Molecular Vision 2019; 25:517-526
Received 13 December 2018 | Accepted 19 September 2019 | Published 21 September 2019
1Structural Biophysics Research Group, School of Optometry and Vision Sciences, Cardiff University, Wales, UK; 2Department of Bioengineering, Imperial College London, London, UK; 3Department of Ophthalmology, Kyoto Prefectural University of Medicine, Hirokoji Kawaramachi, Kamigyo-ku, Kyoto, Japan
Correspondence to: Andrew J Quantock, Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cardiff CF24 4HQ, Wales, UK; +44 (0)29 2087 5064 FAX: +44 (0)29 2087 4859; email: QuantockAJ@cf.ac.uk
Purpose: Increased resistance of aqueous humor drainage from the eye through Schlemm’s canal (SC) is the basis for elevated intraocular pressure in glaucoma. Experimental evidence suggests that the bulk of outflow resistance lies in the vicinity of the inner wall endothelial lining of SC and the adjacent juxtacanalicular tissue (JCT). However, there is little understanding of how this resistance is generated, and a detailed understanding of the structure-function relationship of the outflow pathway has not been established yet. In the present study, regional variations in the ultrastructure of the JCT and the inner wall of SC were investigated in three dimensions.
Methods: With the use of serial block face scanning electron microscopy (SBF-SEM), the volume occupied by the electron lucent spaces of the JCT compared to that occupied by the cellular and extracellular matrix was investigated and quantified. The distribution of giant vacuoles (GVs) and pores in the inner wall endothelium of SC was further examined.
Results: With increasing distance from the inner wall of SC, the volume of the electron lucent spaces increased above 30%. In contrast, the volume of these spaces in immediate contact with the inner wall endothelium was minimal (<10%). Circumferential variability in the type and distribution of GVs was observed, and the percentage of GVs with pores varied between 3% and 27%.
Conclusions: These studies provide a detailed quantitative analysis of the ultrastructure of JCT and the distribution of GVs along the circumference of SC in three dimensions, supporting the non-uniform or segmental aqueous outflow.
Elevated intraocular pressure (IOP) associated with primary open-angle glaucoma is caused by increased aqueous humor outflow resistance [1-3]. While the bulk of outflow resistance lies near the downstream end of the trabecular meshwork (TM) and the inner wall endothelium of Schlemm’s canal (SC) [4-8], there is little understanding of how this resistance is generated, or why this resistance becomes elevated in glaucoma. Understanding the mechanism of resistance generation, therefore, may inform novel approaches to target the mechanisms of outflow resistance generation as a means of reducing IOP in glaucoma.
The inner wall endothelium of SC lies within a biomechanically demanding environment, where it experiences large cellular deformations because of the peculiar nature of aqueous humor flow across the endothelium . Because the flow passes in the basal-to-apical direction across the inner wall, the pressure drop between the IOP and the episcleral venous pressure deforms the endothelial cells lining SC, causing them to form dome-like bulges known as giant vacuoles (GVs) [9-13]. GVs are often associated with pores, which are micron-sized openings through the endothelium. Pores may pass either through transcellular “I” pores or between individual SC cells, paracellular “B” pores [14-17]. GVs and pores together are believed to provide a channel for transendothelial flow [16-22]. Importantly, the number of pores is reduced in glaucomatous eyes [19,20], suggesting that impaired function of the SC barrier may contribute to outflow obstruction associated with glaucomatous ocular hypertension.
Pores on their own, however, are likely insufficient to generate a significant contribution to outflow resistance . Simply put, there are too many SC pores such that their net contribution to the hydrodynamic resistance of the inner wall should be too low to explain the bulk of outflow resistance generation. However, because pores are the pathway for flow across an otherwise continuous endothelium containing tight junctions, pores (or the mouth of a GV associated with a pore) represent local choke points where aqueous humor must converge, or “funnel,” to cross the inner wall [7,22-25]. According to the funneling model, extracellular matrix in the immediate vicinity of a pore or GV mouth would have a dominant effect on the generation of outflow resistance .
Although several investigators have examined the matrix composition in the juxtacanalicular tissue (JCT) [26-30], and quantified open spaces [18,31-37], few investigators have examined the relationship between optically open spaces and pores or GV mouths. The complex three-dimensional (3D) structure of the extracellular matrix upstream of the pore and/or GV may affect outflow resistance generation via funneling, but the structure of this tissue varies with distance from the inner wall, transitioning from a relatively dense (yet discontinuous) inner wall basement membrane to relatively loose ECM in the JCT. It is difficult to capture the 3D structure of this tissue using conventional two-dimensional sections. In this study, we used serial block face scanning electron microscopy (SBF-SEM) to examine the 3D ultrastructure of the JCT surrounding the basal openings of GVs and pores in the inner wall endothelium of SC in post-mortem human eyes.
One ostensibly healthy eye of a 69-year-old female donor with no report of previous ocular disease was obtained from the Bristol Eye Bank. Time of death to enucleation was 18 h, after which the eye was held overnight at 4 °C in a moist sterile chamber, and placed in 4% paraformaldehyde fixative the following morning. Careful dissection of the TM was performed by one of the authors (MU, a glaucoma surgeon) after which the tissue was post-fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 3 h at room temperature. To increase the backscatter electron signal for SBF-SEM, the tissue was infiltrated with 1% osmium tetroxide containing 1.5% potassium ferricyanide in 0.1 M sodium cacodylate for 1 h, and 1% tannic acid for an additional hour. Specimens were then infiltrated and polymerized in Durcupan epoxy resin (Agar Scientific, Stansted, UK). This study was approved by the School of Optometry and Vision Sciences Research and Ethics committee at Cardiff University, UK. Experiments were conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki, the ARVO statement for the use of human subjects, including human material and data, in biomedical research and in line with the requirements of the UK Human Tissue Act.
Following sample embedding, the tissue was first investigated with transmission electron microscopy. Initially, a preliminary histological analysis was performed by obtaining 0.3 µm thick sections using an Ultracut E ultramicrotome (Reichert-Jung, Vienna, Austria). Semithin sections were stained with 1% toluidine blue, and visualized under a Olympus BH-2 light microscope (Tokyo, Japan) to facilitate optimization of the plane of the section, and identification of a suitable area of interest (Figure 1A). Ultrathin, 100 nm thick sections were also cut, and investigated using a JEM 1010 transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kv. An 11-megapixel Orius SC1000 CCD camera (Gatan, Pleasanton, CA) was used to acquire images.
SBF-SEM was then performed using an FEI Quanta FEG 250 scanning electron microscope (Cambridge, UK) fitted with a Gatan 3View® system at the Wellcome Centre for Cell Matrix Research (Faculty of Life Science, University of Manchester, UK). Serial image data sets facilitated volume 3D reconstruction of the human TM nanostructure. The principle behind SBF-SEM is to repeatedly image the tissue block surface alternating with removal of a surface layer, in this case of 125 nm thickness, using a microtome located inside the microscope. A scan resolution of 4,096 × 4,096 pixels2 (Figure 1B) with a magnification of 27 nm/pixel was employed in this study, entailing 500 serial backscatter electron images, at 125 nm intervals, that were collected at 3.8 kv, with a dwell time of 10 µs. The total area of the inner wall of SC analyzed in the present study was 3,170 µm2. An area of 1,588 × 1,844 pixels2 of the inner wall of SC and the JCT was selected for quantitative ultrastructural analysis in the present study (Figure 1C).
The volume occupied by the optically empty spaces of the JCT against the distance from the borders of SC was calculated to investigate the spatial variation under the inner wall of SC. Additionally, we sought to explore the spatial variation along the circumferential direction of SC. To achieve this, we evaluated the areal fraction occupied by optically empty spaces within the JCT at various locations along SC (12.5, 25, 37.5, 50, and 62.5 µm) referenced to the arbitrary starting position of the tissue block sectioning (Figure 2A). At each location, four micrographs were analyzed to determine the volume of the electron lucent spaces (excluding cellular and extracellular matrix components) as a function of distance from the inner wall of SC. To do this, we first identified the basal surface of the inner wall endothelium. This was traced and adjusted manually for each micrograph. Using a macro, we then divided the JCT into six parallel portions that were located 2.7, 5.4, 8.1, 10.8, 13.5, and 16.2 µm from the inner wall (Figure 2B). For each portion, we traced and measured the area of the electron lucent space, and normalized by the total area of the segment.
The borders of the inner wall of SC varied along the circumferential direction of the canal, and within the four micrographs examined at each distance under the inner wall. Therefore, the borders of the canal were traced and adjusted manually for every distance and every micrograph analyzed. A macro was used to create six segments that had a distance of 2.7, 5.4, 8.1, 10.8, 13.5, and 16.2 µm from the borders of the inner wall of SC. For every segment, the total volume of all the electron lucent spaces was determined by manually tracing around the perimeter of each space, calculating its area and then summing the areas. These data were normalized against the area of the box in the occasional cases where the box partially exceeded the edge of the micrograph. Area measurements were converted into volumes by multiplying by the thickness of the cut section (125 nm). The volume of the electron lucent space in the JCT, averaged across all four tissue regions examined and expressed as a percentage, was then plotted as a function of the distance from the inner wall of SC. GVs throughout the entire data set (500 serial micrographs; tissue depth 62.5 µm) were also analyzed to ascertain whether they contained pores, and whether they opened to the JCT or the SC, or both.
Image processing, quantitative analysis, and 3D reconstructions were conducted with the use of Image J software package [38,39]. Selected sequences were presented in three dimensions using the 3D Viewer plugin of Image J.
The average volume occupied by the electron lucent spaces of the JCT at each distance upstream of the inner wall was used for statistical analysis using IBM SPSS Version 21.0 (SPSS Inc., Chicago, IL). The Shapiro-Wilk normality test, with p>0.05 showing normal distribution, was performed, followed by one-way ANOVA and the Holm-Sidak multiple comparisons test.
Transmission electron microscopy clearly identified the inner wall and lumen of Schlemm’s canal, and the underlying JCT and corneoscleral TM (Figure 3). High-resolution analysis of the ultrastructure of the JCT, achieved with SBF-SEM, revealed structural differences as a function of distance from the inner wall of SC (Appendix 1). These structural differences reflected variations in the tissue proportion occupied by the electron lucent, optically empty spaces, compared to that by the TM cells and extracellular matrix components (Figure 3, Appendix 1).
To analyse quantitatively the relationship between the volume occupied by the optically empty spaces and the ultrastructure of the JCT along the canal’s circumference, the data set was subdivided into 12.5 µm thick portions as described in the Methods section (Figure 2). This revealed no difference in the volume occupied by the electron lucent spaces of the JCT compared to that occupied by the cellular and extracellular matrix material as a function of the distance below the borders of SC. Moreover, a measure of the average volume of electron lucent spaces against the cellular and matrix material of the JCT at distances of 2.7, 5.4, 8.1, 10.8, 13.5, and 16.2 µm from the borders of the inner wall of SC was calculated (Figure 4). This showed that the percentage of the volume occupied by the electron lucent spaces at a distance of 2.7 µm from the inner wall of SC was statistically significantly different (7.47±1.04%, p<0.001) compared to more distant regions. At a distance of 5.4 µm and 8.1 µm from the borders of the inner wall, the percentage of the volume occupied by the electron lucent spaces was 26.0±2.2% and 32.8±1.8%, respectively. While the volume occupied by the electron lucent spaces increased statistically significantly within the proximal 8.1 µm (p<0.001) from the inner wall of SC, thereafter the proportion of cellular and extracellular matrix components was enhanced, and therefore, a decrease in the volume of electron lucent spaces was noted. Farther away from the borders of SC, at a distance of 16.2 µm, the volume of electron lucent spaces of the JCT increased again to 33.0±5.0%, p<0.001.
GVs along the circumference of the inner wall of SC were counted and categorized into four types (Appendix 2), based on the endothelial vacuolation cycle as suggested by Tripathi . A total of 40 GVs were tracked and manually segmented throughout the entire z-stack, a total of 500 serial micrographs. In one case, in which a GV was imaged at multiple slices throughout the z-stack and overlapped two SC circumference bins, the GV was excluded from analysis. Therefore, GVs were classified into four categories depending on whether the vacuoles exhibited: 1) no pores, or presented pores toward 2) the JCT region, 3) the lumen of SC, or 4) toward the JCT and SC (Figure 5A). The distribution and type of GV, however, were not evenly distributed throughout the entire circumference of the canal. In particular, a tissue depth of 12.5, 25, 37.5, 50, and 62.5 µm contained 27%, 8%, 22%, 8%, and 3% GVs with pores compared to the total GVs within each region, respectively (Figure 5B). Throughout the entire data set and a total tissue depth of 62.5 µm, the majority of the GVs had pores, toward either the JCT or the lumen of SC, or both (Figure 5C).
The type of GV, whether it had no pores, or pores opening toward the JCT, or the lumen of SC, or both, also appeared to vary within each region (Figure 5C). For instance, at 12.5 µm along the circumference of the canal, all four types of GV were present, whereas at 62.5 µm, only two types could be observed; GV with no pores and GV with pores toward the lumen of the canal.
The predominant drainage pathway for aqueous humor is the conventional outflow pathway which consists of the TM, the JCT, the inner wall endothelium of SC, and the collector channels/aqueous veins [2,8,22,23]. Within the conventional outflow route of aqueous humor drainage, the bulk of outflow resistance is generated by the endothelial lining of SC and the adjacent JCT [6-8,40-42]. The funneling model has emerged as a hydrodynamic model that supports a synergistic outflow resistance generation in which pores in the inner wall , often associated with GVs, affect flow patterns in the conventional outflow route [37,43-48]. Yet, still unknown today are the specific mechanisms and the hydrodynamic details of how outflow resistance is generated and regulated. Despite numerous physiologic, pharmacological, and morphometric studies [49-55] a detailed understanding of the structure-function relationships of the outflow pathway, over large tissue volumes, has not been established. In this study, SBF-SEM was used to investigate a large area of the conventional outflow pathway, with a particular focus on the fine structure of the JCT and the GVs in the inner wall endothelium. Using this approach, a better characterization of regional variations in the ultrastructure of JCT was obtained, supporting the non-uniform or segmental aqueous outflow.
With the reported importance of the JCT in outflow resistance, numerous empirical studies calculated the amount of empty spaces or solid tissue within the JCT, in normal and glaucomatous tissues, including immersion- and perfusion-fixed samples [18,32,56-58]. Of these studies, the variability of the components of the JCT along the circumference of the JCT along the circumference of the eye was highlighted [18,32,56-58], emphasizing that large tissue specimen examination and sample size are crucial. The present study used immersion fixation to prepare the tissue, because others have found that perfusion fixation can lead to an artificial increase in the inner wall pore density [15,58-62]. Moreover, Braakman and associates  recently indicated that immersion fixed samples preserve pores and GVs along the inner wall of SC.
This study exceeded the limitation of two dimensions, and by using SBF-SEM, a powerful technique which enables 3D visualization of large tissue volumes at high resolution, examined the relationship between the volume occupied by the optically empty, electron lucent spaces of the JCT and the distance upstream of the inner wall endothelium. With increasing distance from the inner wall of SC, the volume of the electron lucent spaces increased above 30%. In contrast, the volume of these spaces in immediate contact with the inner wall endothelium was minimal (<10%). In accordance with previous morphometric studies of the JCT [6-8,32], these data suggest that within the JCT, the region in close proximity to the inner wall of SC might be attributable to outflow resistance regeneration. Simply put, the volume of the electron lucent spaces further away from SC is too big to statistically significantly contribute to outflow resistance, unless these spaces contain an unobserved extracellular matrix gel . These findings are also in agreement with an elegant study by Lϋtjen-Drecoll in 1973, which showed a positive correlation between the outflow facility and the area of the electron lucent spaces in the subendothelial region of SC . This is additionally supported by previous theoretical studies which characterized the subendothelial region of the JCT as the “locus generis” of aqueous humor outflow resistance . Of interest, the ultrastructure pattern of JCT is indistinguishable along the circumference of the canal, at least for up to a distance of 50 μm from the arbitrary starting point, examined in this study.
The fact that the subendothelial JCT region is nearly depleted by electron lucent spaces, and the volume of these spaces increases with increasing distance from the canal, is of particular significance. Variation in the volume occupied by the optically empty spaces across the JCT thickness aids the establishment of a segmental outflow which will impact the outflow facility. Therefore, it is reasonable to speculate that this specific tissue ultrastructure acts to increase outflow resistance in the subendothelial region of SC. This particular tissue ultrastructure may further provide a supportive barrier, preventing collapse of SC, caused by uncontrolled outflow. An advantage of this study is that the volume of the empty spaces was measured in three dimensions, as opposed to previous investigations that were limited in two-dimensional area measurements [32,56,57,63]. Although JCT morphometric volume measurements potentially provide excellent accuracy, data consolidation by future studies with bigger sample numbers is a must.
The second part of this study examined the type and distribution of GVs along the circumference of SC. Circumferential variability in the type and distribution of GVs was observed. Taking into consideration that GVs and pores associated with them are thought to represent areas of active fluid drainage, and have been proposed to form preferentially near collector channels , these data reinforce previous experimental evidence supporting that aqueous flow is not evenly distributed throughout the inner wall [17,65-67]. Although the exact mechanism that drives the formation of GVs and pores is not well understood, biomechanical strain [16,17] and pressure drop across the inner wall have been implicated as underlying factors [11,15,19,21,68]. In a recent study, Lai and colleagues, using SBF-SEM demonstrated that a decrease in cellular connectivity in the inner wall and JCT is associated with larger GV formation .
Taken together, these studies provide a detailed quantitative analysis of the ultrastructure of JCT and distribution of GVs along the circumference of SC. To the authors’ knowledge, this correlation between the volume occupied by the electron lucent spaces and the distance upstream of the inner wall provides the first evidence from 3D observations, in our attempts to understand tissue-structure function relationships with respect to aqueous flow across the JCT. Although this study is preliminary, these findings are sufficiently promising to merit further development. Taking into account the variability among human eyes, future work with larger samples is necessary. Clearly understanding the cellular, molecular, and mechanical mechanisms that regulate aqueous outflow, in combination with changes in tissue-specific ultrastructure in health and disease, could accelerate our ability to devise more specific future glaucoma therapies. The opportunity offered by SBF-SEM in studying large tissue volumes at high resolution is highlighted in the present study, revealing the 3D ultrastructure within the TM. We believe this approach will be invaluable in future work to study the conventional aqueous humor outflow pathway in three dimensions in health and disease.
Appendix 1. Supplementary video 1.
Appendix 2. Supplementary video 2.
Supported by a Cardiff University Professor Sir Martin Evans’ President's Studentship to EK, by a BBSRC Project Grant (BB/M025349/1) to AJQ, CK, and RDY, and by collaborative research grants from the Japan Society for the Promotion of Sciences and the Japan Eye Bank.