February 2016
Volume 57, Issue 2
Open Access
Glaucoma  |   February 2016
Morphological Abnormalities of Schlemm's Canal in Primary Open-Angle Glaucoma From the Aspect of Aging
Author Affiliations & Notes
  • Teruhiko Hamanaka
    Department of Ophthalmology Japanese Red Cross Medical Center, Tokyo, Japan
  • Akira Matsuda
    Laboratory of Ocular Atopic Diseases, Department of Ophthalmology, Juntendo University, Tokyo, Japan
  • Tetsuro Sakurai
    Department of Center of General Education, Tokyo University of Science, Suwa, Japan
  • Toshio Kumasaka
    Department of Pathology, Japanese Red Cross Medical Center, Tokyo, Japan
  • Correspondence: Teruhiko Hamanaka, Department of Ophthalmology, Japanese Red Cross Medical Center, 4-1-22 Hiroo Shibuya-ku, Tokyo 150-8935, Japan; hamanaka.teruhiko@gmail.com
Investigative Ophthalmology & Visual Science February 2016, Vol.57, 692-706. doi:10.1167/iovs.15-17127
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      Teruhiko Hamanaka, Akira Matsuda, Tetsuro Sakurai, Toshio Kumasaka; Morphological Abnormalities of Schlemm's Canal in Primary Open-Angle Glaucoma From the Aspect of Aging. Invest. Ophthalmol. Vis. Sci. 2016;57(2):692-706. doi: 10.1167/iovs.15-17127.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose: To evaluate morphological abnormalities of Schlemm's canal (SC) among primary open-angle glaucoma (POAG) patients with a family history of POAG (group A), those without a family history of POAG (group B), and patients with normal-tension glaucoma (group C) from the aspect of aging.

Methods: A total of 160 trabeculectomy specimens from 133 POAG patients were processed for light microscopy using immunohistochemical staining of thrombomodulin and transmission electron microscopy. The following parameters were statistically evaluated: SC length, the percentage of the thrombomodulin-negative area (PTNA) of SC, and the inner-wall SC endothelial cell density (SC-ECD/100 μm).

Results: No significant differences in age were observed among the three groups (group A: 56.71 ± 14.83; group B: 58.13 ± 18.13; group C: 56.61 ± 9.78). Length of SC in the group A patients (198.70 ± 81.65 μm, n = 70 eyes) was significantly shorter than in group B (250.30 ± 70.83 μm, n = 67 eyes), and group C (277.70 ± 65.52 μm, n = 23 eyes) patients. A positive correlation of patient age and SC length was observed in group B (r = 0.45, P = 0.0013), but SC length in group A tended to decrease with aging (r = −0.22, P = 0.07). No significant difference in SC was found between group A and B patients before age 50 years (P = 0.30). Correlations between patient age and increase of PTNA (r = 0.38, P = 0.0013) and patient age and decrease of SC-ECD (r = −0.53, P = 3.95 × 10−6) were observed only in the group B cases.

Conclusions: Our findings suggest that SC in group A may easily collapse during middle age, while SC in group B remains open and SC endothelial cells drop out at around middle age.

Early detection and initiation of treatment is important to prevent the loss of visual function due to glaucoma, which is one of the leading causes of blindness worldwide. Moreover, it may be important to establish strategies for controlling intraocular pressure (IOP) in glaucoma patients via clarification of morphological abnormalities in aqueous outflow routes. It has been reported that in healthy eyes, 49% to 75% of the total aqueous outflow resistance is located between the anterior chamber and Schlemm's canal (SC).1,2 Previous research related to aqueous outflow has focused on juxtacanalicular tissue (JCT), as it is considered to be the primary site of outflow resistance, which causes an increase in IOP. In particular, JCT is reportedly considered to be the site of highest aqueous humor outflow resistance.3 Moreover, it has been reported that the occurrence of juvenile glaucoma is related to abnormality of the JCT.4 On the other hand, although it has been reported that minimum outflow resistance of aqueous humor exists at the SC inner wall,3 SC endothelial cells (ECs) do play some roles in the outflow of aqueous humor. Pathological changes of SC (e.g., closure of SC due to uveitis and primary angle-closure glaucoma [PACG]), may cause severe obstruction of the outflow pathways.57 Aging is known to be one of the leading risk factors for primary open-angle glaucoma (POAG),815 and the results of most epidemiological studies have shown that age is an important risk factor for glaucoma. Several studies have shown a relationship between IOP elevation in POAG patients and the age-related morphological changes (i.e., thickening of the trabecular meshwork [TM]),16 progressive decrease in trabecular cellularity,17 and accumulation of extracellular matrix in JCT.18 Although the findings of previous reports have shown a relationship between aging and the function of SC in healthy eyes,19,20 there has yet to be a study regarding age-related SC abnormalities in the eyes of POAG patients. Gramer et al.21 reported that patients with a family history of glaucoma, including POAG, were significantly younger at diagnosis than patients without a family history of glaucoma. Therefore, we theorized that it would be interesting to investigate morphological abnormalities of SC in POAG from the aspect of aging by comparing the patients with a family history and without a family history of POAG. Blood vessel endothelium expresses anticoagulant thrombomodulin and procoagulant von Willebrand factor; however, expression of these opposing substances is different according to the function of the organs where those blood vessels exist. Thrombomodulin-dominant endothelial cells have been reported in alveolar capillaries22 and SC endothelial cells (SC-ECs).23 Immunohistochemical staining of thrombomodulin and/or CD34 is an excellent marker for identification of SC-ECs.23 Thrombomodulin immunostaining is especially useful for identification of SC-ECs in PACG eyes with partial EC loss.7 
In this study, we evaluated the age-related morphological changes of SC in surgical specimens by light microscopy (LM) thrombomodulin and/or CD34 immunohistochemical staining, as well as by transmission electron microscopy (TEM). 
Materials and Methods
This study involved 160 eyes of 133 POAG patients who underwent trabeculectomy (TLE) surgery from March 1997 to October 2014 at the department of ophthalmology, Japanese Red Cross Medical Center, Tokyo, Japan. The study protocols were approved by the ethics committee of the Japanese Red Cross Medical Center, and all subjects provided prior written informed consent in accordance with the tenets set forth in the Declaration of Helsinki. Exclusion criteria included patients with other types of glaucoma including secondary glaucoma, PACG, exfoliation glaucoma, and patients with a previous history of intraocular surgery other than small-incision cataract surgery without complications. 
A detailed family history of glaucoma was obtained from all participants. To confirm the family history of glaucoma, ophthalmological examinations were carried out at our glaucoma clinic on first-degree relatives with a known history of glaucoma. Patients whose first-degree relatives were unable to undergo the examination were excluded from the study. 
Ophthalmological examinations were also carried out on all of the enrolled patients. Visual field tests were carried out and classified by Aulhorn-Greve classification, and cup-to-disk (C/D) ratio, maximum preoperative IOP, and refractive error (spherical equivalence: SE) were evaluated within 6 months prior to surgery. All TLE operations were performed by one surgeon (TH). In all patients, TM and SC specimens were obtained from the 10 to 2 o'clock upper corneoscleral limbus at the time of surgery. The specimens were then fixed in a mixture of 2.5% or 5% formalin and 1% glutaraldehyde, and subsequently divided into four or five sections. All divided specimens were embedded in paraffin or epon to enable SC to be cut perpendicularly to its longitudinal axis. Histologic analysis was carried out using paraffin sections, as well as an ultrathin section using epon. Paraffin sections were used for LM examination of hematoxylin-eosin (HE) staining, and immunohistochemical staining using antithrombomodulin and anti-CD34 antibodies. The ultrathin section was used for the TEM examination. The methods used for dissecting the TLE specimens and the procedures for LM and TEM were as described in the previous report.7 We measured the length of SC (anterior-to-posterior distance) in the LM observation using HE staining and immunohistochemical staining of thrombomodulin and CD34 (Figs. 1, 2). The percentage of thrombomodulin-negative area (PTNA) of SC was measured using thrombomodulin immunohistochemical staining (Fig. 3). Schlemm's canal EC density (SC-ECD, number of nuclei/100 μm) of the inner wall of SC was counted by immunohistochemical staining of thrombomodulin (Figs. 1, 2) and CD34. The number of Sondermann canals was counted by thrombomodulin immunohistochemical staining (Figs. 1, 2). Length of SC, PTNA, SC-ECD, and the number of Sondermann canals were evaluated using two to three different tissue blocks, and counted in triplicate for one block. Length of SC was measured primarily by thrombomodulin immunostaining; however, in the thrombomodulin negative area of the canal, HE staining (Figs. 3A, inset; 3B, inset; 4) and CD34 immunostaining (Fig. 2A, inset) were helpful for evaluating the actual SC length. The counts were carried out in a masked fashion by two authors (TH, AM). Repeatability was evaluated by interobserver comparison, and the difference was within 10%. 
Figure 1
 
(A, B) Light microscopy images of SC and TM in the eye of familial POAG patient no. 3, a 38-year-old male. Thrombomodulin immunohistochemical staining. Length of SC was measured between the anterior tip (open arrow) and the posterior tip (solid arrow) of the canal. Sondermann's canals were observed in the TM (small black arrows). Inset: serial section stained with HE from the same block. In the other block of the same eye, SC length was shorter (B). The anterior part of the canal (A, star) and the posterior half of the canal (B, star) appeared to be much shorter and showed strong positive thrombomodulin staining. Open arrowheads in A and B indicate the endothelium of the canal. Judging from the photograph of the section stained with HE (B, inset), it was difficult to estimate the length of SC. CC, collector channel; I, iris.
Figure 1
 
(A, B) Light microscopy images of SC and TM in the eye of familial POAG patient no. 3, a 38-year-old male. Thrombomodulin immunohistochemical staining. Length of SC was measured between the anterior tip (open arrow) and the posterior tip (solid arrow) of the canal. Sondermann's canals were observed in the TM (small black arrows). Inset: serial section stained with HE from the same block. In the other block of the same eye, SC length was shorter (B). The anterior part of the canal (A, star) and the posterior half of the canal (B, star) appeared to be much shorter and showed strong positive thrombomodulin staining. Open arrowheads in A and B indicate the endothelium of the canal. Judging from the photograph of the section stained with HE (B, inset), it was difficult to estimate the length of SC. CC, collector channel; I, iris.
Figure 2
 
(A, B) Light microscopy images of SC and TM in the eye of nonfamilial POAG patient no. 23, a 49-year-old male. Thrombomodulin (A, B) and CD34 (A, inset) immunohistochemical staining. Length of SC was measured between the anterior tip (open arrow) and the posterior tip (solid arrow) of the canal in which the anterior part did not show positive for thrombomodulin (A, stars, inset). Note that the positive staining area was almost the same as that in CD34 (A, inset). Figures (A, B) were taken from different blocks of the same specimen. At the border of the positive and nonpositive thrombomodulin area, endothelium of the canal sealed off the nonpositive thrombomodulin area (B, black arrow in inset). Note that the inset is an enlargement of the boxed area in (B). Arrowheads: endothelium of the canal. Small black arrows: Sondermann canal.
Figure 2
 
(A, B) Light microscopy images of SC and TM in the eye of nonfamilial POAG patient no. 23, a 49-year-old male. Thrombomodulin (A, B) and CD34 (A, inset) immunohistochemical staining. Length of SC was measured between the anterior tip (open arrow) and the posterior tip (solid arrow) of the canal in which the anterior part did not show positive for thrombomodulin (A, stars, inset). Note that the positive staining area was almost the same as that in CD34 (A, inset). Figures (A, B) were taken from different blocks of the same specimen. At the border of the positive and nonpositive thrombomodulin area, endothelium of the canal sealed off the nonpositive thrombomodulin area (B, black arrow in inset). Note that the inset is an enlargement of the boxed area in (B). Arrowheads: endothelium of the canal. Small black arrows: Sondermann canal.
Figure 3
 
(A, B) Light microscopy (A, B, inset; C) and TEM (C) images of SC and TM in the eyes of nonfamilial POAG patient no. 54, a 71-year-old male. (A) Right eye. (B, C) Left eye. (A, B) Thrombomodulin immunohistochemical staining. (A, inset; B, inset) HE staining. (C, inset) Toluidine blue staining. Note that SC endothelium in the right eye sealed off the canal (A, arrowhead) from the nonthrombomodulin staining area (double arrows: PTNA in [A] = 45%) and that the strong positive staining of the canal is comparable with that of the Sondermann canal (A, arrow). Intraocular pressure in the left eye (B, C) was elevated to 27 mm Hg despite maximum medication after 3.5 years of good IOP control. Nonthrombomodulin staining areas (double arrows) were also observed ([B] PTNA = 40%), but no sealing of the canal by SC endothelium was observed. The spaces of the TM and JCT seemed to be open and HE staining showed no inflammatory cells (B, inset). In the TEM image (C), dropout of SC endothelium was observed. The degenerated SCE was observed in the inner wall (arrow) and JCT (asterisks) was exposed to the lumen of SC. (C) The TEM image was made from the boxed area in the inset.
Figure 3
 
(A, B) Light microscopy (A, B, inset; C) and TEM (C) images of SC and TM in the eyes of nonfamilial POAG patient no. 54, a 71-year-old male. (A) Right eye. (B, C) Left eye. (A, B) Thrombomodulin immunohistochemical staining. (A, inset; B, inset) HE staining. (C, inset) Toluidine blue staining. Note that SC endothelium in the right eye sealed off the canal (A, arrowhead) from the nonthrombomodulin staining area (double arrows: PTNA in [A] = 45%) and that the strong positive staining of the canal is comparable with that of the Sondermann canal (A, arrow). Intraocular pressure in the left eye (B, C) was elevated to 27 mm Hg despite maximum medication after 3.5 years of good IOP control. Nonthrombomodulin staining areas (double arrows) were also observed ([B] PTNA = 40%), but no sealing of the canal by SC endothelium was observed. The spaces of the TM and JCT seemed to be open and HE staining showed no inflammatory cells (B, inset). In the TEM image (C), dropout of SC endothelium was observed. The degenerated SCE was observed in the inner wall (arrow) and JCT (asterisks) was exposed to the lumen of SC. (C) The TEM image was made from the boxed area in the inset.
To evaluate the statistically significant difference between two groups, the Student's t-test was used. Statistical significance was assessed by use of the Bonferroni multiple comparison procedure, and a P value < 0.017 was considered statistically significant. Pearson's correlation coefficients were calculated to evaluate the correlation between patient age and SC length, PTNA, and SC-ECD. Age matching of SC length, PTNA, and SC-ECD in the three groups was also evaluated. 
Results
Of the total number of subjects, 70 eyes of 55 POAG patients with a family history of the disease (group A), 67 eyes of 56 POAG patients without a family history of the disease (group B), and 23 eyes of 22 normal-tension glaucoma (NTG) patients (group C) were ultimately enrolled in this study. The clinical characteristics of the enrolled patients are summarized in Table 1. In all enrolled patients, glaucoma was under control with the maximum tolerable medication prior to surgery. There was no significant difference of patient age and C/D among the three groups (Table 2). Higher maximum preoperative IOP was noted in groups A and B compared with group C (Table 2). Group C patients showed more advanced visual field defects compared with groups A and B (Table 2). The histologic characteristics and the statistical evaluations of those characteristics are shown in Tables 3 and 4, respectively. No significant difference in the number of Sondermann canals was found among the three groups (Table 4). 
Table 1
 
Clinical Observations of the Patients in the Study
Table 1
 
Clinical Observations of the Patients in the Study
Table 2
 
Statistical Results of Clinical Observations, Comparisons between Two Groups
Table 2
 
Statistical Results of Clinical Observations, Comparisons between Two Groups
Table 3
 
Histological Observations of the Patients in Study
Table 3
 
Histological Observations of the Patients in Study
Table 4
 
Statistical Results of Histological Observations, Comparisons between Two Groups
Table 4
 
Statistical Results of Histological Observations, Comparisons between Two Groups
Length of SC in the group A patients (198.70 ± 81.65 μm) was significantly shorter than that of group B (250.30 ± 70.83 μm) and group C (277.70 ± 65.52 μm) patients (Figs. 1, 2). Histological analysis revealed that SC in the eyes of POAG patients with a family history of the disease (group A) tended to have narrower lumen, even in the cases with a normal SC length (Fig. 1A). Part of SC in group A showed strong immunoreactivity of thrombomodulin, as in the case of the Sondermann canal (Fig. 1). 
Significantly higher PTNA was observed in the samples obtained from the group B patients (26.97% ± 26.60%) compared with that of group A and C patients (16.83% ± 20.62% and 17.70% ± 11.50%, respectively). Variable degrees of PTNA were observed from 0%, to a moderate degree of PTNA (Figs. 2, 3), to 100% (Figs. 4A, 4B). Eight eyes from seven patients showed a gradual increase of IOP (more than 25 mm Hg) after long periods (i.e., more than 3 years) of good IOP control. In these cases, PTNA was more than 40% (Figs. 3A, 3B). We then compared preoperative IOP between the patients showing less than 40% of PTNA and those showing more than 40% of PTNA. We did not find significant differences in group A; however, the patients in group B showing more than 40% of PTNA had significantly higher preoperative IOP (Table 5; Figs. 3A, 3B). In the cases with a small-diameter SC, we also observed prominent shortening (Figs. 5A, 5C) or occlusion with fibroblasts (Figs. 4B, 7C) and fibrotic components (Fig. 5D). It was difficult to differentiate between these findings and the congenital/developmental abnormalities that we found in a relatively young case (Figs. 6A, 6B). 
Figure 4
 
Light microscopy images of SC and TM in the eyes of nonfamilial POAG patient no. 25, a 73-year-old male. (A, B) Thrombomodulin immunohistochemical staining. (A, inset; B, inset) HE staining. (A, B) Images were taken from serial sections of the same block. Although the canal appears open in HE staining (A, inset), thrombomodulin was completely negative throughout the entire length of SC (distance between open and solid arrows, PTNA = 100%). Part of SC became occluded with a replacement of spindle-shaped fibroblasts (arrows) in (B). Preoperative IOP showed 30 mm Hg despite of the maximum medication.
Figure 4
 
Light microscopy images of SC and TM in the eyes of nonfamilial POAG patient no. 25, a 73-year-old male. (A, B) Thrombomodulin immunohistochemical staining. (A, inset; B, inset) HE staining. (A, B) Images were taken from serial sections of the same block. Although the canal appears open in HE staining (A, inset), thrombomodulin was completely negative throughout the entire length of SC (distance between open and solid arrows, PTNA = 100%). Part of SC became occluded with a replacement of spindle-shaped fibroblasts (arrows) in (B). Preoperative IOP showed 30 mm Hg despite of the maximum medication.
Table 5
 
Correlation Between Max Preoperative IOP and PTNA in Groups A and B
Table 5
 
Correlation Between Max Preoperative IOP and PTNA in Groups A and B
Figure 5
 
(A–D) Light microscopy images of a short SC and TM stained with HE (A–C, inset; D) and antibodies against thrombomodulin (A–C; D, inset). (A) Familial POAG patient no. 58, a 46-year-old female. (B, C) Nonfamilial POAG patient no. 49, an 83-year-old male. (D) Familial patient no. 2, a 58-year-old male. The thrombomodulin staining pattern was very strong in some areas of the canal (A, arrowhead) compared with the rest of canal. Note that the staining of the Sondermann canal is also very strong (A, arrows). Although the canal looked normal (B), the canal in the other block appeared to be short (C), or completely collapsed in the other patient (D, stars). Fibrotic change was observed in the supposed region of SC (asterisks).
Figure 5
 
(A–D) Light microscopy images of a short SC and TM stained with HE (A–C, inset; D) and antibodies against thrombomodulin (A–C; D, inset). (A) Familial POAG patient no. 58, a 46-year-old female. (B, C) Nonfamilial POAG patient no. 49, an 83-year-old male. (D) Familial patient no. 2, a 58-year-old male. The thrombomodulin staining pattern was very strong in some areas of the canal (A, arrowhead) compared with the rest of canal. Note that the staining of the Sondermann canal is also very strong (A, arrows). Although the canal looked normal (B), the canal in the other block appeared to be short (C), or completely collapsed in the other patient (D, stars). Fibrotic change was observed in the supposed region of SC (asterisks).
Figure 6
 
(A, B) Light microscopy images of a short SC and TM stained with antibodies against thrombomodulin. Insets show the HE-stained sections. (A) Familial POAG patient no. 32, a 40-year-old female. (B) Nonfamilial patient No. 31, a 17-year-old female. (A, B) The canals appear to be short rather than collapsed after birth, because the areas where there should be a canal (stars, A, B) are compact and were occupied by oval-shaped cells.
Figure 6
 
(A, B) Light microscopy images of a short SC and TM stained with antibodies against thrombomodulin. Insets show the HE-stained sections. (A) Familial POAG patient no. 32, a 40-year-old female. (B) Nonfamilial patient No. 31, a 17-year-old female. (A, B) The canals appear to be short rather than collapsed after birth, because the areas where there should be a canal (stars, A, B) are compact and were occupied by oval-shaped cells.
Figure 7
 
(A–C) Light microscopy and TEM (D) images of SC and TM. Staining with HE (A), thrombomodulin (B), and toluidine blue (C) in nonfamilial POAG patient no. 39, an 81-year-old female. The anterior part of the canal (A, star) showed thrombomodulin negative and SC occlusion (B, star). Replacement of fibrotic tissue with spindle-shaped fibroblasts (C, arrows) and drop out of SC endothelium (D, asterisk) were observed at the corresponding area of negative thrombomodulin (B, star). (D) Transmission electron microscopy image was made from the boxed area in inset C.
Figure 7
 
(A–C) Light microscopy and TEM (D) images of SC and TM. Staining with HE (A), thrombomodulin (B), and toluidine blue (C) in nonfamilial POAG patient no. 39, an 81-year-old female. The anterior part of the canal (A, star) showed thrombomodulin negative and SC occlusion (B, star). Replacement of fibrotic tissue with spindle-shaped fibroblasts (C, arrows) and drop out of SC endothelium (D, asterisk) were observed at the corresponding area of negative thrombomodulin (B, star). (D) Transmission electron microscopy image was made from the boxed area in inset C.
In the cases showing constant positive thrombomodulin in SC-ECs, no abnormal TEM findings were observed. On the other hand, in the cases with a partial loss of thrombomodulin positive staining (Figs. 3A, 3B, 7B), dissociation of the intercellular junction (Fig. 8C), EC loss (Figs. 3C, 7D), and partial occlusion of SC (Figs. 4B, 7C, 7D, stars) was found. Twelve eyes (10 eyes from group A and two eyes from group B) showed SC occlusion with fibrotic changes of its lumen (Figs. 5D, 7C) and loss of the ECs (Fig. 7D). 
Figure 8
 
(A, B) Light microscopy. (C) Transmission electron microscopy images of SC and TM with LM inset (toluidine blue staining). Staining of HE (A) and thrombomodulin immunohistochemical staining (B) in familial POAG patient no. 21, a 69-year-old male. Irregular shape of SC was observed in HE stain (A). The anterior parts of SC showed thrombomodulin negative (B, thick arrows). Discontinuity of the endothelium (C, arrows) was observed at the corresponding area of negative thrombomodulin (B, thick arrows). (C) The transmission electron microscopy image of was made from the boxed area in inset. Note that the shortened Schlemm's canal (SC in B) showed the same strong positive thrombomodulin as the Sondermann canal (thin arrow, B).
Figure 8
 
(A, B) Light microscopy. (C) Transmission electron microscopy images of SC and TM with LM inset (toluidine blue staining). Staining of HE (A) and thrombomodulin immunohistochemical staining (B) in familial POAG patient no. 21, a 69-year-old male. Irregular shape of SC was observed in HE stain (A). The anterior parts of SC showed thrombomodulin negative (B, thick arrows). Discontinuity of the endothelium (C, arrows) was observed at the corresponding area of negative thrombomodulin (B, thick arrows). (C) The transmission electron microscopy image of was made from the boxed area in inset. Note that the shortened Schlemm's canal (SC in B) showed the same strong positive thrombomodulin as the Sondermann canal (thin arrow, B).
Significantly lower SC-ECD was observed in the samples obtained from group B patients (1.14 ± 0.63/100 μm) compared with those obtained from the patients in groups A and C (P = 0.0024, 1.55 ± 0.91/100 μm and P = 0.01, 1.76 ± 1.04/100 μm, respectively; Table 4). A positive correlation was found between SC length and PTNA in group A; however, no correlation was found in groups B and C (Figs. 9A–C). A positive correlation between patient age and increase of PTNA (r = 0.38, P = 0.0013) was observed only in group B cases (Figs. 10A–C). Moreover, a significant correlation between patient age and decrease of SC-ECD (r = −0.53, P = 3.95 × 10−6) was observed only in group B cases (Figs. 11A–C). A positive correlation of patient age and SC length was observed in group B (r = 0.45, P = 0.0013), while SC length in group A tended to decrease with age (r = −0.22, P = 0.07, Figs. 12A, 12B). 
Figure 9
 
(A–C) Graphs showing the correlation between PTNA and SC length in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents PTNA (%), and the horizontal axis represents the length of SC. Significant correlation was found between PTNA and SC length in group A (r = 0.27, P = 0.02), but not in group B (r = 0.27, P = 0.07) and group C (r = 0.31, P = 0.15).
Figure 9
 
(A–C) Graphs showing the correlation between PTNA and SC length in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents PTNA (%), and the horizontal axis represents the length of SC. Significant correlation was found between PTNA and SC length in group A (r = 0.27, P = 0.02), but not in group B (r = 0.27, P = 0.07) and group C (r = 0.31, P = 0.15).
Figure 10
 
(A–C) Graphs showing the correlation between PTNA and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents PTNA (%), the horizontal axis represents age. Significant correlation was found between PTNA and age in group B (r = 0.39, P = 0.0013), but not in group A (r = −0.005, P = 0.97) and group C (r = 0.39, P = 0.06).
Figure 10
 
(A–C) Graphs showing the correlation between PTNA and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents PTNA (%), the horizontal axis represents age. Significant correlation was found between PTNA and age in group B (r = 0.39, P = 0.0013), but not in group A (r = −0.005, P = 0.97) and group C (r = 0.39, P = 0.06).
Figure 11
 
(A–C) Graphs showing the correlation between SC-ECD and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents SC-ECD (number/100 μm), and the horizontal axis represents age. Significant correlation was found between SC-ECD and age in group B (r = −0.53, P = 3.95 × 10−6), but not in group A (r = 0.05, P = 0.70) and group C (r = −0.34, P = 0.11).
Figure 11
 
(A–C) Graphs showing the correlation between SC-ECD and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents SC-ECD (number/100 μm), and the horizontal axis represents age. Significant correlation was found between SC-ECD and age in group B (r = −0.53, P = 3.95 × 10−6), but not in group A (r = 0.05, P = 0.70) and group C (r = −0.34, P = 0.11).
Figure 12
 
(A–C) Graphs showing the correlation between SC length and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents SC length, and the horizontal axis represents age. Significant correlation was found between SC length and age in group B (r = 0.45, P = 0.0013), but not in group A (r = −0.22, P = 0.07) and group C (r = 0.31, P = 0.16).
Figure 12
 
(A–C) Graphs showing the correlation between SC length and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents SC length, and the horizontal axis represents age. Significant correlation was found between SC length and age in group B (r = 0.45, P = 0.0013), but not in group A (r = −0.22, P = 0.07) and group C (r = 0.31, P = 0.16).
In this study, 14 pairs of eyes in group A and 11 pairs of eyes in group B were used, and the interclass correlation coefficient (ICC) of each parameter was investigated in those pairs of eyes. It was impossible to evaluate ICC in group C due to there being only two samples. Age (group A: ICC = 0.985, P = 2.3 × 10−12; group B: ICC = 0.995, P = 9.9 × 10−13); C/D (group A: ICC = 0.817, P = 6.3 × 10−5; group B: ICC = 0.830, P = 2.5 × 10−4); and SE (A: ICC = 0.983, P = 5.3 × 10−12; group B: ICC = 0.975, P = 8.5 × 10−9) in both groups, the number of Sondermann canals (ICC = 0.489, P = 0.029) in group B, and VF (ICC = 0.494, P = 0.045), preoperative IOP (ICC = 0.565, P = 0.023), and PTNA (ICC = 0.776, P = 0.001) in group B showed significantly similar values. We also investigated whether the results of each parameter of groups A and B in the use of a single eye (use of right [R] or left [L] eye) of paired eyes differs from those in which both eyes of the pair were used. No significant difference was found in each of the clinical parameters between groups A and B in the paired eyes in which a single eye was used, as observed in the paired eyes in which both eyes were used (Table 2). All histological parameters, except the number of Sondermann canals, showed the same significant difference or tendency (SC length: P = 0.03 [R], 1.9 × 10−3 [L]; PTNA: P = 0.10 [R], 0.06 [L]; SC-ECD: P = 0.02 [R], 0.08 [L]) between groups A and B as those in the use of both eyes (Table 4). 
Age matching of SC length, PTNA, and SC-ECD in the three groups was evaluated by dividing every 5 years (e.g., 40–45 years: 40 years or older and younger than 45 years). No significant differences in SC length were found between groups A and B in the age ranges of 40 to 45 years (P = 0.54) years and 45 to 50 (P = 0.09) years. In addition to this, no significant differences in SC were found between groups A and B before age 50 years (P = 0.30). However, a short and narrow SC was observed in some eyes of group B before age 50 years (Fig. 6B), which was a characteristic abnormality in group A. In all 5-year age ranges older than 50 years, the average SC length in group A was found to be shorter than those of groups B and C. Length of SC in group A was significantly shorter compared with that of group B only in the age range of 75 to 80 years (P = 0.00; A: n = 8; B: n = 6) or tended to be shorter in the age range of 60 to 65 years (P = 0.02; A: n = 4; B: n = 12) and 70 to 75 years (P = 0.02; A: n = 9; B: n = 10). Moreover, SC length in group A was found to be significantly shorter compared with that of group C in the age range of 50 to 55 years (P = 0.01; A: n = 8; C: n = 7) and 65 to 70 years (P = 0.01; A: n = 8; C: n = 3). However, it was difficult to evaluate age matching in group C in some age ranges, because the number of the eyes (n = 1–7) in each age range was very small. Although no significance differences were found in the age matching of PTNA among the three groups, the eyes in group B showed a tendency of decreasing PTNA in individuals older than 65 years when compared with those in group A (age range: 65–70 years, P = 0.06; age range: 70–75 years, P = 0.05; age range: 75–80 years, P = 0.09). In group B, SC-ECD was also found to significantly be (age range: 65–70 years, P = 0.01) or tend to be (age range: 70–75 years, P = 0.02; age range: 75–80 years, P = 0.08) lower compared with those in group A. 
Discussion
In this study, no significant differences were found in regard to age, sex, C/D, and refractive errors among the three groups. Moreover, no significant difference in preoperative IOP was found between groups A and B. Significantly advanced visual field defects were observed in group C (patients with NTG) compared with groups A and B, suggesting that TLE was spared in cases of NTG unless showing advanced defects in their visual fields. 
There have been two conflicting reports concerning the length of SC. Rohen et al.24 reported that there was no difference of SC length between normal control eyes and POAG eyes. On the other hand, it has been reported that SC length was significantly shorter in POAG eyes than in normal eyes,25 and that there was a significantly higher percentage of collapse of SC in POAG eyes compared with normal eyes.26 Moreover, Allingham et al.25 reported that the average SC length in normal control eyes was 264 ± 55 μm, which is comparable to SC length in group C in this present study (277.7 ± 65.52 μm). Thus, we theorized that the length of SC in group C was within the normal range. Length of SC in group A was found to be significantly shorter than that in groups B and C, and no significant difference was found between groups B and C (Tables 3, 4). From these findings, we concluded that the eyes from POAG patients with a family history of the disease (group A) had a shorter SC length, thus suggesting that SC might more easily collapse with age. 
There have been several reports regarding the immaturity of SC in congenital glaucoma and developmental glaucoma. Broughton et al.27 reported that the morphology of SC in congenital glaucoma patients was normal. On the other hand, Perry et al.28 reported that SC in congenital glaucoma patients was short or locally obliterated. Since it has been reported that SC development is accomplished between embryonal weeks 27 and 40,29 we considered that our cases with a very short and fine SC like the Sondermann canal (Figs. 1A, 1B, 5A, 5C) or with a short SC (Figs. 6A, 6B), not only in group A but also in group B, were a congenital anomaly of SC development. Advancements in bioimaging technologies applied to neonatal eyes or genetic studies concerning blood vessel development might possibly help to clarify whether or not abnormal SC development causes POAG. 
Bill et al.30 reported that SC endothelium has been estimated to generate approximately 10% of the total trabecular resistance; however, their conclusion was based on the assumption that SC morphology was normal. Allingham et al.25 reported that SC dimensions play an important role in influencing outflow facility in normal eyes. Moreover, Ainsworth et al.19 reported that the decrease in the number of ECs with age is closely linked to an age-related reduction in the size of SC, notably, a reduction in the meridional length of the canal. A previous report demonstrated that SC filtration function is significantly influenced by age, due to the reduced endothelial inner-wall outflow facility.20 These reports indicated that age-related morphological alteration of SC could affect outflow facility and control of IOP in POAG patients. 
In this study, TEM observation of SC ECs showed dissociation of the intercellular junctions, degenerative changes, and EC loss at the thrombomodulin-negative area of SC endothelium (Figs. 3, 4, 7, 8). Therefore, we considered that thrombomodulin or CD34 (Fig. 2A, inset) immunohistochemical staining is useful to evaluate the functional aspects of SC ECs.7,23 Moreover, we found that the patients in group B had a higher PTNA and significantly smaller SC-ECD compared with those in groups A and C (Tables 3, 4). 
We observed the thrombomodulin-negative area in all three groups in variable degrees (Figs. 24, 7, 8). We also considered that the abnormalities of the TM made its ECs more susceptible to age-related changes. Johnson et al.31 reported that pore density of the inner-wall endothelium in glaucomatous eyes is less than one-fifth of that found in normal eyes. Moreover, Allingham et al.32 reported that pores in POAG eyes appeared to be more unevenly distributed than in normal eyes. These reports suggested that POAG patients have abnormalities of the TM, which made its ECs outflow less, thus resulting in more susceptibility to age-related changes. This idea is consistent with the findings of Allingham et al.,25 who reported that an increase in flow resistance within the TM could promote closure and final collapse of SC. In this study, the correlations of the increase of PTNA and decrease of SC-ECD with the increase of patient age were observed in group B (Figs. 10, 11). These findings indicate that SC EC loss and degenerative changes became apparent with aging in group B patients at around middle age. We did wonder why there were no correlations between patient age and PTNA, as well as patient age and SC-ECD, in the group A patients in this present study. One possible explanation is that SC (Figs. 1A, 1B) like the Sondermann canals or slit-like SC (Fig. 5A) in the group A patients tended to collapse directly (Figs. 5D) without leaving de-endothelialized lumen of SC (Figs. 3, 4), which was significantly evident in group B. The other is that severe abnormalities of the TM may cause rapid SC collapse even if SC is normal. 
The accumulation of extracellular matrix in JCT is an important age-related change in the TM18; however, we considered that a greater than 40% EC loss in SC induces more prominent effects on the elevation of IOP (Figs. 3, 4). Our results showed that higher preoperative IOP was observed in the patients with higher PTNA (greater than 40%, Table 5). This result also supported our idea that high PTNA (which indicates a high rate of EC loss) may induce negative effects on the control of IOP. 
Schlemm's canal ECs were found to seal off the de-endothelialized lumen of SC at the border (Figs. 2B, inset arrow; 3A, arrowhead). Schlemm's canal lumen without ECs became ghost vessels (Fig. 4A), replaced by coarse connective tissue (Figs. 5D, 7C, 7D) and finally occluded (Figs. 4B, 5D, 7C, 7D, 8B, arrows). A thrombomodulin-negative SC (Figs. 3A, 3B) or ghost vessels with loss of SC ECs (Fig. 4) may ultimately become occluded and replaced by fibrotic tissue (Figs. 4B, 5D, 7C). We observed another type of short SC, which occurred in a very fine SC similar to the Sondermann canal (Figs. 5A, 5C). It was difficult to clearly distinguish between shorter SC length and an acquired closure of SC in our cases, because our cases were not neonatal ones. However, the short SCs with rather round-shaped TM cells (Figs. 6A, 6B, asterisks) which were different from spindle-shaped fibroblast (Figs. 4B, arrows; 7C, arrows) in the supposed region of SC could be regarded as abnormal SC development or an immature SC, as opposed to a collapsed SC. Age-related changes of cerebrovascular ECs have been reported, including pathological changes of cytoplasm33 and loss of the ECs themselves.34 Since SC originates from blood vessels,28 the age-related changes of ECs in SC are consistent with the report of cerebrovascular ECs. 
We also investigated age matching of histological parameters (SC length, PTNA, and SC-ECD) by dividing every 5 years. The average SC length in group A was found to be shorter than that of groups B and C in all 5-year age ranges over the age of 50 years, yet significant differences were only found in the age-range of 75 to 80 years in the comparison between groups A and B, and 50 to 55 years and 65 to 70 years in the comparison between groups A and C. On the other hand, no significant differences in SC length were found between groups A and B in the age ranges of 40 to 45 years, 45 to 50 years, and younger than 50 years. Some eyes with a short SC before the age of 50 years in group B (Fig. 6B) may be one of the reasons for no significant difference in SC length between groups A and B. However, SC length in group A tended to decrease with age (r = −0.22, P = 0.07, Fig. 12A), while SC length in group B significantly increased with age (r = 0.45, P = 0.0013, Fig. 12B). Length of SC in group B patients was significantly longer than that in group A (Table 4). In the eyes with longer SC in group B, timing of the TLE may have been delayed, thus explaining the significant increase of SC length with age. Together with the data of no significant difference of SC length before the age of 50 in the age-matching of groups A and B, there may be a different mode of aging in the morphological abnormalities of SC between groups A and B. It is possible that SCs in group A collapse more easily when compared with those in group B, and we speculate that there may be two reasons for this. The first reason is that SC, when narrow like the Sondermann canal (Figs. 1A, 1B) or slit like SC (Fig. 5A), in POAG with a family history may easily collapse without the process of SC EC dropout. On the other hand, the lumen of SC in POAG without a family history (group B) tended to remain open even if SC loses SC ECs (Figs. 3, 4A). The other reason is that abnormalities in JCT or the trabecular meshwork in group A may cause a more rapid collapse, resulting in a shorter SC according to aging (Fig. 5D). 
The age-related PTNA and SC-ECD of group B was more evident in age matching when compared with group A. PTNA significantly increased or tended to increase over the age of 65. SC-ECD in Group B also significantly decreased or tended to decrease over the age of 65. Therefore, PTNA (Fig. 10B) and SC-ECD (Fig. 11B) might be the best parameters for the assessment of aging in relation to SC. 
We investigated ICC of the pairs of eyes in groups A and B, and how the use of paired eyes affected those results, as discussed above. Some of the clinical and histological parameters showed significantly similar values in the paired eyes; however, use of the paired eyes did not affect those results in the comparison of groups A and B. The Sondermann canal data was found to be unstable in the use of the paired eyes in groups A and B. The winding routes of the Sondermann canal in the TM might explain the varied results obtained when counting the number of canals in the paraffin sections. 
It should be noted that this present study did have several limitations. First, we cannot deny the possibility that the group B patients may have included familial glaucoma cases, since the patients may not have been aware of the complete family history and future occurrence of glaucoma in their relatives. Some of the nonfamilial cases showed a shorter SC length, like the Sondermann canal (Figs. 5B, 5C, 6B), which is a characteristic change of familial cases. Second, using the TLE specimens, we could only observe approximately 10% of the complete outflow structure, so there might be a bias from the limited area of observation. In fact, most of the cases showed small intertissue block deviations in SC length within the one tissue sample (Figs. 2A, 2B); however, some samples showed relatively large intertissue block deviations of SC length (Figs. 1A, 1B, 5B, 5C). Third, due to the relatively advanced age of the patients in this study that had undergone TLE, the number of samples aged younger 40 years was not sufficient for studying age matching in the three groups. Finally, all the cases in our study received the maximum tolerable antiglaucoma eye drop medication, so these treatments may have induced some morphological changes in the outflow structures. Allingham et al.25 reported that obstructive changes were observed even in SC of normal eyes without the history of glaucoma medication. Therefore, it is reasonable to consider that aging could induce the loss and degenerative changes of SC ECs, although we could not exclude the effects of antiglaucoma eye drops. 
In summary, the significantly shorter SC in group A suggests that SC may be more easily to collapse without the process of SC EC loss, de-endothelialized lumen, and then collapse, which were the characteristic changes of those in group B. In primary open angle glaucoma patients without a family history of the disease, prominent loss and degenerative changes of SC ECs were observed before collapse of SC. Schlemm's canal aging might progress more slowly in group B. The findings of this study may prove useful for the strategies of controlling IOP in glaucoma patients, the choice of surgical methods, and the prediction of surgical outcome. Future advancements in bioimaging technologies, including in vivo confocal microscopy and high-resolution optical coherence tomography, may enable us to better detect the morphological abnormalities in SC or the changes of SC ECs. 
Acknowledgments
The authors thank Kyoko Hamanaka for feedback and John Bush for editing the manuscript. 
Disclosure: T. Hamanaka, None; A. Matsuda, None; T. Sakurai, None; T. Kumasaka, None 
Grant Information: None to report. 
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Figure 1
 
(A, B) Light microscopy images of SC and TM in the eye of familial POAG patient no. 3, a 38-year-old male. Thrombomodulin immunohistochemical staining. Length of SC was measured between the anterior tip (open arrow) and the posterior tip (solid arrow) of the canal. Sondermann's canals were observed in the TM (small black arrows). Inset: serial section stained with HE from the same block. In the other block of the same eye, SC length was shorter (B). The anterior part of the canal (A, star) and the posterior half of the canal (B, star) appeared to be much shorter and showed strong positive thrombomodulin staining. Open arrowheads in A and B indicate the endothelium of the canal. Judging from the photograph of the section stained with HE (B, inset), it was difficult to estimate the length of SC. CC, collector channel; I, iris.
Figure 1
 
(A, B) Light microscopy images of SC and TM in the eye of familial POAG patient no. 3, a 38-year-old male. Thrombomodulin immunohistochemical staining. Length of SC was measured between the anterior tip (open arrow) and the posterior tip (solid arrow) of the canal. Sondermann's canals were observed in the TM (small black arrows). Inset: serial section stained with HE from the same block. In the other block of the same eye, SC length was shorter (B). The anterior part of the canal (A, star) and the posterior half of the canal (B, star) appeared to be much shorter and showed strong positive thrombomodulin staining. Open arrowheads in A and B indicate the endothelium of the canal. Judging from the photograph of the section stained with HE (B, inset), it was difficult to estimate the length of SC. CC, collector channel; I, iris.
Figure 2
 
(A, B) Light microscopy images of SC and TM in the eye of nonfamilial POAG patient no. 23, a 49-year-old male. Thrombomodulin (A, B) and CD34 (A, inset) immunohistochemical staining. Length of SC was measured between the anterior tip (open arrow) and the posterior tip (solid arrow) of the canal in which the anterior part did not show positive for thrombomodulin (A, stars, inset). Note that the positive staining area was almost the same as that in CD34 (A, inset). Figures (A, B) were taken from different blocks of the same specimen. At the border of the positive and nonpositive thrombomodulin area, endothelium of the canal sealed off the nonpositive thrombomodulin area (B, black arrow in inset). Note that the inset is an enlargement of the boxed area in (B). Arrowheads: endothelium of the canal. Small black arrows: Sondermann canal.
Figure 2
 
(A, B) Light microscopy images of SC and TM in the eye of nonfamilial POAG patient no. 23, a 49-year-old male. Thrombomodulin (A, B) and CD34 (A, inset) immunohistochemical staining. Length of SC was measured between the anterior tip (open arrow) and the posterior tip (solid arrow) of the canal in which the anterior part did not show positive for thrombomodulin (A, stars, inset). Note that the positive staining area was almost the same as that in CD34 (A, inset). Figures (A, B) were taken from different blocks of the same specimen. At the border of the positive and nonpositive thrombomodulin area, endothelium of the canal sealed off the nonpositive thrombomodulin area (B, black arrow in inset). Note that the inset is an enlargement of the boxed area in (B). Arrowheads: endothelium of the canal. Small black arrows: Sondermann canal.
Figure 3
 
(A, B) Light microscopy (A, B, inset; C) and TEM (C) images of SC and TM in the eyes of nonfamilial POAG patient no. 54, a 71-year-old male. (A) Right eye. (B, C) Left eye. (A, B) Thrombomodulin immunohistochemical staining. (A, inset; B, inset) HE staining. (C, inset) Toluidine blue staining. Note that SC endothelium in the right eye sealed off the canal (A, arrowhead) from the nonthrombomodulin staining area (double arrows: PTNA in [A] = 45%) and that the strong positive staining of the canal is comparable with that of the Sondermann canal (A, arrow). Intraocular pressure in the left eye (B, C) was elevated to 27 mm Hg despite maximum medication after 3.5 years of good IOP control. Nonthrombomodulin staining areas (double arrows) were also observed ([B] PTNA = 40%), but no sealing of the canal by SC endothelium was observed. The spaces of the TM and JCT seemed to be open and HE staining showed no inflammatory cells (B, inset). In the TEM image (C), dropout of SC endothelium was observed. The degenerated SCE was observed in the inner wall (arrow) and JCT (asterisks) was exposed to the lumen of SC. (C) The TEM image was made from the boxed area in the inset.
Figure 3
 
(A, B) Light microscopy (A, B, inset; C) and TEM (C) images of SC and TM in the eyes of nonfamilial POAG patient no. 54, a 71-year-old male. (A) Right eye. (B, C) Left eye. (A, B) Thrombomodulin immunohistochemical staining. (A, inset; B, inset) HE staining. (C, inset) Toluidine blue staining. Note that SC endothelium in the right eye sealed off the canal (A, arrowhead) from the nonthrombomodulin staining area (double arrows: PTNA in [A] = 45%) and that the strong positive staining of the canal is comparable with that of the Sondermann canal (A, arrow). Intraocular pressure in the left eye (B, C) was elevated to 27 mm Hg despite maximum medication after 3.5 years of good IOP control. Nonthrombomodulin staining areas (double arrows) were also observed ([B] PTNA = 40%), but no sealing of the canal by SC endothelium was observed. The spaces of the TM and JCT seemed to be open and HE staining showed no inflammatory cells (B, inset). In the TEM image (C), dropout of SC endothelium was observed. The degenerated SCE was observed in the inner wall (arrow) and JCT (asterisks) was exposed to the lumen of SC. (C) The TEM image was made from the boxed area in the inset.
Figure 4
 
Light microscopy images of SC and TM in the eyes of nonfamilial POAG patient no. 25, a 73-year-old male. (A, B) Thrombomodulin immunohistochemical staining. (A, inset; B, inset) HE staining. (A, B) Images were taken from serial sections of the same block. Although the canal appears open in HE staining (A, inset), thrombomodulin was completely negative throughout the entire length of SC (distance between open and solid arrows, PTNA = 100%). Part of SC became occluded with a replacement of spindle-shaped fibroblasts (arrows) in (B). Preoperative IOP showed 30 mm Hg despite of the maximum medication.
Figure 4
 
Light microscopy images of SC and TM in the eyes of nonfamilial POAG patient no. 25, a 73-year-old male. (A, B) Thrombomodulin immunohistochemical staining. (A, inset; B, inset) HE staining. (A, B) Images were taken from serial sections of the same block. Although the canal appears open in HE staining (A, inset), thrombomodulin was completely negative throughout the entire length of SC (distance between open and solid arrows, PTNA = 100%). Part of SC became occluded with a replacement of spindle-shaped fibroblasts (arrows) in (B). Preoperative IOP showed 30 mm Hg despite of the maximum medication.
Figure 5
 
(A–D) Light microscopy images of a short SC and TM stained with HE (A–C, inset; D) and antibodies against thrombomodulin (A–C; D, inset). (A) Familial POAG patient no. 58, a 46-year-old female. (B, C) Nonfamilial POAG patient no. 49, an 83-year-old male. (D) Familial patient no. 2, a 58-year-old male. The thrombomodulin staining pattern was very strong in some areas of the canal (A, arrowhead) compared with the rest of canal. Note that the staining of the Sondermann canal is also very strong (A, arrows). Although the canal looked normal (B), the canal in the other block appeared to be short (C), or completely collapsed in the other patient (D, stars). Fibrotic change was observed in the supposed region of SC (asterisks).
Figure 5
 
(A–D) Light microscopy images of a short SC and TM stained with HE (A–C, inset; D) and antibodies against thrombomodulin (A–C; D, inset). (A) Familial POAG patient no. 58, a 46-year-old female. (B, C) Nonfamilial POAG patient no. 49, an 83-year-old male. (D) Familial patient no. 2, a 58-year-old male. The thrombomodulin staining pattern was very strong in some areas of the canal (A, arrowhead) compared with the rest of canal. Note that the staining of the Sondermann canal is also very strong (A, arrows). Although the canal looked normal (B), the canal in the other block appeared to be short (C), or completely collapsed in the other patient (D, stars). Fibrotic change was observed in the supposed region of SC (asterisks).
Figure 6
 
(A, B) Light microscopy images of a short SC and TM stained with antibodies against thrombomodulin. Insets show the HE-stained sections. (A) Familial POAG patient no. 32, a 40-year-old female. (B) Nonfamilial patient No. 31, a 17-year-old female. (A, B) The canals appear to be short rather than collapsed after birth, because the areas where there should be a canal (stars, A, B) are compact and were occupied by oval-shaped cells.
Figure 6
 
(A, B) Light microscopy images of a short SC and TM stained with antibodies against thrombomodulin. Insets show the HE-stained sections. (A) Familial POAG patient no. 32, a 40-year-old female. (B) Nonfamilial patient No. 31, a 17-year-old female. (A, B) The canals appear to be short rather than collapsed after birth, because the areas where there should be a canal (stars, A, B) are compact and were occupied by oval-shaped cells.
Figure 7
 
(A–C) Light microscopy and TEM (D) images of SC and TM. Staining with HE (A), thrombomodulin (B), and toluidine blue (C) in nonfamilial POAG patient no. 39, an 81-year-old female. The anterior part of the canal (A, star) showed thrombomodulin negative and SC occlusion (B, star). Replacement of fibrotic tissue with spindle-shaped fibroblasts (C, arrows) and drop out of SC endothelium (D, asterisk) were observed at the corresponding area of negative thrombomodulin (B, star). (D) Transmission electron microscopy image was made from the boxed area in inset C.
Figure 7
 
(A–C) Light microscopy and TEM (D) images of SC and TM. Staining with HE (A), thrombomodulin (B), and toluidine blue (C) in nonfamilial POAG patient no. 39, an 81-year-old female. The anterior part of the canal (A, star) showed thrombomodulin negative and SC occlusion (B, star). Replacement of fibrotic tissue with spindle-shaped fibroblasts (C, arrows) and drop out of SC endothelium (D, asterisk) were observed at the corresponding area of negative thrombomodulin (B, star). (D) Transmission electron microscopy image was made from the boxed area in inset C.
Figure 8
 
(A, B) Light microscopy. (C) Transmission electron microscopy images of SC and TM with LM inset (toluidine blue staining). Staining of HE (A) and thrombomodulin immunohistochemical staining (B) in familial POAG patient no. 21, a 69-year-old male. Irregular shape of SC was observed in HE stain (A). The anterior parts of SC showed thrombomodulin negative (B, thick arrows). Discontinuity of the endothelium (C, arrows) was observed at the corresponding area of negative thrombomodulin (B, thick arrows). (C) The transmission electron microscopy image of was made from the boxed area in inset. Note that the shortened Schlemm's canal (SC in B) showed the same strong positive thrombomodulin as the Sondermann canal (thin arrow, B).
Figure 8
 
(A, B) Light microscopy. (C) Transmission electron microscopy images of SC and TM with LM inset (toluidine blue staining). Staining of HE (A) and thrombomodulin immunohistochemical staining (B) in familial POAG patient no. 21, a 69-year-old male. Irregular shape of SC was observed in HE stain (A). The anterior parts of SC showed thrombomodulin negative (B, thick arrows). Discontinuity of the endothelium (C, arrows) was observed at the corresponding area of negative thrombomodulin (B, thick arrows). (C) The transmission electron microscopy image of was made from the boxed area in inset. Note that the shortened Schlemm's canal (SC in B) showed the same strong positive thrombomodulin as the Sondermann canal (thin arrow, B).
Figure 9
 
(A–C) Graphs showing the correlation between PTNA and SC length in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents PTNA (%), and the horizontal axis represents the length of SC. Significant correlation was found between PTNA and SC length in group A (r = 0.27, P = 0.02), but not in group B (r = 0.27, P = 0.07) and group C (r = 0.31, P = 0.15).
Figure 9
 
(A–C) Graphs showing the correlation between PTNA and SC length in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents PTNA (%), and the horizontal axis represents the length of SC. Significant correlation was found between PTNA and SC length in group A (r = 0.27, P = 0.02), but not in group B (r = 0.27, P = 0.07) and group C (r = 0.31, P = 0.15).
Figure 10
 
(A–C) Graphs showing the correlation between PTNA and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents PTNA (%), the horizontal axis represents age. Significant correlation was found between PTNA and age in group B (r = 0.39, P = 0.0013), but not in group A (r = −0.005, P = 0.97) and group C (r = 0.39, P = 0.06).
Figure 10
 
(A–C) Graphs showing the correlation between PTNA and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents PTNA (%), the horizontal axis represents age. Significant correlation was found between PTNA and age in group B (r = 0.39, P = 0.0013), but not in group A (r = −0.005, P = 0.97) and group C (r = 0.39, P = 0.06).
Figure 11
 
(A–C) Graphs showing the correlation between SC-ECD and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents SC-ECD (number/100 μm), and the horizontal axis represents age. Significant correlation was found between SC-ECD and age in group B (r = −0.53, P = 3.95 × 10−6), but not in group A (r = 0.05, P = 0.70) and group C (r = −0.34, P = 0.11).
Figure 11
 
(A–C) Graphs showing the correlation between SC-ECD and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents SC-ECD (number/100 μm), and the horizontal axis represents age. Significant correlation was found between SC-ECD and age in group B (r = −0.53, P = 3.95 × 10−6), but not in group A (r = 0.05, P = 0.70) and group C (r = −0.34, P = 0.11).
Figure 12
 
(A–C) Graphs showing the correlation between SC length and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents SC length, and the horizontal axis represents age. Significant correlation was found between SC length and age in group B (r = 0.45, P = 0.0013), but not in group A (r = −0.22, P = 0.07) and group C (r = 0.31, P = 0.16).
Figure 12
 
(A–C) Graphs showing the correlation between SC length and age in groups A through C. (A) Group A. (B) Group B. (C) Group C. The vertical axis represents SC length, and the horizontal axis represents age. Significant correlation was found between SC length and age in group B (r = 0.45, P = 0.0013), but not in group A (r = −0.22, P = 0.07) and group C (r = 0.31, P = 0.16).
Table 1
 
Clinical Observations of the Patients in the Study
Table 1
 
Clinical Observations of the Patients in the Study
Table 2
 
Statistical Results of Clinical Observations, Comparisons between Two Groups
Table 2
 
Statistical Results of Clinical Observations, Comparisons between Two Groups
Table 3
 
Histological Observations of the Patients in Study
Table 3
 
Histological Observations of the Patients in Study
Table 4
 
Statistical Results of Histological Observations, Comparisons between Two Groups
Table 4
 
Statistical Results of Histological Observations, Comparisons between Two Groups
Table 5
 
Correlation Between Max Preoperative IOP and PTNA in Groups A and B
Table 5
 
Correlation Between Max Preoperative IOP and PTNA in Groups A and B
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