Giant Vacuoles Are Found Preferentially near Collector Channels

Christine E. Parc; Douglas H. Johnson; Harilaos S. Brilakis

Abstract

purpose. To determine whether giant vacuoles form preferentially near collector channels or over regions of optically empty space within the juxtacanalicular tissue (JCT).

methods. To assess the relationship between giant vacuoles and collector channels, six eyes were perfused with phosphate-buffered saline (PBS) at 20 mm Hg and then fixed by perfusion. Serial sections were cut in the frontal plane and light microscopy used to count the number of giant vacuoles per length of Schlemm’s canal. The number of giant vacuoles between two adjacent collector channels was determined. To assess the relationship between giant vacuoles and the ultrastructure of the JCT, an additional seven eyes were perfused with PBS at 10 mm Hg, fixed by perfusion, and examined by transmission electron microscopy. The ultrastructural components of the JCT were quantitated with an image analysis system.

results. Twice as many giant vacuoles were present in regions underlying collector channels as in regions between channels (giant vacuoles per histologic section: 14.0 ± 1.7 versus 7.3 ± 0.8, P = 0.01). Giant vacuoles occurred on both the inner and outer walls of the canal but were more numerous on the inner wall (9.1 ± 1.0 versus 2.6 ± 0.4, P < 0.001). No significant increase in optically empty space was found in the JCT regions underlying giant vacuoles compared with regions with no vacuoles (50.7% ± 2.3% versus 47.3% ± 2.5%, P = 0.09). Examination of the amount of optically empty space immediately adjacent (within 1 μm) to the inner wall endothelial cells of the canal did not reveal a significant difference between regions under vacuoles and regions without giant vacuoles.

conclusions. Giant vacuoles are found preferentially near collector channels, indicating that aqueous flow across the inner wall is sensitive to downstream pressure. The variability in giant vacuole distribution noted in previous studies is in part due to the distance of the vacuoles from the collector channels. No distinct findings in the JCT were associated with the presence of giant vacuoles.

Afundamental question in the study of aqueous outflow is that of which factors control aqueous drainage into Schlemm’s canal. At least half the normal aqueous outflow resistance occurs in the trabecular meshwork,¹ ² ³ ⁴ and all the increased resistance to aqueous outflow in primary open-angle glaucoma occurs in the trabecular meshwork.¹ The most likely anatomic barriers to outflow are the continuous endothelial lining of Schlemm’s canal and the underlying juxtacanalicular tissue (JCT). The mechanism of aqueous entry into the canal across the endothelial cells is unknown. Those proposed include both intercellular and transcellular routes.⁵ ⁶ ⁷ ⁸ ⁹ Transcellular aqueous flow probably occurs through giant vacuoles in the endothelial lining of the canal. Giant vacuoles are pressure-sensitive structures, increasing in number as intraocular pressure increases.⁶ ⁷ ⁸ The significance of giant vacuoles has been controversial, however, and an alternate route of aqueous entry into the canal, the intercellular pathway, has been suggested.⁹ Whether aqueous passes directly through giant vacuoles, or whether the vacuoles serve to stretch the endothelial cells and loosen the intercellular junctions, allowing increased intercellular flow, the pressure-sensitive nature of the vacuoles indicates that their presence may serve as a marker for regions of active aqueous flow. This is supported by a recent study that found that most giant vacuoles are short-lived structures once perfusion pressure decreases, the majority lasting less than 3 minutes.¹⁰

All studies of giant vacuoles comment on the marked variability in their distribution.⁷ ⁸ ¹¹ ¹² ¹³ ¹⁴ Some regions of Schlemm’s canal have a few vacuoles, whereas other regions contain many. If giant vacuoles represent areas of active fluid drainage, this variability indicates that not all the inner wall functions simultaneously in draining aqueous. Why, then, do giant vacuoles form where they do? Three explanations are possible: They may form over aqueous pathways within the JCT and meshwork; they may form in regions of lower downstream resistance, such as near the entry of collector channel into Schlemm’s canal; or they may form in regions of greatest pull or stretch on the inner wall, as may occur with contraction of the ciliary muscle.

We sought to determine whether giant vacuoles form preferentially near two obvious anatomic features: collector channel ostia, and optically empty space within the JCT.

Materials and Methods

The study was conducted in two parts, using different sets of eyes and examining the relationship between giant vacuoles and collector channels and the relationship between giant vacuoles and the ultrastructural characteristics of the JCT.

Tissue Processing

Collector Channels.

Six normal human eyes were enucleated at autopsy within 24 hours of death (mean time: 13.3 ± 7.7 hours; range, 5–23 hours). The average donor age was 65.3 ± 9.5 years (range, 50–72 years). Eyes were first perfused with Dulbecco’s phosphate-buffered saline (PBS; Sigma, St. Louis, MO) with 5.5 mM glucose for 2 hours before perfusion with fixative. This initial saline perfusion was performed to establish fluid flow through the trabecular meshwork, allowing any cyclic giant vacuole formation to reach an equilibrium before fixation. Studies in our laboratory have found fewer giant vacuoles in eyes that were immediately perfused with fixative compared with eyes perfused initially with saline (unpublished). Eyes were then fixed by intracameral perfusion with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Fixation continued overnight. The perfusion pressure for both the initial PBS perfusion and also the fixative was 20 mm Hg. This pressure would correspond to an in vivo pressure of 29 mm Hg and was specifically chosen for this study to maximize the number of giant vacuoles. Vacuole counts increase with increased intraocular pressure,⁶ ⁷ ⁸ and larger numbers of vacuoles give a greater statistical power to the study. In addition, this pressure was chosen to minimize the variability in vacuole counts around the circumference of the eye. The coefficient of variation (CV) is larger in eyes fixed at lower pressures.⁶ ⁷ ⁸ Higher perfusion pressures were not chosen, because the canal can collapse at higher pressures.

Eyes were dissected into quadrants, and wedges from each quadrant were rinsed overnight in 0.1 M phosphate buffer, dehydrated in ascending concentrations of alcohol, and embedded in JB4 plastic (Ted Pella, Inc., Redding, CA). A preliminary study of histologic section thickness compared 2-, 5-, and 10-μm-thick sections and found that 5-μm-thick sections provided adequate resolution of histologic details while minimizing the number of serial sections required. At least one tissue block from each eye had serial sections made in the frontal plane to encompass all of Schlemm’s canal from anterior to posterior. This required 40 to 60 5-μm-thick sections. Sections were stained with 1% toluidine blue and examined until two adjacent collector channel ostia entering Schlemm’s canal were found on the same histologic section (Fig. 1) . At least six serial sections were required to encompass the entire opening of the two collector channel ostia. Giant vacuoles were counted on each of these histologic sections.

Frontal sections were used because it was possible to find a histologic section that contained the entry of two adjacent collector channels with this plane of sectioning (Fig. 2) . It was then simple to count the number of giant vacuoles in these sections, because their precise relationship to the collector channel ostia could be determined. Approximately 30 collector channels are present in each eye,¹⁵ spaced approximately 1 to 1.5 mm apart. Use of conventional sagittal sections would require serial sectioning of this entire 1-mm distance, entailing 1000 serial 1-μm sections, or 200 serial 5-μm sections. Frontal sections, however, can be at least 1 mm wide (actually, 3 mm wide in our series) and therefore cover the distance between collector channels in a single section. Thus, serial sectioning of only the 300-μm anterior to posterior width of the canal was required or approximately 60 sections each 5-μm thick. Note that the anterior to posterior dimension of the canal is actually the width when the circumferential nature of the canal is considered; the canal is approximately 36 mm long (circumference).¹⁶

JCT Characteristics.

Seven normal human eyes were enucleated at autopsy within 12 hours of death. The average age of the donor eyes was 79.9 ± 10.0 years (range, 69–96 years). Eyes were first perfused with PBS for at least 3 hours at 10 mm Hg before fixation overnight at a pressure of 10 mm Hg in the fixative described earlier. The mean (± SEM) time from death to fixation was 22 ± 5 hours. After fixation, eyes were bisected at the equator and the anterior segments dissected into quadrants. Wedges of the limbal region including the trabecular meshwork were taken from one quadrant of each eye, dehydrated in ascending alcohols, and embedded in Araldite 502 (Ted Pella, Redding, CA). Thin sections were cut for transmission electron microscopy and mounted on 135 hexagonal mesh grids. Grids were stained with uranyl acetate and lead citrate and examined by microscope (model 1200; JEOL, Peabody, MA) electron microscope.

Morphologic Analysis

Collector Channels.

Giant vacuoles were counted using the method of Grierson and Lee ⁸ : light microscopy at ×1000 magnification, with giant vacuoles defined as “smooth-walled, round, oval, reniform or crescentic spaces,” either within one cell or forming at the junction of two cells of the endothelial lining of Schlemm’s canal (Fig. 3) . Vacuoles were counted on both the inner wall and the outer wall of the canal. Giant vacuoles were easily identified by light microscopy at this magnification, as reported in numerous studies.⁶ ⁷ ⁸ ¹⁰ ¹¹ ¹² ¹³ ¹⁷ Transmission electron microscopy was not required to identify giant vacuoles and indeed would have required thousands of serial sections to complete this study. Scanning electron microscopy is unsuitable for counting giant vacuoles, because it is impossible to distinguish a giant vacuole from a bulge caused by a nucleus.¹⁸ In addition, pores within giant vacuoles are not a reliable marker to distinguish vacuoles from nuclei, because pores may be hidden between cells, and not all vacuoles have pores.¹⁸

The relationship between giant vacuoles and collector channels was determined first by measuring the distance between the two collector channel ostia with a graticule (chord length from the middle of the ostium of each collector channel). This distance was then subdivided into 22-μm segments and the number of vacuoles in each 22-μm segment recorded.

The size of the collector channels was determined by measuring the ostia of the lumen at its entry into Schlemm’s canal and also the luminal diameter 20 μm from the canal. The anterior–posterior dimension of the lumen was determined by counting the number of 5-μm sections in which a patent lumen was visible.

JCT Characteristics.

The JCT was defined as the tissue underlying Schlemm’s canal, extending from Schlemm’s canal endothelial cells to the empty space adjacent to the first trabecular lamella. Schlemm’s canal endothelial cells and giant vacuoles were not included in the JCT area.

The protocol used has been described in several other studies.¹⁹ ²⁰ In brief, one quadrant from each eye had orientation micrographs of the juxtacanalicular region taken at ×600 and four micrographs at ×2500: one photograph of the anterior canal region, two of the middle, and one of the posterior region. Multiple quadrants were not examined from each eye because the comparison of interest was between regions underlying giant vacuoles and adjacent regions without vacuoles. In this situation, the immediately adjacent, nonvacuole region serves as the control for the nearby vacuole region. The study was not intended to determine the average composition of the JCT, which would require examination of multiple quadrants for each eye.¹⁹ Micrograph negatives were digitized and quantitatively analyzed (KS400 Image Analysis System; Carl Zeiss, Thornwood, NY). The amount of basement membrane, tendon and sheath material, cytoplasm, and miscellaneous elements of the entire JCT region were traced by hand. The total amount of optically empty space was determined by subtracting the amount of solid tissue. The computer was then programmed to draw lines 1 μm, 3 μm, 5 μm, and 7 μm from the inner wall and analyze the tissue within these areas. These lines were drawn because the entire juxtacanalicular region ranges from 7 μm to 10 μm thick, and it is possible that the regions nearest the inner wall are the most important in aqueous outflow.²¹ ²² ²³

Giant vacuoles were categorized into two types. The most common vacuoles were completely encircled by cytoplasm and appeared as intracytoplasmic, membrane-lined, empty spaces at least 2 μm in diameter (resembling a signet ring; termed type I vacuoles). Type II vacuoles were similar to type I but had a basal opening to the underlying JCT. This opening was termed a meshwork pore by Grierson and Lee.⁷ Regions of the inner wall that appeared elevated off of the underlying basement membrane (ballooning²⁴ ) were not considered to be giant vacuoles. To be classified as vacuoles, the structures had to have some cytoplasmic infolding at their basal aspect (Fig. 4) .

The boundaries of the giant vacuoles along the inner wall were marked and the area directly under the vacuoles analyzed separately by the computer program as subregions within the JCT (Fig. 3) . For the purposes of this study, it was assumed that aqueous flow would be directly toward the inner wall of the canal through the JCT, with minimal lateral or circumferential flow within the juxtacanalicular region.

Statistical Analysis

Collector Channels.

Giant vacuoles were counted on each 5-μm-thick section and their location in relation to the collector channel ostia recorded. The distance between collector channel ostia was divided into thirds, and the number of vacuoles in each third was recorded. Vacuoles in the middle third were farthest away from a collector channel ostium (Fig. 3) . A paired two-tailed t-test was used to compare the mean number of vacuoles in the region under a collector channel ostium with the number of vacuoles in the middle third distance between adjacent collector channel ostia. This comparison was made for vacuoles along the inner wall, the outer wall, and both combined. Reproducibility was determined by recounting giant vacuoles on 20 randomly selected sections at least 1 week after the initial examination. The mean of these vacuole counts was within 9% of the initial count.

JCT Characteristics.

Measurements from each of the four micrographs per tissue section were combined for each section of one eye, keeping vacuole and nonvacuole subregions separate. The amount of optically empty space, basement membrane material, tendon and sheath material (SD plaque),²⁵ cytoplasm, and miscellaneous material (pigment granules, for example) was normalized in comparison with the sample area. Data from all eyes were combined to determine the range of values, mean, SD, and CV (CV = ς/x). Values are expressed as mean ± SEM. Comparisons were made using a paired, two-tailed t-test for regions under giant vacuoles and regions without vacuoles.

The study had a 90% power of detecting a 6.8% difference in the amount of optically empty space underlying giant vacuoles and regions with no giant vacuoles.

Results

Collector Channels

Twice as many giant vacuoles were present in regions underlying collector channel ostia as in the region between channel ostia (giant vacuoles per histologic section: 14.0 ± 1.7 versus 7.3 ± 0.8, P = 0.01; mean ± SEM). Expressed another way, the middle third region between adjacent collector channel ostia contained 21% ± 0.9% of the total number of vacuoles, whereas each region under a collector channel ostia contained 40% ± 4.9% of the vacuoles (mean ± SEM, P = 0.02). The null hypothesis predicts that each region should contain equal numbers, or 33%, of the vacuoles.

Figure 5 shows the distribution of vacuoles between collector channels for one eye, summing all histologic sections for that eye. Although the statistical analysis of vacuoles was performed for the vacuoles between collector channel ostia, vacuoles distal to this region were also counted. These counts are also included in Figure 5 to allow the pattern of vacuole distribution to be visualized. However, because the location of other collector channels was not known, these distal vacuoles were not included in the statistical analysis. Note that the pattern of vacuole distribution resembles a sine wave, with the peaks under the collector channel ostia.

Giant vacuoles were present on both the inner and outer walls of the canal but were more numerous on the inner wall (9.1 ± 1.0 versus 2.6 ± 0.4, P < 0.001, includes all regions). Figure 6 shows the distribution of giant vacuoles on the inner and outer walls. Approximately 20% of the vacuoles were found on the outer wall. The distribution of vacuoles among the serial sections from a quadrant was less variable along the inner wall than the outer wall (CV = 18.8% ± 9.5% versus 53.2% ± 33.2%, P = 0.04).

The size of the collector channel did not appear to make a difference in the number of vacuoles. Neither the size of the luminal entry (ostium) into Schlemm’s canal, nor the size of the lumen within the sclera 20 μm from the entry into the canal was correlated with the number of vacuoles (data not shown).

Schlemm’s canal was open and had not collapsed in all histologic sections. Collapse of the canal can occur at higher perfusion pressures.⁶ ¹² ¹³ In a preliminary study of human eyes perfused at 40 mm Hg in our laboratory, canal collapse was evident in regions between collector channels (Johnson, unpublished data, 1999).

JCT Characteristics

Analysis of the entire thickness of the JCT revealed no significant difference between the amount of optically empty space underlying giant vacuoles and that in areas without giant vacuoles (50.7% ± 2.3% versus 47.3% ± 2.5%, mean ± SEM; P = 0.09; Fig. 4 ). No significant difference in the amount of optically empty space was found between type I and type II vacuoles (Table 1) . No significant difference in the amount of basement membrane material was noted between areas of the JCT underlying vacuoles in regions without giant vacuoles. Similarly, no significant differences were found between the amount of tendon sheath material or cytoplasm in regions underlying giant vacuoles and in regions without vacuoles (data not shown).

Analysis of the subregions of the JCT that were defined by the computer at either 1, 3, 5, or 7 μm from the inner wall of the canal did not reveal significant differences in regions underlying vacuoles and in regions without vacuoles. No significant increase in the amount of optically empty space was found underlying giant vacuoles, even in the 1-μm layer closest to the canal (Table 1) . No significant difference was found between type I and type II vacuoles for the amount of optically empty space or basement membrane material in this 1-μm layer or any of the other layers.

Considering regions with and without giant vacuoles together, less optically empty space was present in the 1-μm layer adjacent to the canal than in the regions farther away from the canal (41.1% ± 2.8% versus 48.6% ± 2.2%, P = 0.001). More basement membrane material was present in this layer adjacent to the canal than in the remainder of the juxtacanalicular region, as expected from qualitative observation of this region (21.1% ± 4.4% versus 7.9% ± 2.3%, P = 0.004).

Discussion

Larger numbers of giant vacuoles were found in regions near collector channel ostia than in regions between collector channels. Giant vacuoles thus seem to form preferentially near collector channel ostia. If giant vacuoles are markers of aqueous flow across the inner wall, it suggests that aqueous flow into Schlemm’s canal is not evenly distributed throughout the inner wall but occurs preferentially in certain areas. Such segmental flow of aqueous through the meshwork may be the cause of the patchy distribution of pigment often observed clinically during gonioscopy. This is consistent with a study that found that segmental pigmentation of the meshwork was related to collector channels.²⁶ These results imply that studies assessing the histologic findings in glaucoma might have different outcomes if areas of higher aqueous flow were examined (near collector channel ostia) rather than regions of lower aqueous flow.

The relationship between giant vacuole numbers and collector channel entry into Schlemm’s canal gives the first bit of anatomic evidence for understanding aqueous flow across the inner wall of the canal. Flow across the inner wall is sensitive to the downstream pressure. Giant vacuoles do not form entirely randomly along the inner wall but form preferentially in regions of the greatest pressure decrease. This pressure decrease appears in regions of collector channel ostia, as aqueous flows along a pressure gradient from the canal into the collector channels and then to the aqueous veins.

The association between the collector channel and giant vacuoles may also explain the large variability found in previous studies in the distribution of giant vacuoles around the circumference of the eye.⁶ ⁷ ⁸ ¹¹ ¹⁴ Most studies of giant vacuoles find a marked variability in vacuole numbers among histologic sections, with CVs ranging from 37% to 97%.⁶ ⁷ ⁸ ¹¹ ¹⁴ Of interest, the present study found that histologic sections near collector channel ostia, but not containing the ostium itself, still had higher numbers of vacuoles than regions farther from the collector channels (Figs. 3 4) . Thus without knowing the distance of the histologic section from the nearest collector channel ostium, the variability in giant vacuole numbers among sections cannot be understood. Size of the collector channel ostia did not appear to influence the results. Without a direct cannulation of the collector channels; however, the pressure and flow within collector channels of different sizes cannot be assessed.

If giant vacuoles were relatively stagnant structures and remained formed even when a pressure drop across the cell no longer existed, these study results would be less meaningful in understanding aqueous flow across the inner wall. In this scenario, giant vacuoles could remain for some time where they had formed, even if the pressure decline no longer existed. Giant vacuoles would then represent a time exposure of aqueous flow, rather than a snapshot of pressure conditions across the inner wall. In a previous study, however, giant vacuoles were found to be short-lived structures once perfusion pressure decreased to zero, with the majority disappearing within 3 minutes.¹³ In that study, 75% of the predicted number of giant vacuoles had disappeared within the first 3 minutes after intraocular pressure was reduced to zero, and by 120 minutes, vacuole numbers were only 10% of predicted values.¹⁰ Thus, the expected short survival time of a giant vacuole once perfusion pressure decreases suggests that the giant vacuoles are markers of active aqueous flow conditions.

An alternate but less likely interpretation of our finding that giant vacuoles form preferentially near collector channel ostia could instead relate to upstream pathways within the JCT of the trabecular meshwork. If such pathways existed, they would have to terminate exactly opposite the ostium of collector channels to explain the relationship with giant vacuoles that we found. It is unlikely that such coincidences would be so consistent as to produce the statistical significance found in this study. The second part of the study examined the JCT to determine whether any aqueous flow pathways, evidenced by the amount of optically empty space underlying giant vacuoles, were present. No significant difference was found in the amount of optically empty space in regions with and without vacuoles. Recent work using a different method of tissue processing, the quick-freeze, deep-etch technique, suggests that the optically empty space within the JCT contains extracellular material not visualized with conventional electron microscopy.²⁷ Such material could reduce the permeability of these presumed empty spaces, in keeping with our finding no preferential formation of giant vacuoles over these spaces. The amount of basement membrane material and other extracellular components was not significantly different between the JCT region underlying vacuoles and the regions without vacuoles.

Together, these studies indicate that giant vacuoles, which are known to be pressure-sensitive structures, form preferentially, but not exclusively, near collector channel ostia, presumably regions of lower downstream pressure.

Submitted for publication August 6, 1999; revised February 28, 2000; accepted March 31, 2000.

Commercial relationships policy: N.

Corresponding author: Douglas H. Johnson, Department of Ophthalmology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. [email protected]

Figure 1.

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Micrograph of frontal section of Schlemm’s canal with two collector channels. Inset: Giant vacuoles (arrowheads) in a different section than in the main photograph. Toluidine blue; magnification, ×400. AC, anterior chamber; SC, Schlemm’s canal; CC, collector channels; S, sclera.

Figure 2.

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Frontal sectioning of Schlemm’s canal.

Figure 3.

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Transmission electron micrograph of both types of giant vacuoles (GV). Type I appear as empty spaces (ES) within cells, completely encircled by cytoplasm. Type II are not completely encircled by cytoplasm. For purposes of this study, vacuoles were differentiated from bulges or uneven portions of the inner wall by size (vacuoles must be at least 2μ m in diameter) and by the presence of some cytoplasmic infolding at their basal aspect. Regions below giant vacuoles are delineated by parallel lines. Original magnification,× 6250.

Figure 4.

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Transmission electron micrograph showing optically empty space (ES) and tissue components within the JCT. BMM, basement membrane; N, nucleus; JCT, juxtacanalicular tissue; GV, giant vacuole (I and II denote vacuole types). Original magnification, ×6250.

Figure 5.

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Giant vacuole counts on inner and outer walls of eye UM53-97R. Each region is 22 μm in length. CC, collector channel; GV, giant vacuole.* Middle third has fewer vacuoles (P = 0.01).

Figure 6.

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Giant vacuole counts on both inner (IW) and outer (OW) walls of all six eyes. More vacuoles are found on the inner wall. GV, giant vacuole; CC, collector channel. *Middle third has fewer vacuoles (P = 0.01).

Table 1.

View Table

Juxtacanalicular Tissue and Giant Vacuoles

Table 1.

Juxtacanalicular Tissue and Giant Vacuoles

	Giant Vacuole Type					No Giant Vacuoles	Total
		I + II	I	II		No Giant Vacuoles	Total
Optically empty space
Total (% of total JCT)	50.7 ± 2.3	50.3 ± 3.6	50.4 ± 2.7	47.3 ± 2.5	48.6 ± 2.2
1 μm (% of 1-μm region from SC)	36.7 ± 2.6	36.6 ± 2.4	35.1 ± 3.1	39.7 ± 3.1	41.1 ± 2.8
Basement membrane material
Total (% of total JCT)	8.1 ± 2.3	8.7 ± 3.8	7.1 ± 2.1	7.1 ± 2.3	7.9 ± 2.3
1 μm (% of 1-μm region from SC)	19.7 ± 3.5	20.7 ± 3.8	14.1 ± 4.0	19.3 ± 4.7	21.1 ± 4.4

SC, Schlemm’s canal. Data are means ± SEM.

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