Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Investigative Ophthalmology & Visual Science, Vol. 33, No. 5, April 1992 Copyright © Association for Research in Vision and Ophthalmology The Relationship Between Pore Density and Outflow Facility in Human Eyes R. Rand Allingham, Annelies W. de Kater, C. Ross Ethier, P. John Anderson, Ellen Hertzmark, David L. Epstein The inner wall (IW) endothelial lining of Schlemm's canal was examined in six normal human eyes and four eyes with primary open angle glaucoma (POAG). Outflow facility was measured using constant pressure perfusion at 15 mmHg, eyes were fixed at 15 mmHg, and the IW endothelial lining was isolated and examined by scanning electron microscopy. Pore density, pore diameter, and bulge density were recorded by quadrant, and pore size and density were used to estimate IW endothelial facility, resistance, and hydraulic conductivity (facility per unit area). In POAG eyes, pores were less common (489 ± 172 vs 1437 ± 423 pores/mm2; P < .005) and appeared to be more unevenly distributed than in normal eyes. A regional analysis of pore density (by quadrant) failed to detect a significant difference between quadrants of normal or POAG eyes. Pore density was correlated with measured outflow facility in normal eyes alone (P < .02) and when normal eyes were pooled with POAG eyes (P < .001). The percentage of total resistance attributed to the IW endothelium was 5.8% in normals compared to 9.5% in POAG eyes. This indicates there is a greater pressure drop across the IW endothelium in POAG eyes, suggesting that an intrinsic difference in IW endothelial function exists between normal and glaucomatous eyes. However, this difference alone does not account for the decreased outflow facility in POAG eyes. IW endothelial hydraulic conductivity is markedly higher than that of other vascular endothelia. We hypothesize that this may protect the IW endothelial lining of Schlemm's canal from mechanical stress induced by the relatively high rate of transcellular fluid flow. Invest Ophthalmol Vis Sci 33:1661-1669,1992 The majority of aqueous humor exits the anterior chamber via the conventional outflow system, ie, the trabecular meshwork (TM). Perfusion studies have suggested that pathways exist for aqueous flow through the TM and into the lumen of Schlemm's canal.1"4 Garron et al reported the presence of "giant vacuoles" within the inner wall (IW) endothelial lining of Schlemm's canal.5 Holmberg first described transcellular pathways that ranged from 0.5-1.5 nm in diameter. These were later named pores.6 Tripathi subsequently proposed a mechanism for aqueous flow through the pores of the giant vacuoles into Schlemm's canal.7-8 From the Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, Massachusetts; and Biomedical Institute and Mechanical Engineering, University of Toronto, Toronto, Ontario, Canada. This work is supported in part by NEIEY01894; by a grant from National Glaucoma Research, a program of the American Health Assistance Foundation, Rockville, Maryland; and by a grant from the Massachusetts Lion's Eye Research Fund, Boston, Massachusetts. Submitted for publication: July 15, 1991; accepted October 31, 1991. Reprint Requests: R. Rand Allingham, MD, University of Texas Southwestern Medical Center at Dallas, 5161 Harry Hines Blvd., Dallas, TX 75235-8895. In a series of elegant perfusion experiments, Grant determined that the site of greatest resistance to aqueous outflow in normal and glaucomatous human eyes resided within the outer TM, made up of the corneoscleral meshwork, juxtacanalicular tissue, and inner endothelial lining of Schlemm's canal.910 In normal eyes, the outer TM accounted for approximately 75% of total resistance to aqueous outflow. Recently, using a microcanulation technique, Maepea and Bill determined that the outer TM contributes 90% of total resistance in nonhuman primate eyes.11 To localize the main sites of resistance within the TM, Bill and Svedbergh examined the IW endothelial lining of Schlemm's canal of normal human eyes with scanning electron microscopy (SEM).12 By analyzing pore density and pore size, these investigators estimated that the contribution of the endothelial lining of Schlemm's canal accounted for no more than 10% of total outflow resistance in the normal human eye. In a similar investigation in nonhuman primate eyes, Moseley et al estimated the IW endothelial resistance contributed 10-24% of total resistance.13 There has not been a similar investigation of the endothelial lining of Schlemm's canal in eyes with primary open angle glaucoma (POAG). In addition, in previous studies, aqueous outflow facility was not 1661 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933167/ on 05/10/2017 1662 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992 measured. Therefore, we have performed a morphometric analysis of the IW endothelial lining of Schlemm's canal in normal and glaucomatous human eyes by SEM, and have correlated measured outflow facility with morphologic appearance and calculated resistance of the IW endothelial lining of Schlemm's canal. Material and Methods Six normal human autopsy eyes from three donors (donor ages 62-84) and four POAG eyes from two donors (donor ages 73-88) were obtained from the National Disease Research Interchange, Philadelphia, PA, or the Foundation for Glaucoma Research, San Francisco, CA. The glaucoma eyes had a well documented history of POAG, including elevated intraocular pressure, glaucomatous visual field loss, and optic atrophy. Specific glaucoma treatment in all four POAG eyes consisted of a combination of pilocarpine, timolol, and dipivefrin. There was no history of ocular surgery, laser treatment, or ocular disease other than POAG in the eyes studies. All donor eyes were enucleated within 3 hr of death, stored in a moist chamber at 4° C, and perfused within 18 hr postmortem. Perfusion Technique Eyes were placed in a bath of Sorensen's phosphate buffered solution at 24°C. A 23 G inflow needle was inserted through the limbus, threaded through the pupil, and inserted into the posterior chamber, avoiding contact with the lens or ciliary body. The inflow needle was connected to a reservoir containing Dulbecco's phosphate buffered saline with 5.5 mM glucose at a height equivalent to 15 mmHg. A second 23 G outflow needle was inserted through the limbus, left in the anterior chamber, and attached to a reservoir containing the same solution at a height corresponding to 12 mmHg. The anterior chamber was exchanged with 0.5 cc of Dulbecco's phosphate buffered saline and the outflow needle was clamped. Outflow facility was measured using constant pressure perfusion at 15 mmHg.9 The eyes were perfused for 60 min or until the outflow facility was constant for at least 30 min, whichever was longer. The anterior chamber was exchanged with 0.5 cc of a fixative solution containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M Sorensen's phosphate buffered solution (pH 7.3), after which the outflow needle was clamped. The eyes then were further perfused at 15 mmHg with thisfixativefor an additional 60 min, after which they were partially bisected at the equator and immersion fixed in the same fixative solution overnight. Vol. 33 Tissue Preparation and Examination After fixation, the eyes were bisected and the posterior segment was examined for evidence of glaucomatous optic nerve atrophy. The lens was dissected from the anterior segment and two meridional wedges were taken from each quadrant. The IW of Schlemm's canal was isolated in the following manner. Meridional wedges were placed on edge and held in position by pins placed through the cornea anteriorly and the sclera posteriorly. Using an operating microscope, an incision was made into the anterior portion of Schlemm's canal, which was opened by the application of gentle traction on the ciliary body, allowing access to the posterior margin of the canal without damaging the inner endothelial surface. The resulting specimen thus consisted of iris, ciliary body, and the outer TM with the IW endothelial lining exposed. Specimens were dehydrated through graded alcohols, critical point dried, mounted on stubs, sputter coated, and examined using a JEOL JSM 35 or JEOL 6400 SEM (JEOL USA, Inc. Applications Laboratory, Peabody, MA). The IW endothelium of Schlemm's canal was photographed at X1800 using Polaroid (Cambridge, MA) 55 film. Ten micrographs were taken of each quadrant (40 per eye). Each micrograph was examined in a masked fashion for numbers of pores, pore diameter, and "bulges" (representing giant vacuoles or endothelial cell nuclei). Each micrograph was analyzed independently by two of us (RRA and AWdK) until interobserver variability was less than 10%. Openings in the endothelial lining were considered pores rather than artifactual tears if the edges were smooth and regular. Pore diameters were measured using a X10 measuring magnifier (Peak, Japan) with a reticle calibrated to 0.1 mm. The largest diameter was recorded for oval pores. Counts of bulges were repeated in a masked fashion until interobserver comparisons were within 10%. Pore density, bulge density, and calculated IW endothelial facility data were obtained for each quadrant to analyze regional variability. Whole eye pore density was calculated by averaging all quadrant pore densities for a given eye. (This method implicitly assumes that pore density is uniform in each quadrant.) Bulge density was calculated in the same manner. Pore density distributions were used to calculate IW endothelial facility for that quadrant, as described below. Quantitative Analysis The resistance to aqueous outflow contributed by the endothelial lining of Schlemm's canal was calculated in the manner described by Bill and Sved- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933167/ on 05/10/2017 No. 5 PORE DENSITY IN NORMAL AND GLAUCOMATOUS HUMAN EYES / Allinghom er ol bergh.l2 Briefly, the resistance toflowthrough a "pore invagination unit" is estimated as a function of pore diameter using elementary fluid mechanics. Pores are then grouped according to diameter, and all pores within a given size grouping are assigned a resistance based on the mean diameter within that group. The exception to this procedure are pores with diameters ^2 jum, which are assigned resistances corresponding to a diameter of 2 jum. This was considered to be a reasonable upper limit of the diameter of the abluminal opening to the giant vacuole. We used the same pore size groupings as Bill and Svedbergh. IW endothelial facility is calculated by multiplying the mean pore density in a given size range by facility per pore (defined as the reciprocal of the resistance per pore invagination unit), summing over all pore size ranges, and multiplying by total IW area considered. For whole eye calculations, the IW endothelial surface was taken to be 11 mm2. For quadrant analysis, it was a quarter of this area.14 In addition to IW facility, we also calculated the hydraulic conductivity of the IW endothelium, defined as IW facility per unit area (units: fi\/ (min • mmHg • cm2). This standard quantity measures the intrinsic ease with which fluid crosses a barrier— in this case, the IW endothelium. Statistical Analysis Data, for whole eyes and quadrants, were analyzed using a regression analysis program that took into account intrapatient correlations as described by Lipsitz 1663 and Harrington.15 Values are given as means ± one standard deviation. Results Morphology The endothelial surface of the IW of Schlemm's canal consisted of a continuous lining of elongated cells generally oriented circumferentially within Schlemm's canal, ie, parallel to the longitudinal axis of the canal (Fig. 1). Cells were 100-150 /*m or more long and typically 4-7 ^m wide. Commonly, one or more discrete bulging structures, presumably giant vacuoles or cell nuclei, were seen protruding at various intervals from a single endothelial cell. Many of these structures were fusiform in shape, measuring 5-7 fim long and 4-5 fim wide. Some bulges had small discrete openings, or pores, typically ranging from 0.3-2 /xm in diameter (Fig. 2). Pores appeared to be randomly distributed in normal eyes. Pores were typically located on bulges, but were occasionally seen on flatter regions of the inner endothelial lining. Although the IW endothelium in eyes of patients with POAG appeared similar to normals in certain areas of the IW, differences were noted. The circumferential orientation of the endothelial cells was less apparent. Although the appearance of the pores was similar to those in normal eyes, they were frequently observed to be clustered, rather than randomly distributed (Fig. 3). Bulging structures seemed to vary more in shape and size in POAG eyes. In addition, regions Fig. 1. Overview SEM of the inner wall endothelial lining in a normal eye perfused at 15 mm Hg. Elongated endothelial cells contain one or more bulging structures, representing nuclei or vacuoles. A pore (arrow) is seen in a flat section of the inner wall endothelium. Original magnification X650. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933167/ on 05/10/2017 1664 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992 Vol. 33 Figs. 2-4. Fig. 2. Higher magnification SEM of a normal eye. A pore (arrow) is observed at the base of a bulging structure. Original magnification X35OO. Fig. 3. SEM of the inner wall endothelium in a POAG eye. The appearance of the bulges in the inner wall lining is less uniform. A cluster of pores (arrows) is present. Original magnification X35OO. Fig. 4. SEM of the inner wall endothelial lining in a glaucomatous eye depicting an area devoid of bulges and pores. Suchflatareas were more commonly seen in POAG eyes than in normal eyes. Original magnification X3100. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933167/ on 05/10/2017 PORE DENSITY IN NORMAL AND GLAUCOMATOUS HUMAN EYES / Allingham er al No. 5 1665 Table 1. Data from individual eyes No. 8864 8867 8901 8872 8928 1 OD OS OD OS OD OS OD OS OD OS Age Sex Nl vs. POAG Measured facility (fil/min X mm Hg) 84 M 62 F 70 F 73 M 88 F Nl Nl Nl Nl Nl Nl POAG POAG POAG POAG 0.15 0.21 0.25 0.27 0.33 0.29 0.13 0.12 0.05 0.13 Calculated IWE* facility (\d/min X mm Hg) Area IW analyzed 2.89 3.09 6.83 4.64 5.06 4.21 1.62 1.11 0.54 1.32 1.02 X 10s 9.7 X 10" 7.4 X 10" 5.6 X 10" 9.1 X 10" 9.2 X 10" 8.7 X 10" 8.6 X 10" 1.03 X 105 1.04 X 10s (nm2) IWE = inner wall endothelium of Schlemm's canal. where the IW wasflatwere noted more commonly in POAG eyes (Fig. 4). Few pores were seen in the flattened regions. Quantitative Analysis Whole eye analysis: Age, sex, measured outflow facility, calculated IW endothelial facility, and area of IW endothelium analyzed by SEM were tabulated for normal and POAG eyes (Table 1), and a comparison between normal eyes and POAG eyes was made (Table 2). Measured outflow facility was 0.25 ± 0.06 /A/ min • mmHg in normal eyes compared to 0.11 ± 0.04 /Lil/min • mmHg in POAG eyes (P < .004). Mean pore density was 1437 ± 423 pores/mm2 in normal eyes and was 489 ± 172 pores/mm2 in POAG eyes (P < .004). Calculated IW endothelial facility was 4.45 ± 1.44 and 1.15 ± 0.46 /il/min • mmHg, in normal and POAG eyes, respectively (P < .02). A histogram of pore counts in each pore diameter group for normal and POAG eyes was made (Fig. 5). The relative distribution of pores per pore diameter group was similar between normal and POAG eyes. Mean pore size, bulge density, and area of IW endothelium analyzed per eye were not significantly different between normal and POAG eyes. Regression analysis was performed examining the relationship between pore density and measured outflow facility in normal eyes, POAG eyes, and in a combined analysis that included both groups (Fig. 6). Pore density was related to measured outflow facility in normal eyes (P < .02) and in the combined group (P < .001). However, this relationship did not reach statistical significance in the POAG group when analyzed alone (P < .27). No statistically significant relationship was found between pore diameter or bulge density and measured outflow facility in these groups. The relationship between measured outflow resistance in whole eyes and calculated IW endothelial resistance (reciprocal of calculated IW facility) was examined. As measured total resistance increased, calculated IW resistance increased for normal and POAG eyes (Fig. 7). However, the rate of increase (as measured by the slope of the regression lines in Fig. 7) was different for POAG and normal eyes. Specifically, the percentage resistance attributed to the IW endothelium in normal eyes was 5.8 ± 1.2%, while in POAG eyes it was 9.5 ± 1.2% (P < .02). Because normal and POAG eyes were perfused at the same constant pressure, this implies that the pressure drop across the IW endothelium was different in normal and POAG eyes Table 2. Comparison normal vs. POAG (Whole eyes) POAG (± STD) Normal (± STD) Mean measured outflow facility (/nl/min X mm Hg) Mean pore density (per mm2) Calculated IW facility (jtl/min X mm Hg) Calculated IW resistance (% of measured total resistance) Mean pore diameter (nm) Mean area of IW Analyzed (/xm2) Mean bulge density (per mm2) P 0.25 ± 0.06 1437 ± 423 0.11 ± 0.04 489 ± 172 0.004 0.004 4.45 ± 1.44 1.15 ±0.46 0.02 5.8 ± 1.2 0.91 ±0.12 8.5 ± 1.7 X 10" 7.3 ± 1.0 X 103 9.5 0.85 9.5 6.7 0.02 0.46 0.42 0.45 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933167/ on 05/10/2017 ± 1.2 ±0.12 ± 0.98 X 10" ± 1.5 X 103 Vol. 33 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992 1666 IW Endothelial Resistance vs. Measured Outflow Resistance Mean Pore Count By Pore Diameter .1-.3 .4-.7 .8-1.1 12-1.6 1.6-1.9 2.0-2.3 2.4-2.7 2.8-3.1 3.2-3.5 >3.5 Pore Diameter (urn) Fig. 5. Histogram of the number of pores per eye within each pore diameter grouping in normal and POAG eyes. Vertical bars are ± STD. (0.87 mmHg vs. 1.43 mmHg for normal and POAG eyes, respectively). There was a statistically significant difference between the calculated hydraulic conductivity of the IW endothelium in normal eyes compared to POAG eyes. The hydraulic conductivity of the IW endothelium was 40.5 ±13.1 /il/(min • mmHg • cm2) in normal eyes and 10.4 ± 4.1 /il/(min • mmHg • cm2) in POAG eyes (P < .02). Regional analysis: Quadrant pore densities were calculated by pooling data from corresponding quadrants from individual eyes. Mean quadrant pore densities in normal eyes ranged from 1262 ± 464 pores/ mm2 in the superonasal quadrant to 1523 ± 759 pores/mm2 in the inferotemporal quadrant (Fig. 8). In POAG eyes, mean quadrant pore densities ranged Outflow Resistance (mmHg x min/ul) Fig. 7. Graph of calculated IW endothelial resistance versus total measured outflow resistance in normal and POAG eyes. The slope was greater in POAG eyes than in normals (P < 0.02). from 373 ± 127 pores/mm2 in the inferotemporal quadrant to 630 ± 564 pores/mm2 in the superonasal quadrant. There were no significant differences in regional mean pore density among quadrants in normal eyes, POAG eyes, or when combined. The pore density was significantly lower in three of four quadrants of POAG eyes when compared to the same region in normal eyes (P < .05), the exception being the superonasal quadrant (P < .11). Comparisons of quadrant-calculated IW endothelial facility were made in normal and POAG eyes. In normal eyes, calculated IW endothelial facility per Regional Pore Density By Quadrant Pore Density vs. Measured Outflow Facility Q Normal • POAG 2 D .8 - D D 1.6 Q 1.4 - I t '* 1.2 - <§ 0.8 S D 1 - S. oe - 0.4 D 0.8 - 02 - • 0.8 - IT 0.4 - SN ST Total IN IT SN ST o Normal • 0.2 - POAG 0 Outflow Facility (ul/mln/mmHg) Fig. 6. Graph of the relationship between pore density and measured outflow facility. The relationship was statistically significant in normal eyes alone (P < 0.02) and when data were pooled with POAG eyes (P< 0.001). Fig. 8. Bar graph of regional pore density per quadrant comparing normal with POAG eyes. There were no significant differences in regional pore density among quadrants of normal or POAG eyes or when data were combined. Pore density was significantly lower in POAG eyes when compared with the corresponding region in normal eyes, with one exception, the superonasal quadrant (P < 0.11). Total is the overall mean pore density. IN, IT, SN, and ST are inferonasal, inferotemporal, superonasal, and superotemporal, respectively. Vertical bars are ± STD. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933167/ on 05/10/2017 No. 5 PORE DENSITY IN NORMAL AND GLAUCOMATOUS HUMAN EYES / Allingham er ol quadrant ranged from 1.5 ± 0.8 ^l/min«mmHg in the superonasal quadrant to 0.8 ± 0.5 /ul/min • mmHg in the superotemporal quadrant. In POAG eyes, the calculated IW endothelial facility per quadrant ranged from 0.2 ± 0.1 jtl/min • mmHg in the superonasal quadrant to 0.5 ±0.3 ^1/min • mmHg in the inferonasal quadrant. There were no significant differences between the calculated IW endothelial facility per quadrant in normal eyes and POAG eyes. The IW endothelial facility per quadrant was significantly lower in POAG eyes than in the respective normal eyes except for the inferotemporal quadrant (P < .06). Discussion Pore/Facility Correlation The endothelial lining of Schlemm's canal previously has been estimated to contribute up to 24% of total outflow resistance in normal human and primate eyes.1213 In addition, it has been shown that pore and giant vacuole density increase with increased perfusion pressure.16"21 In this study, we have demonstrated that a relationship exists between measured outflow facility and pore density in normal eyes alone and when combined with POAG eyes. This relationship did not reach statistical significance in POAG eyes when analyzed alone, possibly because of the small number, the relatively lower outflow facility of POAG eyes, or pathological changes in the IW endothelium. Although the postmortem changes may have influenced the results of this study—and may influence the results of any study that uses donor tissue—we made every effort to prevent artifactual changes. Donor eye tissue was harvested soon after death. Measurements of outflow facility and perfusion fixation were performed within 18 hr. Furthermore, the difference observed in pore density between normal and glaucomatous eyes, and the noted relationship between pore density and outflow facility, cannot be explained by postmortem time differences. The possibility that pore formation could be occurring postmortem also was considered. We examined specimens from emersion fixed eyes that were otherwise identically prepared and found pore density to be near zero (R. Allingham, A. de Kater, D. Epstein, Howe Laboratory of Ophthalmology, Boston, MA, unpublished data). Therefore, we believe that fluid flow through the trabecular meshwork was responsible for the formation of endothelial pores observed in this study. There are at least three possible reasons for the positive correlation between outflow facility and pore density. (1) The endothelial lining itself is responsible for a majority of total resistance in the human eye. (2) 1667 The endothelial lining of Schlemm's canal is responsible for "modulating" total resistance in the eye. This implies that the endothelial lining could alter fluid flow resistance "upstream" within the TM, perhaps by altering the resistance of flow pathways within the juxtacanalicular region. (3) The IW endothelium is a passive resistor to aqueous outflow, and pore density is modulated by a variable such as fluid flow or pressure drop across the IW endothelial lining. In our study, the mean calculated resistance of the inner endothelial lining of Schlemm's canal was 5.8% of total measured resistance in normal eyes and 9.5% in POAG eyes. These values are consistent with those of Bill and Svedbergh.12 Therefore, it appears unlikely that the IW endothelium by itself produces a majority of total aqueous outflow resistance and explanation (1) can be eliminated. Explanation (2) is similar to (1) in that it attributes a controlling role on facility to the endothelial lining, although in this case by interaction with other tissues "upstream." The present study does not allow us to rule out this mechanism. Even if it eventually proves not to be a dominant factor, hydraulic interactions of the IW endothelium with the juxtacanalicular region still may contribute significantly to overall resistance. Explanation (3) rests on the widely accepted idea that the pores in the IW endothelium open in response to transmural pressure. The reduction in pore number in POAG eyes can be seen as a consequence of lower facility causing a lower fluid flow rate. This could produce a lower pressure drop across the IW endothelium (with perfusion pressure held constant). However, as noted above, not only was the overall flow resistance higher in POAG eyes but the IW resistance was proportionately greater. The calculated IW endothelial transmural pressure drop is 64% higher for POAG eyes (0.87 mmHg for normal eyes and 1.43 mmHg for POAG eyes). The higher transmural pressure in the presence of lower flow clearly implies that the reduction in pore number was more than could be accounted for by lower fluid flow alone. Therefore, the IW endothelium in POAG eyes appears to be more resistant to pore formation. There is not enough data in this study to characterize the pressure/pore formation process. A similar study that analyzes pore density after perfusion at different levels of constant flow (rather than constant pressure) may clarify this and other questions. Evidence that supports an intrinsic difference between the IW endothelium of Schlemm's canal in normal and POAG eyes comes from tracer studies. De Kater et al perfused normal and POAG autopsy eyes with cationic ferritin (CF).22 In normal eyes, the abluminal cell surface of giant vacuoles was consis- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933167/ on 05/10/2017 1668 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992 tently decorated. However, in POAG eyes, CF staining was invariably absent at this location. These investigators concluded that an alteration of the electrical charge was present in this region or CF entry was blocked from entering the vacuoles in POAG eyes. In a study that examined the distribution of sialic acid moieties associated with the endothelial lining of normal and glaucomatous eyes, Tripathi et al found an increased density of sialic acid on luminal and abluminal surfaces in POAG specimens compared to normal eyes.23 Increased levels of sialic acid, which is believed to stabilize cell membranes, could induce abnormally high cell rigidity, ultimately affecting the process of vacuolization as well as pore formation. In addition, sialic acid is known to interact with a variety of extracellular matrix molecules that could directly or indirectly affect the IW endothelium. This study suggests that there also may be a functional alteration of the IW endothelium in POAG. The effect of medical therapy on the function of the IW endothelium of glaucoma eyes is not known. Regional Variability Regional differences in aqueous outflow have been suggested by some investigators.24'25 We were unable to detect a statistically significant difference in regional pore density or calculated IW endothelial facility in normal or POAG eyes. However, this does not rule out the possibility that regional variation exists. In fact, we observed substantial variation in pore and bulge distribution within segments of normal and glaucomatous eyes, although this was not localized to a specific quadrant. Interestingly, de Kater et al found evidence for regional variation in flow in POAG eyes.22 Qualitatively, we found that glaucomatous eyes appear to have greater variability in pore distribution than normal eyes. Pores were more clustered in POAG, suggesting the presence of localized areas of increased aqueous outflow. In addition, there appeared to be a greater number of areas where the IW endothelium was flat with few pores. Flattened regions may represent areas where Schlemm's canal was collapsed. If so, pore formation (and related aqueous flow) through these areas appears to have been diminished. These findings lend further support to the impression that there is greater regional variability in aqueous outflow in POAG eyes. Preliminary evidence suggests there may be more giant vacuoles in normal eyes than in POAG eyes.26 In the present study, we observed no apparent relationship between "bulges" and outflow facility. However, SEM is not well suited to analyzing giant vacuole formation because it is not possible to clearly dis- Vol. 33 tinguish between giant vacuoles and IW endothelial cell nuclei. Comparison With Other Endothelia In this study, the calculated hydraulic conductivity of the IW endothelium of normal eyes was 40.5 ±13.1 jul/(min • mmHg • cm2), while in POAG eyes it was 10.4 ± 4.1 /il/(min • mmHg • cm2). In the circulatory system, the primary region for fluid flow across the endothelial lining is within the capillaries. The hydraulic conductivity of several different vascular endothelia has been calculated (Table 3).27 Interestingly, the hydraulic conductivity of the IW endothelium of Schlemm's canal in normal eyes is 30 times greater than that of fenestrated renal capillary endothelium and far greater than other types of vascular endothelia. The hydraulic conductivity of POAG IW endothelium is lower than that of normals, but is still nearly an order of magnitude greater than fenestrated renal capillary endothelium. We speculate that high hydraulic conductivity may be essential to IW endothelial cell survival. In the absence of low resistance transcellular endothelial pores, stresses on the IW endothelial lining, induced by aqueous flow, might prove excessive, causing disruption of cell-to-cell and cell-to-matrix attachments. Ultimately, the underlying IW endothelial basement membrane could be exposed to the contents of Schlemm's canal. If blood were to reflux into Schlemm's canal, platelet aggregation could occur on exposed basement membrane surfaces, resulting in obstruction of aqueous pathways within the juxtacanalicular tissues or occlusion of Schlemm's canal itself.28 Of course, if blood were to reflux through these areas into the anterior chamber, visual acuity also might be adversely affected. Table 3. Comparison between hydraulic conductivity of IW endothelium of Schlemm's canal and capillary endothelium Endothelium IW endothelium, Schlemm's canal Normal POAG Fenestrated capillaries* Renal (frog, mammal) Intestinal mucosa (mammal) Nonfenestrated capillaries* Frog mesentery Dog lung Cat hind leg Tight junction* Rabbit brain Lp (fil/(min x mm Hg X cm2) 40.5 ± 13.1 (STD) 10.4 ±4.1 (STD) 1.2 1.0 X 10"' 4.0 X 10"3 2.7 X 10"3 2.0 X 10"3 2.4 X 10"5 * Values for hydraulic conductivity derived from Renkin.28 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933167/ on 05/10/2017 No. 5 PORE DENSITY IN NORMAL AND GLAUCOMATOUS HUMAN EYES / Allinghom er ol Key words: Schlemm's canal, outflow facility, endothelial pores, scanning electron microscopy, flow resistance, hydraulic conductivity, primary open angle glaucoma, trabecular meshwork, aqueous humor outflow system. Acknowledgments The authors would like to thank Victor Merritt and Aliakbar Shahsafaei for excellent technical assistance, Pat Basler for secretarial assistance, Mark Johnson, for stimulating discussions on the dynamics of fluid flow, and Robert Glynn, for suggestions regarding the statistical analysis. The authors are grateful to JEOL USA, Inc. Applications Laboratory, Peabody, MA, for the use of the JEOL 6400. 14. 15. 16. 17. 18. References 1. Karg SJ, Garron KL, Feeney L, and McEwen WK: Perfusion of human eyes with latex microspheres. Arch Ophthalmol 61:68, 1959. 2. Feeney L and Wessig S: Outflow studies using an electron dense tracer. Trans Am Acad Ophthalmol Otolaryngol 70:791, 1966. 3. Grierson I, Lee WR, and Abraham S: The appearance of the outflow apparatus of the eye after staining with ruthenium red. Acta Ophthalmol 55:827, 1977. 4. Barany E: Pore size and passage of paniculate matter through the trabecular meshwork. Doc Ophthalmol 13:41, 1959. 5. Garron L, Feeney ML, Hogan MJ, and McEwen WK: Electron microscopic studies of the human eye. Am J Ophthalmol 46:27, 1958. 6. Holmberg A: Thefinestructure of the inner wall of Schlemm's canal. Arch Ophthalmol 62:956, 1047, 1959. 7. Tripathi RC: Ultrastructure of Schlemm's canal in relation to aqueous outflow. Exp Eye Res 7:335, 1968. 8. Tripathi RC: Mechanisms of the aqueous outflow across the trabecular wall of Schlemm's canal. Exp Eye Res 11:116,1971. 9. Grant WM: Further studies on facility offlowthrough the trabecular meshwork. Arch Ophthalmol 60:523, 1958. 10. Grant WM: Experimental aqueous perfusion in enucleated human eyes. Arch Ophthalmol 69:783, 1963. 11. Maepea O and Bill A: The pressures in the episcleral veins, Schlemm's canal and the trabecular meshwork in monkeys: Effects of changes in intraocular pressure. Exp Eye Res 49:645, 1989. 12. Bill A and Svedbergh B: Scanning electron microscopic studies of the trabecular meshwork and the canal of Schlemm—an attempt to localize the main resistance to outflow of aqueous humor in man. Acta Ophthalmol 50:295, 1972. 13. Moseley H, Grierson I, and Lee WR: Mathematical modelling 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 1669 of aqueous humour outflow from the eye through the pores in the lining endothelium of Schlemm's canal. Clin Phys Physiol Meas4:47, 1983. McEwen WK: Application of Poiseuille's law to aqueous flow. Arch Ophthalmol 60:290, 1958. Lipsitz SR and Harrington D: Analyzing correlated binary data using SAS. Comput Biomed Res 23:268, 1990. Grierson I and Lee WR: Changes in the monkey outflow apparatus at graded levels of intraocular pressure: A qualitative analysis by light microscopy and scanning electron microscopy. Exp Eye Res 39:505, 1984. Grierson I and Lee WR: Changes in the monkey outflow apparatus at graded levels of intraocular pressure: A qualitative analysis by light microscopy and scanning electron microscopy. Exp Eye Res 19:21, 1974. Grierson I and Lee WR: Light microscopic quantitation of the endothelial vacuoles in Schlemm's canal. Am J Ophthalmol 84:234, 1977. Grierson I and Lee WR: Pressure-induced changes in the ultrastructure of the endothelium lining Schlemm's canal. Am J Ophthalmol 80:863, 1975. Ainesworth JR and Lee WR: Effects of age and rapid high-pressure fixation on the morphology of Schlemm's canal. Invest Ophthalmol Vis Sci 31:745, 1990. Johnstone MA and Grant WM: Pressure-dependent changes in the structures of the aqueous outflow system of human and monkey eyes. Am J Ophthalmol 75:365, 1973. de Kater AW, Melamed S, and Epstein DL: Patterns of aqueous humor outflow in glaucomatous and nonglaucomatous human eyes. A tracer study using cationized ferritin. Arch Ophthalmol 107:572, 1989. Allingham RR, de Kater AW, Shahsafaei A, and Epstein DL: Relationship between outflow facility and giant vacuoles of Schlemm's canal in normal and glaucomatous human eyes, abstract. Invest Ophthalmol Vis Sci 31(Suppl):338, 1990. Ellingsen BA and Grant WM: Trabeculotomy and sinusotomy in enucleated human eyes. Invest Ophthalmol Vis Sci 11:21, 1972. Campbell DG, Boys-Smith JW, and Woods WD: Variation of pigmentation and segmentation of pigmentation in primary open angle glaucoma, abstract. Invest Ophthalmol Vis Sci 25(Suppl):122, 1984. Raviola G and Raviola E: Paracellular route of aqueous outflow in the trabecular meshwork and canal of Schlemm. A freeze-fracture study of the endothelial junctions in the sclerocorneal angle of the macaque monkey eye. Invest Ophthalmol Vis Sci 21:52, 1981. Renkin EM: Multiple pathways of capillary permeability. Circ Res 41:735, 1977. Hamanaka T and Bill A: Role of blood in Schlemm's canal, abstract. Invest Ophthalmol Vis Sci 31(Suppl):337, 1990. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933167/ on 05/10/2017