted the tracer size (20 nm) and injection protocol (45 minutes post-injection) from previous investigations into conventional outflow patterns^25,29 to maintain consistency and comparability with existing research. The protocol was optimized for studying the conventional outflow²⁵ but might not be ideal for investigating unconventional outflow and the relationship of tracer intensity in the SCS and CB. Further studies with various post-injection times and tracer sizes are warranted to establish a more comprehensive understanding of uveoscleral outflow. 

In summary, this study utilized a novel quantitative method by measuring tracer intensity in two separate images at each of the 12 clock-hour positions in each eye to investigate the flow patterns of unconventional and conventional outflows and their interaction. Our work, for the first time, to the best of our knowledge, demonstrates the segmental flow pattern in both outflows with varied statistical relationships at different clock-hour positions, highlighting the complexity of aqueous humor drainage. These findings provide a foundational understanding of the patterns of uveoscleral outflow, their interaction with conventional outflow, and their potential implications for glaucoma treatment strategies. Future studies should explore the entire unconventional outflow patterns with different tracer sizes and perfusion/post-injection durations; investigate morphologic, proteomic, and genetic distinctions characterizing the high and low flow regions; and identify optimal locations for placement of drainage devices and drug administration. 

Acknowledgments

Supported by a grant from the BrightFocus Foundation (G 2022013S); by a Boston University School of Medicine Wing Tat Lee award; by the Rifkin Family Glaucoma Research Fund; by a grant from the National Institutes of Health (EY022634); and by the Massachusetts Lions Eye Research Fund. 

Disclosure: H.-L. Li, None; R. Ren, None; H. Gong, None