Histopathology of the Trabecular Meshwork and Schlemm's Canal in Primary Angle-Closure Glaucoma

Abstract

Purpose.: To investigate the detailed histopathology of trabecular meshwork changes associated with primary angle closure glaucoma (PACG).

Methods.: Thirty trabecular blocks obtained from trabeculectomy (TLE) of 25 PACG patients were embedded in paraffin for immunohistochemical staining of thrombomodulin, CD68, D2-40, and epon for transmission electron microscopy. Eleven TLE blocks obtained from normal-tension glaucoma patients were used as a control. Histologic changes of outflow routes were analyzed by comparing the existence of iridotomy, gonioscopy-evaluated angle closure, intraocular pressure (IOP), episodes of acute attack, visual field defect classified by Aulhorn-Greve, anterior chamber depth, lens thickness, and axial length.

Results.: Occlusion of the Schlemm's canal (SC) of <150 μm was observed in 11 eyes, which significantly correlated with gonioscopy-evaluated angle closure (T = 19.33 > χ² (f,α) = 9.488). Moderate correlation between SC occlusion and IOP before TLE was also observed (correlation coefficient: −0.540). Slightly negative or no correlations were found between SC occlusion and the other parameters. Thinned SC endothelium at the junction or degenerated SC endothelium and various degrees of SC occlusion and fusion of the trabecular beams where trabecular cells degenerated with damaged mitochondria were the general findings in the PACG eyes involved in this study.

Conclusions.: Persistent trabecular-iris contact or peripheral anterior synechia may block aqueous outflow resulting in a progressive process of SC endothelial damage and subsequent SC occlusion, as well as trabecular cell damage possibly due to impairment of mitochondrial function and subsequent fusion of the trabecular beams. These changes may be the reason for residual glaucoma after laser iridotomy or cataract surgery.

There is a very high prevalence rate of primary angle-closure glaucoma (PACG) in East Asian countries, and patients with PACG need to undergo continued medical assessment to detect any progression of the disease. ¹ Elevation of intraocular pressure (IOP) in cases of PACG occurs due to mechanical blockage of aqueous outflow by direct contact of the iris root with the trabecular meshwork. IOP elevation is usually resolved by releasing the trabecular-iris contact after laser iridotomy (LI), iridoplasty, goniosynechiolysis, or cataract surgery. However, in some cases, IOP remains elevated or increases again after such surgeries, and trabeculectomy (TLE) is often necessary. Despite the enormous amount of published clinical research on PACG, there has been little study into the pathology of this disease. Previous reports have speculated that fusion of the trabecular meshwork tissue is responsible for the increased IOP in cases of PACG. ^2,3 Narrowing or occlusion of Schlemm's canal (SC) in PACG cases has also been reported. ^2,4 However, there have been no histologic studies combined with clinical observation. In this study, we investigated the correlation between the clinical data and the histologic changes of TLE specimens of eyes with PACG addressing the following questions: Is there any correlation between the pathologic changes of SC and clinical findings? What are the reversible or irreversible changes in the outflow of PACG eyes? What is the residual glaucoma after LI or cataract surgery? 

Methods

Thirty TLE specimens obtained from 25 consecutive patients with PACG or primary angle closure and 11 TLE specimens obtained from 11 consecutive patients with normal tension glaucoma (NTG) who visited Japanese Red Cross Medical Center from 1996 to 2010 were retrospectively reviewed as to each patient's clinical record, and the TLE specimens were examined histologically. The TLE specimens obtained from the NTG patients were used as a control. TLE specimens of angle closure glaucoma eyes combined with pseudoexfoliation or due to a reason other than primary angle closure were excluded from this study. This study was approved by the Institutional Review Board of the Japanese Red Cross Medical Center, and prior informed consent was obtained from all patients for the use of the TLE specimens. The diagnosis of PACG was defined as the presence of glaucomatous cupping with a vertical cup ratio of > 0.7 and/or notching with compatible visual field loss, in association with the presence of at least 180° of the angle in which the posterior trabecular meshwork was not visible on static gonioscopy and raised IOP and/or peripheral anterior synechia (PAS) were observed by indentation gonioscopy. In this study, NTG was defined as the presence of glaucomatous cupping with a vertical cup ratio of > 0.7 and/or notching or saucerization with compatible visual field loss and IOP < 21 mm Hg. Diurnal eye pressure was checked before the start of medication or after discontinuing the previous glaucoma medication for at least 2 months in all NTG patients. During the TLE surgery, a square shaped flap (5 × 5 mm) was made parallel to the limbus. After the flap was raised, a rectangle cut of the deep sclera (1.5 × 3.5 mm) adjusted to include the gray zone at the center and to be parallel to the limbus was made by use of a razor blade to allow the aqueous humor to escape gradually with meticulous care to avoid a sudden loss of the anterior chamber. After cutting both sides and anterior sides of the TLE specimens, the basal side was cut and freed from the sclera. Then, the uveal part was cut using scissors by pinching the corner of the scleral part of the TLE specimen with forceps. The TLE specimens were immediately immersed in a mixture of 2.5% formalin and 1% glutaraldehyde overnight and then dissected into three to five pieces. During the dissection, the TLE specimens were cut by placing the uveal side up to avoid mechanical compression to the specimens. The divided pieces of all specimens were then alternately chosen for paraffin and epon embedding. For the embedding, the specimens were allowed to sink completely into the embedding plate in order for the entire cut surface of the specimen to come into contact with the bottom of the embedding plate. One to three pieces of each specimen were embedded in paraffin for hematoxylin-eosin (HE) staining and immunohistochemical staining of thrombomodulin for detecting SC endothelium and collector channels, CD68 immunostaining for detecting macrophages and monocytes, and D2-40 (Podoplanin; Covance, Emeryville, CA) staining for detecting trabecular cells in accordance with the previously reported methods by Watanabe et al. ⁵ The remaining two pieces from each TLE specimen were embedded in epon, cut into thin sections, stained with toluidine blue, and observed by light microscopy. Next, ultrathin sections cut from the epon-embedded sections were stained with uranium acetate and lead citrate, and then observed by transmission electron microscopy (TEM). The meridional length of the SC in each TLE specimen was then determined by the average diameter in the paraffin and epon sections of each block. Average diameters < 30 μm were regarded as zero. 

In regard to the clinical data (Table 1), the gonioscopic appearance and range of the PAS index were recorded. The locations of the TLE were judged as either closed, transitional, or open areas according to the record of the gonioscopic observation. Episodes of acute attack were judged by whether the patients had pain and redness in the affected eye before undergoing the TLE. Goldmann and Humphrey visual field tests were performed on each patient within 3 months before them undergoing the TLE, and severity of visual field deterioration was then judged according to the Aulhorn-Greve classification. ⁶ Anterior chamber depth (ACD), lens thickness, and axial length were examined by an ultrasound A scan before the TLE or cataract surgery. All clinical data were obtained after LI but before the TLE. The correlation between the length of the SC obtained by histologic study and the clinical parameters as listed above was analyzed by the Student's t-test and the Pearson correlation coefficient. 

Table 1.

 

List of Patients and Associated Clinical Observations

Patient Number (Sex)	(1) Age at TLE (Eye)	(2) Anterior Segment Parameters	(3) PAS Index (%)	(4) IOP	(5) VF
1 (M)*	77	2.16,5.42,22.41	91.7	27	6
2 (F)*	75 (R)	2.05,5.45,22.15	100	30	3
2 (F)*	77 (L)	2.10,5.45,22.36	33.3	21	2
3 (M)*	57	1.89,4.88,22.07	83.3	28	3
4 (M)*	66	1.99,5.14,23.01	100	38	4
5 (M)	68 (R)	2.38,5.10,21.96	0	25	1
5 (M)*	69 (L)	2.47,4.89,22.93	83.3	31	2
6 (M)	55	—,—,25.03	100	46	5
7 (F)	74	2.05,5.28,22.37	83.3	35	5
8 (M)	60	1.45,4.81,20.98	100	34	6
9 (F)	74	1.99,5.49,20.97	100	25	3
10 (F)	68	2.61,4.84,23.60	33.3	18	5
11 (F)	74	2.50,5.74,21.90	58.3	20	6
12 (M)	71	2.44,4.41,24.90	83.3	26	5
13 (F)	69	2.31,4.93,21.22	83.3	28	2
14 (M)	73	2.05,5.36,21.61	83.3	19	5
15 (F)	62 (R)	2.19,5.22,21.79	33.3	21	5
15 (F)	63 (L)	1.98,5.56,21.85	50.0	20	5
16 (F)	82	1.80,5.44,21.95	100	32	6
17 (M)	71	2.74,4.72,25.0	33.3	23	2
18 (F)	70	2.05,4.72,22.03	75.0	26	0
19 (F)*	79	1.22,5.72,22.01	100	42	5
20 (F)	55 (R)	2.78,4.65,23.47	0	24	3
20 (F)	56 (L)	2.77,4.45,23.04	0	32	2
21 (M)	57	1.73,5.66,20.85	83.3	32	5
22 (M)*	77	1.94,4.03,21.77	100	30	6
23 (M)	59	2.17,4.76,22.16	58.3	28	3
24 (F)	76	2.69,4.99,22.62	66.7	46	3
25 (M)	68 (R)	2.50,4.90,22.20	50	52	6
25 (M)	68 (L)	2.50,4.88,22.31	100	50	5

 Clinical observations: (1) Patient age at time of TLE; (2) Anterior segment parameters: anterior chamber depth (mm), lens thickness (mm), and axial length (mm) examined by ultrasound A scan; (3) PAS index (%) prior to TLE; (4) IOP before TLE; all eyes received the maximum amount of medication, with or without oral acetazolamide; and (5) VF was evaluated by Aulhorn-Greve classification. F, female; L, left; M, male; R, right; VF, visual field.

*  Presence of acute attack.

Results

Eleven TLE specimens from 11 eyes of 11 PACG patients were excluded from the results because eight specimens showed incomplete preservation of the trabecular meshwork and SC and because three eyes showed recurrent high IOP spikes, possibly due to a large amount of lens cortex remaining after cataract surgery. Three NTG patients were excluded from the results because their TLE specimens showed incomplete preservation of the trabecular meshwork and SC. The clinical data of all PACG eyes is listed in Table 1. The eyes were divided into three groups according to the surgeries that were performed before undergoing the TLE: group A (nine eyes of eight patients), no surgery; group B (16 eyes of 13 patients), LI; and group C (five eyes of five patients) phacoemulsification (PEA) with intraocular lens (IOL) implantation (Table 2). Group C included 4 eyes which had received LI before PEA+IOL implantation (patients 6, 9, 23, and 25 [R eye]). 

Table 2.

 

Surgeries before Trabeculectomy and Histological Results of Trabecular Meshwork and the Schlemm's Canal

Patient Number	(1) LI-TLE (mo)	(2) Type of Surgery	(3) Location of TLE	(4) Histological Findings of TM		(5) LSC
				JCT	TM (CD68)
1*	1.5	B	PAS	Compact	+	35
2 R*	1	B	PAS	Compact-open	+	309
2 L	1.8	B	TRS	Open	+	216
3*	119	B	PAS	Compact	+	0
4*	1.5	B	PAS	Compact	+	307
5 R	72	B	OPN	Open	−	257
5 L*	62	B	PAS	Compact	−	218
6	84	C	PAS	Compact-open	−	108
7	—	C	TRS	Open	−	139
8	1	B	PAS	Compact	−	0
9	45	C	PAS	Open	+	266
10	—	A	TRS	Compact	−	274
11	—	A	PAS	Compact	−	206
12	43	B	PAS	Compact	−	254
13	3	B	PAS	Compact	−	250
14	—	A	PAS	Compact	−	301
15 R	—	A	OPN	Compact	−	187
15 L	—	A	TRS	Compact	−	280
16	—	A	PAS	Compact	−	164
17	—	A	OPN	Compact	−	214
18	89	B	PAS	Compact	−	190
19*	4	B	PAS	Compact	+	0
20 R	3.5	B	OPN	Compact	−	114
20 L	7.5	B	OPN	Open	−	331
21	4	B	PAS	Compact	−	183
22*	—	A	PAS	Compact	+	301
23	95	C	PAS	Open	−	129
24	1	B	PAS	Compact-open	−	0
25 R	11	C	PAS	Compact	−	68
25 L	—	A	PAS	Compact	−	41

 Type of surgery and histological results: (1) LI-TLE, period (months) between laser iridotomy and trabeculectomy; (2) types of surgeries were divided into three categories, (A) PEA + IOL + TLE (no surgery prior to TLE), (B) LI → PEA + IOL + TLE, and (C) (LI →) PEA + IOL → TLE, including five eyes which had received LI prior to PEA + IOL implantation (patients 6, 9, 23 and 25 [R eye]); (3) gonioscopic observation at the area of the TLE was divided into three parts: at the area of PAS, the transitional part (TRS), and open (OPN). Note: Both eyes of patient 20 did not receive cataract surgery at the time of TLE; (4) morphologic appearance (open or compact) in the juxta-canalicular tissue (JCT) observed by light and transmission electron microscopy, and immunohistochemical staining of CD68 in the TM: existence of CD68-positive cells; and (5) Length of the Schlemm's Canal (LSC); LSC less than 30 μm was regarded as 0 μm.

*  Presence of acute attack.

SC and Collector Channels

The meridional length of the examined SCs varied from open (normal condition) (Fig. 1), partly occluded (Fig. 2), or almost completely occluded (Fig. 3, Table 2). The length of the canal was sometimes difficult to determine in eyes with a partly or almost occluded canal, which were clearly demonstrated by immunohistochemical staining of thrombomodulin (Figs. 2b, 4b, inset). In most of the eyes, the length of SC in each of the three pieces obtained from the same sample were similar, however, a large variation of > 150 μm was observed in three eyes (patients 2 [right eye], 15 [left eye], and 18). Thinning of the junction (Fig. 4a), separation of the junction and degeneration of the endothelium of the canal (Fig. 4a), and defect of the endothelium of the canal (Fig. 4b) were observed in the specimens with an occluded SC. Adjacent to the occluded canal, the JCT became compact with swollen trabecular cells (Figs. 3c, 4a). Normal-size canals also showed thinning of the junction (Figs. 5b, 6b) or separation of the junction (Fig. 5b) in the two trabeculectomy specimens taken from the gonioscopically open area, but the JCT of those two specimens were found to be different. In the eye of patient 15 which had undergone no surgery before the TLE, the JCT was compact with swollen trabecular cells (Fig. 6b). However, in the right eye of patient 5 that had undergone LI 72 months before the TLE, the JCT was open with a mixture of normal and degenerated trabecular cells (Fig. 5c). Moreover, SC in that eye was locally occluded by infiltration of melanocyte and pigment-laden trabecular cells (Figs. 5a, 5b). In the eyes in which the SC was occluded, the occluded area was replaced by pigment-laden trabecular cells, melanocytes, wide-banded collagen, and elastic fibers (Fig. 4b). Blood serum was found in all SCs that were almost occluded in the area where macrophage infiltration into the JCT or escaping melanocytes from the trabecular meshwork into the canal (Fig. 4b) were observed. Blood serum was also found in the normal-size SCs (Figs. 1, 5b) in the area where thinning or separation of the junction or degeneration of the endothelium of the canal with compact JCT was observed. The blood component was not observed in the normal endothelium of SC where the JCT looked normal with open spaces. 

Figure 1.

 

Light (upper inset) and TEM images of the endothelium of the SC and JCT in the eye of patient 4 with acute attack. TLE was performed 1.8 months after the acute attack. IOP before TLE: 38 mm Hg; gonioscopy of the TLE area: closed; type of surgery: LI. The size of the SC was of normal diameter (upper inset, light microscopy, toluidine blue staining) but it contained blood serum (open star). The JCT became compact with swollen trabecular cells and infiltration of macrophage (M). The trabecular-cell mitochondria appeared to be pyknotic (lower inset; increased magnification of boxed area). Numerous melanin granules existed around the collector channels (arrows in upper inset).

Figure 2.

 

Light (a–c, inset in d) and TEM (d) images of the trabecular meshwork and SC in the eye of patient 7 without acute attack. IOP before TLE: 27 mm Hg; gonioscopy of TLE area: transitional; type of surgery: PEA + IOL implantation. (a) HE staining. (b) Thrombomodulin immunohistochemical staining. (c) D2-40 immunohistochemical staining. (d, inset) Toluidine blue staining. The TEM image (d) was made from boxed area (d, inset). Although the SC length was difficult to determine in the HE-staining photograph (a), the photograph of thrombomodulin immunohistochemical staining clearly demonstrated the size of the moderately occluded SC (b). Trabecular cells throughout the entire meshwork area expressed D2-40. The spaces of the trabecular meshwork were normally open and trabecular cells did not show swelling, as is shown in Figure 1.

Figure 3.

 

Light (a, b) and TEM (c, d) images of the trabecular meshwork and the occluded SC in the eye of patient 3 with acute attack. The TEM image of (c) was made from boxed area in (b). IOP before TLE: 28 mm Hg; gonioscopy of the TLE area: closed; type of surgery: LI. (a) D2-40 immunohistochemical stain. (b) Toluidine blue staining. (d) Increased magnification of the boxed area in (c). Most of the trabecular cells did not express D2-40 except those in the anterior part of the meshwork (a, arrow). The SC was almost occluded (b) and the JCT trabecular meshwork became compact (b, c) with swollen trabecular cells (c). The trabecular-cell mitochondria showed pyknosis (d).

Figure 4.

 

Light (insets) and TEM images of the trabecular meshwork and the SC in the eyes of patients without (patient 16, a) and with (patient 19, b) acute attack. IOP before TLE: 32 mm Hg (patient 16) and 42 mm Hg (patient 19). Type of surgery: no surgery (patient 16) and LI (patient 19); gonioscopy of TLE area: closed in the eyes of both patients (a, left inset: D2-40 immunohistochemical staining; b, right inset: toluidine blue staining). The TEM images of (a) and (b) were made from the boxed areas (right inset, a, b). (b, left inset) Thrombomodulin immunohistochemical staining. (b, right inset) Toluidine blue staining. The SC became moderately (a) or almost (b) occluded. SC endothelium became very thin at the junction (a), degenerated (a, arrows), or disappeared (b) where melanocytes (solid star) penetrated into the canal. The JCT in (a) became compact with swollen trabecular cells. Trabecular cells in the middle and posterior part of the meshwork did not express D2-40 (a, left inset). The occluded part of the canal (b, open stars) was replaced by melanocytes, wide-banded collagen fibers, and elastic fibers. Thrombomodulin immunohistochemical staining detected collector channels (CC) but not the SC.

Figure 5.

 

Light and TEM (a, inset: toluidine blue staining) images of the trabecular meshwork and SC in the right eye of patient 5 without acute attack. IOP before TLE: 25 mm Hg; gonioscopy of the TLE area: open; type of surgery: LI. The shallow anterior chamber became deeper and the whole angle became visible by gonioscopy after the LI that was performed 72 months before the TLE. The TEM images of (a) and (c) were made from the boxed areas (A) and (C), respectively, shown in the inset of (a). The SC was open (a, inset). In the posterior part of the canal (b: increased magnification of the boxed area in a), the SC was locally occluded by infiltration of melanocytes (M in a) and pigment-laden trabecular cells (T in a and b) where the junction of the endothelium of the canal became very thin (b, black thin arrows) or separated (b, white thick arrow). Part of the SC was collapsed in the area indicated by the open star in (a). In the middle part of the SC (c), the spaces of the trabecular meshwork appeared as normally open and there were a few degenerated trabecular cells (open arrows) with impaired nuclei of the intermediate stage between margination and lysis (open star) among normal trabecular cells. Abundant pigment granules were also noted in the trabecular meshwork (a, inset).

Figure 6.

 

Light (a) and TEM (b, c) images of the trabecular meshwork and the SC in the right eye of patient 15 without acute attack. IOP before TLE: 21 mm Hg; gonioscopy of the TLE area: open; type of surgery: no surgery. (a) Toluidine blue staining. The TEM image (b) was made from the boxed area shown in (a). (c) Increased magnification of the boxed area in (b). Even in the specimen taken from the area of the gonioscopically open angle, the JCT spaces became compact with swollen trabecular cells (b). The SC endothelium became very thin at the junctions. The mitochondria of the trabecular cells in the JCT appeared normal (c).

Collector channels were normally open in all TLE samples, however, the accumulation of a large amount of melanocytes was observed around the collector channels (Fig. 1, inset) and the outer wall of the canal in patients 4, 7, 9, 11, and 22, whose eyes showed a greater-than stage 3 visual field defect (VFD) by Aulhorn-Greve classification. Among these patients, patients 7, 11, and 22 had not undergone LI. 

Occlusion of SC of <150 μm was observed not only in eyes with acute attack (three of seven eyes), but also in eyes without acute attack (7 of 23 eyes). There was no statistical difference in the length of SC between the eyes with and without acute attack (P = 0.330, Student's t-test). The length of SC was divided into the following three stages (Table 3): normal (>150 μm; 19 eyes), moderately occluded (between 50 and 150 μm; 5 eyes), and severely occluded (<50 μm; 6 eyes), and was significantly correlated with the area of gonioscopic observation (open, transitional, and closed or PAS) as evaluated by analysis with a 2 × 2 contingency table (T = 19.33 > χ² (f, α) = 9.488; Table 3). No statistical correlation was found between the length of the SC and lens thickness (correlation coefficient: −0.002672), axial length (correlation coefficient: −0.12476) and existence of iridotomy before TLE (T = 1.17 < χ² (f, α) = 5.991). Slightly negative correlations were found between the length of SC and the following parameters: VFD, ACD, and PAS index before TLE (correlation coefficient: −0.249, −0.204, and −0.220, respectively). There was a moderate correlation between the length of the SC and IOP (correlation coefficient: −0.539). The length of the SC in the PACG eyes (−2178 ± 106 μm; Table 4) was significantly shorter than that in the NTG eyes (Table 4, 1; 253 ± 66 μm) (P = 0.01195, Student's t-test). The mean age of the PACG patients (Table 4, 2; mean age: 68.8 ± 8.3 years) was significantly greater than that of the NTG patients (Table 4, 1; mean age: 54.5 ± 11.9 years) (P = 0.003845, Student's t-test). 

Table 3.

 

Correlation between LSC and Gonioscopic Appearance

Gonioscopy	LSC ≥ 150 μm	50 μm ≤ LSC < 150 μm	LSC < 50 μm	Total (Eyes)
Open	4	1	0	5
Transitional	3	1	0	4
Closed	12	3	6	21
Total	19	5	6	30

 The eyes were divided into three groups according to the gonioscopic appearance (open, transitional, and closed [PAS]). The length of SC was also divided into three groups according to the severity of occlusion (normal: ≥150 μm; moderately occluded: 50 μm ≤ LSC < 150 μm; and severely occluded: <50 μm). The length of SC was significantly correlated with the gonioscopic appearance (open, transitional, and closed) at the area of the TLE as evaluated by analysis with a 2 × 2 contingency table (T = 19.33 > χ² (f, α) = 9.488).

Table 4.

 

Patient Age at TLE and the Meridional LSC

Patient Number (Sex)	Patient Age at TLE	LSC
NTG patients
1 (M)	51	322
2 (M)	47	196
3 (M)	48	264
4 (M)	51	361
5 (M)	44	174
6 (F)	64	178
7 (M)	64	227
8 (F)	78	232
9 (F)	67	353
10 (M)	40	227
11 (M)	45	245
Mean ± SD	54.5 ± 11.9	252.6 ± 66.0
PACG patients
Mean ± SD	68 ± 8.3	178 ± 106.4

 The LSC in the eyes of the PACG patients was significantly shorter than that of NTG-patient eyes (P = 0.01195, Student's t-test). The average age of the PACG patients was significantly greater than that of the NTG patients (P = 0.003845, Student's t-test).

JCT

In some of the TLE specimens taken from both the gonioscopically open and closed areas, the trabecular meshwork in the JCT was found to have become compact with swollen trabecular cells. The mitochondria of trabecular cells in the JCT of these eyes appeared normal (Fig. 6c) or as having become pyknotic (Figs. 1, inset, 3c). Those JCT trabecular cells did not express D2-40 (Fig. 3a). TLE specimens were obtained mostly from the gonioscopically closed (PAS) area, but some were obtained from the transitional (4 specimens, Table 2) or open area (5 specimens, Table 2). Compact JCT with swollen trabecular cells even in the gonioscopically open area was observed in two TLE specimens (patients 15 and 17, Fig. 6b). However, trabecular cells in the JCT appeared normal (Fig. 7a) and were found to express D2-40 (Fig. 7b) in the TLE specimens obtained from the gonioscopically closed (PAS) area. These normal appearances in the JCT of TLE specimens taken from the gonioscopically transitional (Fig. 2) or closed (Fig. 7) area were observed mostly in the eyes which had previously undergone lens extraction with IOL implantation. The normal appearances of trabecular cells in the JCT were also observed in two eyes without PAS (patients 5 [R eye] and 20 [L eye]; Fig. 5c). 

Figure 7.

 

Light micrographs of the trabecular meshwork and SC in the eye of patient 23 without acute attack. IOP before TLE: 28 mm Hg; gonioscopy of the TLE area: closed; type of surgery: LI and PEA + IOL implantation. (a) Toluidine blue staining. (b) D2-40 immunohistochemical staining. Despite of the specimen being taken from the gonioscopically closed area, JCT and the spaces of trabecular meshwork seemed to be open (a) and the trabecular meshwork and JCT showed strongly positive with D2-40 (b).

Trabecular Meshwork

The spaces of trabecular meshwork were open in the three eyes (patients 5 [R eye] and 20 [R and L eyes]) had received LI (Fig. 5c). These eyes were found to be gonioscopically open and had a few degenerated trabecular cells surrounded by normal trabecular cells. Open spaces of the trabecular meshwork were also observed in most of the eyes that had previously undergone cataract surgery (Figs. 2d, 7a, 7b). These open spaces of trabecular meshwork expressed D2-40 (Figs. 2c, 7b). On the other hand, in the eyes with no spaces present between trabecular meshwork constituents, severe damage of the trabecular meshwork was located in the posterior part of the meshwork, where disappearance of trabecular cells (Fig. 8a) and degenerated trabecular cells with swollen mitochondria with absent cristae were observed (Figs. 8b, 8c). Disappearance of the trabecular cells was found to have resulted in fusion of the beams (Fig. 8b). Trabecular cells in the posterior part of the meshwork did not express D2-40 (Fig. 8a), however, trabecular cells in the anterior part of the meshwork had nearly normal mitochondria (Fig. 8f) and expressed D2-40 (Figs. 3a, 4a, left inset, 8d). 

Figure 8.

 

Light (a, d) and TEM (b, c, e, f) images of the trabecular meshwork and the SC in the eye of patient 18 without acute attack. IOP before TLE: 26 mm Hg; gonioscopy of TLE area: closed; type of surgery: LI. (a, d); D2-40 immunohistochemical staining. Trabecular cells in the posterior part of the trabecular meshwork did not express D2-40 (a) and had degenerated mitochondria with cloudy swelling (open stars in c; c is an increased magnification of the boxed area in b) and disappeared cristae (c). There was a fusion of trabecular beams (b, open stars) with disappearance of trabecular cells. On the other hand, trabecular cells in the anterior part of the meshwork clearly expressed D2-40 (d). Although there were trabecular beams without covering trabecular cells (e, stars), the trabecular meshwork appeared less damaged compared with that in the posterior part (b). The mitochondria appeared almost normal with slightly swollen cristae (f; an increased magnification of the boxed area in e). M, melanocyte.

The anterior part of the trabecular meshwork expressed D2-40 in all TLE specimens taken from the gonioscopically closed area. However, the staining pattern of D2-40 in the middle or posterior part of the meshwork of that same TLE sample was found to be different (Figs. 9c–d, 10d, 10f, 10h). These different staining patterns of D2-40 within the same sample seemed to be correlated with different stages of SC damage. In the area where the SC endothelium showed a complete positive reaction to thrombomodulin staining (Fig. 9a), the anterior part of trabecular meshwork stained strongly with D2-40 (Fig. 9c). However, in the other area where SC showed only small parts of positive reaction with thrombomodulin staining (arrowheads in Fig. 9b), the anterior part of the trabecular meshwork became fused and showed less positive with D2-40 (Fig. 9d). Similar results were observed in the different degrees of the canal occlusion with no or slightly positive reaction with thrombomodulin (Figs. 10c, 10e, 10g), ranging from the entire space of the canal remaining (Figs. 10c, 10d), to moderate (Figs. 10e, 10f) and complete disappearance of the space of the canal (Figs. 10g, 10h). D2-40 reactivity around the canal and trabecular meshwork seemed to correspond with the remaining size of the spaces of the canal (Figs. 10d, 10f, 10h). Fusion of the trabecular meshwork was also more evident in areas with complete disappearance of the canal (Fig. 10h) compared with areas where the entire space of the canal was preserved (Fig. 10d). 

Figure 9.

 

Light micrographs of the trabecular meshwork and SC in the eye of patient 6 without acute attack. IOP before TLE: 46 mm Hg; gonioscopy of the TLE area: closed; type of surgery: LI and PEA + IOL implantation. (a, b) Thrombomodulin immunohistochemical staining. (c, d) D2-40 immunohistochemical staining. (e, f) Control staining. Images a, c, e, and b, d, f were serial sections from two pieces embedded in paraffin. The canal expressed thrombomodulin (arrowheads in a) and the anterior part of the meshwork seemed to be open where the trabecular cells showed positive with D2-40 (c). However, in the other piece, only small parts of the canal expressed thrombomodulin (arrowheads in b) and the trabecular meshwork became compact where the trabecular cells showed less positive with D2-40 (d). Note the abundant melanin granules throughout the trabecular meshwork in the control staining of both pieces (e, f).

Figure 10.

 

Light micrographs of the trabecular meshwork and the SC stained with HE (a, b), thrombomodulin (c, e, g) and D2-40 (d, f, h) in the eye of patient 24 without acute attack. IOP before TLE: 46 mm Hg; gonioscopy of the TLE area: closed; type of surgery: LI. This TLE sample was divided into five pieces, among which, three pieces were embedded in paraffin. Images b–d, e–f, a, and g–h were serial sections from three different pieces. Anterior displacement of ciliary processes was observed (a). The canal looked normally open in (b), (c), and (d), which were serial sections from the same piece. However, the inner wall of the endothelium of the canal completely disappeared (b) where they did not express thrombomodulin (c). Slight positive staining in the remaining open spaces of the canal (arrowhead in e) and no positive staining of thrombomodulin in the completely occluded canal except the area indicated by ‘SC’ (g) were observed in the other pieces. Even in the same sample, D2-40 reactivity in JCT and the trabecular meshwork was different from piece to piece (d, f, h). D2-40 reactivity seemed to diminish in accordance with the disappearance of the open spaces of the canal. The iris was attached in some pieces (a, e–h) but separated in the other piece (c, d). Note the thick pigmentation on the surface of the trabecular meshwork (arrows in c). Large magnification of the rectangular area shown in the inset of (b). CM, ciliary muscle; CP, ciliary process.

Melanocytes and pigment-laden trabecular cells were found not only in the TLE specimens obtained from the gonioscopically closed area (Figs. 4a, 4b, 8e, 9), but also in those obtained from the open area (Fig. 5a). PAS were not present in some of the specimens taken from the gonioscopically closed area. However in the other areas even in the same specimens, PAS or apposition of the iris to the trabecular meshwork (Figs. 2a, 2b, 10a, 10e–h) were observed. PAS with anterior displacement of ciliary processes (Fig. 10a) or dispersed trabecular meshwork in different directions (Figs. 3a, 8d) were observed in specimens taken from the gonioscopically closed area. 

Macrophage or CD68-positive cells were found in the eyes which had an acute attack < 1.5 months before the TLE (patients 1, 2 [R eye], and 4; Fig. 1), as well as in an eye that had undergone LI within 1.8 months before the TLE (patient 2 [L eye]), eyes with a severely occluded SC (patients 3, 19, and 22). 

NTG Control Eyes

In the NTG control eyes, SC was normally open (Fig. 11a) and showed strongly positive with antibodies against thrombomodulin (Figs. 11c–e). Different sizes of SC were observed in different pieces taken from the same samples (Figs. 11c–e; 11c, 174 μm; 11d, 225 μm; 11e, 281 μm), however, those of a size < 150 μm were not observed in all NTG specimens (Table 4). D2-40 was positive throughout the JCT of the entire trabecular meshwork (Figs. 11b, 11f). However, in some pieces, D2-40 was negative in the posterior part of the trabecular meshwork because the scleral spur extremely protruded anteriorly (Fig. 11f). Anterior rotation of the iris and ciliary body during the preparation processes caused the iris to become close to the trabecular meshwork, however, in the iris root, there was remaining space (asterisk in Fig. 11a) which was not observed in any specimens of the PACG eyes if the iris remained attached to the trabecular meshwork (Figs. 2a, 2b, 10a, 10e–h). 

Figure 11.

 

Light micrographs of the trabecular meshwork and the SC in the control eyes of normal tension glaucoma. Serial sections of HE stain (a) and D2-40 immunohistochemical staining (b) from patient 1. Thrombomodulin immunohistochemical staining (c–e) and D2-40 staining (f) from patient 10. (c–e) Sections from three different pieces of the TLE specimen. (e–f) Serial sections from the same piece. The canal was normally open (a, 322 μm) and the entire trabecular meshwork and JCT expressed D2-40 strongly positive (b). Thrombomodulin staining in the different pieces from the same sample of patient 10 showed different sizes of the canal (c, 174 μm; d, 225 μm; e, 281 μm; average, 227 μm). The canals (double arrows in c and d) were separated by septum in some sections. Prominent protrusion of the scleral spur (SS in f) into the trabecular meshwork was observed in some sections of TLE samples in the control eyes. Note that the iris almost attached to the trabecular meshwork, however, space remained in the bottom of the angle (asterisk in a), which was not observed in PACG eyes if the iris remained attached to the trabecular meshwork (see Figs. 2a, 2b, 10a). The open arrowheads and solid arrowhead in (c) indicate the Sondermann's canal and the collector channel, respectively. SS, scleral spur.

Discussion

The narrowing or occlusion of SC in cases of PACG has been described in previous reports, ^{2 –4} however, all histologic studies and textbooks put a focus on the changes of the trabecular meshwork when discussing the reason for the increased IOP in PACG cases. The occlusion of SC that was observed in the samples in this present study seemed to be one of the most serious damages to the aqueous outflow system, and was found to occur in eyes with or without an acute attack (P = 0.330). Moderate correlation between the length of SC and IOP (correlation coefficient: −0.539) also supports the notion that severity of glaucoma in the outflow routes may partly depend on the severity of SC damage. There was no meaningful correlation between the length of the canal, Aulhorn-Greve classification, and ACD. In the eyes which showed SC occlusion < 150 μm, there were one, one, and nine eyes from the gonioscopically open, transitional, and occluded areas, respectively (Table 3). Moreover, in the eyes which showed SC occlusion < 50 μm, there were zero, zero, and six eyes from the gonioscopically open, transitional, and occluded areas, respectively (Table 3). Moreover, a significant correlation was found between the TLE samples obtained from three different gonioscopic observations (the open, transitional, and closed [PAS] areas of the angle) and three different sizes of SC (≧ 150 μm; 50 μm ≦ × <150 μm; and <50 μm; T = 19.33 > χ² (f, α) = 9.488) (Table 3). This finding strongly suggests that PAS or trabecular-iris contact are causal factors in occlusion of the canal. The SC size of the NTG patients (252 ± 66 μm; range, 174 and 361 μm; Table 4) was similar to that of normal eyes (264 ± 55 μm) in a previous report. ⁷ In addition, the trabecular meshwork and SC in NTG eyes is believed to function normally. Therefore, it seemed possible to use the TLE samples from NTG patients as a control. Although the average age of our NTG-sample patients was younger (mean age, 54.5 ± 11.9 years) than that of our PACG-sample patients (mean age, 68.0 ± 8.3 years), a SC size of <150 μm can be regarded as occluded. However, the presence of a wide range of differences with >150 μm of the canal in the same sample that was observed in three eyes (patients 2 [R eye], 15 [L eye], and 18) in this present study may represent the transition from an open to an occluded area. Thinning of the area of junction (Figs. 4a, 5b, 6b), separation (Fig. 5b), degeneration (Fig. 4a), and dropout (Figs. 4b, 10b) in the endothelium of the SC may be a progressive process which may lead to an occlusion of the canal. In addition, the open spaces of different sizes of SC within the same sample that did not express thrombomodulin (Figs. 10c, 10e) may be transitional stages to complete SC occlusion (Fig. 10g). Infiltration of melanocytes or pigment-laden trabecular cells into such an impaired area of the canal via the JCT may also play a role in the initiation of SC occlusion (Fig. 5a), followed by the entire canal finally being occluded due to the replacement by the fibrotic tissue (Fig. 4b). These serial changes of the endothelium of the canal were observed not only in the TLE specimens with occlusion of the SC, but also in those with a normal size canal (Fig. 6). The damaged area of the canal was always accompanied by swelling of the trabecular cells in the JCT, except in some eyes that had undergone LI (Fig. 5) or cataract surgery (Fig. 7) before the TLE. It now appears to be probable that the trabecular-iris contact or PAS causes blockade of aqueous outflow in the trabecular meshwork, which may result in swelling of the trabecular cells in the JCT. Blood reflux might have occurred at the time of perforation into the anterior chamber during the surgery, however, blood serum was not observed in the TLE samples which had normal SC endothelium with open JCT. Blood serum found in the eyes of different-size SCs with compact JCT (Figs. 1, 5) also suggested that the absence of aqueous outflow in the trabecular meshwork caused reflux of the blood component into the canal. 

Accumulation of melanocytes around the collector channels or outer wall of the canal was observed in 5 eyes with (patient 4, Fig. 1, inset) or without an acute attack. Those melanocytes seemed to have infiltrated into these areas from the iris root via the septum or occluded area of the canal (Figs. 4b, 5a). It seemed to be unlikely that the accumulation of melanocytes in these areas was caused by previous LI treatment, because the eyes of patients 7, 11, and 22 had not undergone LI before TLE. The evidence of the accumulated melanocytes around the collector channels indicated that there had been a long persistence of PAS or trabecular-iris contact, which may cause infiltration of melanocytes from iris stroma into the trabecular meshwork. Although the collector channel was not always observed in the TLE specimens, the finding that the visual field had severely deteriorated to a greater-than stage 3 Aulhorn-Greve classification in all eyes also supports the hypothesis that PAS or trabecular-iris contact may have existed for a long period, thus resulting in longstanding or intermittent IOP elevation. 

Interestingly, in the TLE samples obtained from the gonioscopically occluded area, D2-40 reactivity in the posterior part of the meshwork tended to be negative (Figs. 3a, 4a, 8a) but differed according to the damage of the SC endothelium (Figs. 9c, 9d, 10d, 10f, 10h). This finding indicates that trabecular cells need contact with the aqueous humor to express D2-40. Therefore, D2-40 might be useful not only for evaluating altered or abnormal functional status of the trabecular meshwork in early- or late-onset primary open angle glaucoma, ⁵ but also for evaluating trabecular-cell function. Pyknotic mitochondria were observed in swollen trabecular cells of the JCT (Fig. 1, inset, 3d). Mitochondrial pyknosis is not too well-recognized and is occasionally cited in the literature as surrounding tissue of the coagulative necrosis in the kidney and liver of rats after starvation carcinogenic diets and necrogenic diets. ⁸ The findings of this present study suggest that the swelling of trabecular cells in the JCT which did not express D2-40 and had pyknotic (Fig. 3a) or normal (Fig. 6c) mitochondria may not be a lethal reaction, but actually a reversible change. On the other hand, the spaces of the JCT with D2-40-positive trabecular cells in the eyes that had undergone cataract surgery before TLE were found to be much wider (Figs. 2, 7) than those in the eyes that had not undergone cataract surgery (Figs. 1, 3, 4, 6, 8a, 8b, 10g, 10h). It is also interesting that the spaces of the trabecular meshwork were open even in the specimen taken from the gonioscopically closed area of the eye that had previously undergone cataract surgery (Fig. 7, 9c). This finding indicates that cataract surgery caused increased aqueous outflow via the anterior border of PAS or trabecular-iris contact into the trabecular meshwork, possibly by reducing the force that was pushing the iris toward the anterior direction. The finding that the trabecular cells in the posterior part of the meshwork did not express D2-40 may correspond to the observation of degenerated trabecular cells with impaired mitochondria in the trabecular cells at the same area (Figs. 8b, 8c). These changes may be a lethal reaction of the trabecular cells, resulting in the disappearance of those cells in the posterior part of the meshwork and fusion of the beams. 

In this present study, it was somewhat difficult to judge from the histologic observations whether the so-called PAS were indicated by the trabecular-iris contact or by the fusion of iris tissue into the trabecular meshwork. Differing images of the relation of the iris root and trabecular meshwork were observed even in the same sample (Fig. 10). Some of the specimens indicated PAS because the fusion of iris tissue into the trabecular meshwork remained (Fig. 10a), but other specimens seemed to only indicate the possibility of PAS because the trabecular beams stretched in an abnormal direction (Figs. 3a, 8d) or because pigment remained on the surface of the trabecular meshwork facing the anterior chamber side (Fig. 10c) due to the preparation process. These observations suggested that the fusion of the iris into the trabecular meshwork was not firmly attached to the meshwork in the histologic examination. Attachment of iris tissue to the trabecular meshwork in the area of PAS in PACG seemed to be much weaker than that of PAS in neovascular glaucoma. Interestingly, swelling of the trabecular cells in the JCT was also observed in the TLE specimens taken from the gonioscopically open area (Fig. 6b). In that eye, intermittent trabecular-iris contact might have actually existed before the TLE. Sihota et al. ³ observed extensive trabecular changes even in areas without PAS in their study involving the histologic examination of 16 trabeculectomy specimens with PACG, and they described that gonioscopic evaluation of the extent of PAS may not truly reflect the extent of trabecular damage in cases of acute and chronic PACG. Therefore, a method of evaluation more accurate than gonioscopy, such as anterior-segment optical coherence tomography (OCT), should be used for assessing the range of PAS or trabecular-iris contact. There are several possible reasons as to why no significant relationship was found between the PAS index and the occlusion of the canal. One possible reason is that the trabecular-iris contact might have been eliminated by LI in some of the eyes, thus resulting in a decreased possibility of damaging SC. Another possible explanation is that only a small part of the outflow routes can be seen in the TLE sample, despite the fact that there is a variation of the size of the canal. ⁷ A third possible reason may be the above-mentioned lack of consistent observation between gonioscopical and histologic results. However, the PAS index may be an important tool for predicting outflow function, because it is believed that angle closure greater than 50% may cause IOP elevation. In this present study, there were nine eyes which had 50% or less PAS index (Table 1). Of those nine eyes, there were three eyes with no PAS (patients 5 and 20 [R and L eyes]) which showed a few degenerated trabecular cells surrounded by normal trabecular cells (Fig. 5c). Each of those three eyes had undergone LI > 3.5 months before the TLE. Those findings appear to indicate that trabecular-iris contact might have existed before LI at the area of the TLE, because abundant pigment granules were observed in the trabecular meshwork (Fig. 5a, inset). In addition, the findings strongly suggest that persistence of trabecular-iris contact before LI also caused serious damage such as degenerated trabecular cells and endothelium of the canal (Fig 5), which may be the primary reason for residual glaucoma after LI. It is believed that PAS of > 50% in PACG may be the cause of IOP elevation. This concept might be partly based on the fact that the trabecular meshwork has enough space to spare for the total amount of aqueous outflow. However, 50% of PAS may not reflect 50% of the normal function in the whole circumference of the trabecular meshwork, but > 50% of the normal function of the trabecular meshwork, because circumferential flow in the trabecular meshwork may be expected in the beginning area of PAS. Although there is some controversy on the extent to which circumferential flow does occur and that segmental flow has been observed, the concept of circumferential flow in the trabecular meshwork is based on the observation reported by Flocks ⁹ of the circumferential orientation of the trabecular beams, and this concept was also applied in the histologic study of PACG reported by Lee. ² Therefore, the trabecular meshwork and SC in the eyes of PACG which show high IOP despite of <50% PAS may have serious damage not only in gonioscopically occluded but also open areas, as was experienced in the right eye of patient 25 in this study. 

Macrophages or CD68-positive cells were observed in the eyes with acute attack within 1.5 months before TLE (Fig. 1), in eyes that underwent LI within 1.8 months before TLE, and in eyes with a severely occluded canal. All these forms of macrophage infiltration may be caused by degeneration of trabecular cells, iris-tissue, and cell debris. Macrophages infiltrating into the trabecular meshwork causes IOP elevation. Therefore, one can expect that the high IOP that develops soon after the acute attack or LI will be lowered to some degree in accordance with the disappearance of macrophage infiltration within a few months after the acute attack or LI. 

In conclusion, IOP elevation in eyes with PACG may be primarily caused by reduced aqueous outflow due to PAS or trabecular-iris contact. However, the swelling and/or degeneration of the trabecular cells and endothelial damage of SC, which may cause more serious changes such as fusion of the trabecular meshwork and occlusion of the canal, respectively, may be the primary reason for residual glaucoma after reducing the amount of PAS or trabecular-iris contact by LI or cataract surgery. Although, our cohort was mostly composed of advanced cases of PACG, all these changes may be caused by a persistence of trabecular-iris contact or PAS formation. Melanocyte and macrophage infiltration and the persistence of degenerated trabecular cells may also contribute to IOP elevation after acute attack, LI, and cataract surgery. Irreversible changes such as occlusion of SC and fusion of the trabecular beams may take an extremely long time to develop, however, trabecular-iris contact or PAS should be relieved as early as possible to avoid irreversible changes in the outflow routes. 

The authors thank Tatsuo Tsuboi for assistance with the evaluation of the statistical data, Guss Gazzard for providing useful and invaluable comments, and John Bush for reviewing the manuscript. 

Footnotes

 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2010.

Footnotes

 Disclosure: T. Hamanaka, None; K. Kasahara, None; T. Takemura, None