October 2011
Volume 52, Issue 11
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Glaucoma  |   October 2011
Otago Glaucoma Surgery Outcome Study: Electron Microscopy of Capsules around Molteno Implants
Author Affiliations & Notes
  • Alex G. Dempster
    From the Southern Community Laboratories Ltd., Dunedin, New Zealand;
  • Anthony C. B. Molteno
    Medicine Department, University of Otago Dunedin School of Medicine, Dunedin, New Zealand; and
  • Tui H. Bevin
    Medicine Department, University of Otago Dunedin School of Medicine, Dunedin, New Zealand; and
  • Andrew M. Thompson
    Eye Department, Dunedin Hospital, Dunedin, New Zealand.
  • Corresponding author: Anthony C. B. Molteno, Ophthalmology, Medicine Department, University of Otago Dunedin School of Medicine, PO Box 913, Dunedin 9054, New Zealand; acb.molteno@otago.ac.nz
Investigative Ophthalmology & Visual Science October 2011, Vol.52, 8300-8309. doi:10.1167/iovs.11-7772
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      Alex G. Dempster, Anthony C. B. Molteno, Tui H. Bevin, Andrew M. Thompson; Otago Glaucoma Surgery Outcome Study: Electron Microscopy of Capsules around Molteno Implants. Invest. Ophthalmol. Vis. Sci. 2011;52(11):8300-8309. doi: 10.1167/iovs.11-7772.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To report the ultrastructure of cells and extracellular matrix components in Molteno implant capsules examined by scanning and transmission electron microscopy.

Methods.: Ultrastructural features including cytology, distribution of apoptotic cells, collagens, basement membranes, elastic fibrils, and glycoproteins were examined by scanning and transmission electron microscopy. Findings were correlated with the clinical features of 31 specimens of glaucomatous eyes treated with Molteno implants 0.3 to 14.9 years previously.

Results.: Capsules showed two layers: an outer, moderately cellular vascular layer of normal-appearing cells and collagen and an inner, avascular, hypocellular layer of altered cells and collagen. Cells included fibroblasts, myofibroblasts, and tissue histiocytes that showed features indicating metabolic activity, with swelling, vacuolation, and apoptosis, and the formation of numerous membrane-bound vesicles. These features, together with alteration and disintegration of extracellular matrix, increased with time after surgery.

Conclusion.: The results support those in previous light microscopic studies and indicate that the normal life cycle of capsules in both primary and secondary glaucoma include continual outer surface renewal balanced by inner surface degeneration associated with apoptosis and breakdown of tissue matrix components which become more marked over time.

The success of aqueous shunt surgery for glaucoma depends on the formation and maintenance of a thin permeable bleb capsule. Histologic and ultrastructural studies of capsules from experimental animals and humans report a characteristic histologic pattern. 1 7 It consists of a moderately cellular fibrous capsule with a layer of small blood vessels on its outer surface that merges into the overlying subconjunctival connective tissue. The inner layers of the capsule are avascular and show a decreased number of variably degenerated cells with disorientation and lysis of collagen fibrils. 5  
Early electron microscopic studies of capsules demonstrate cells resembling fibroblasts, myofibroblasts, and macrophages, with loosening and separation of collagen fibrils toward the inner surface of the capsule. In some cases, an incomplete layer of fibroblast-like cells lines the inner surface. 3,6,7  
Histologic examination of 75 ocular specimens that had been treated with Molteno implants between 4 days and 23 years previously (from the Otago Glaucoma Surgery Outcome Study) were examined and showed that, without aqueous flow (first stage of two-stage insertion), the episcleral plates of Molteno implants were encapsulated by a very thin (20–60 μm), avascular, collagenous layer. The second stage of two-stage insertion with delayed drainage of aqueous and early temporary postoperative IOP increase to 25 to 35 mm Hg, produced thin (190–250 μm), permeable capsules with less fibrovascular than fibrodegenerative components. Insertion of nonligatured implants with immediate aqueous flow produced thicker capsules (300–600 μm) composed of an outer fibrovascular layer and an inner fibrodegenerative layer of approximately equal thickness. Three-stage insertion of modified Molteno implants with temporary externalization of aqueous flow onto the conjunctival surface and postoperative IOP not exceeding 12 mm Hg, produced the thickest (375–700 μm), most heavily fibrosed, and most impermeable capsules composed entirely of dense fibrovascular tissue without a fibrodegenerative layer, even though the extraocular tissues over the plate of the implant were exposed to reduced amounts of aqueous at pressures that did not exceed 12 mm Hg. 8  
Subsequent more detailed cytologic and immunohistochemical examination of 10 bleb capsules surrounding Molteno implants from noninflamed eyes, with good IOP control supplemented the previous findings. The thin external capsule layer was cellular, with fairly numerous small blood vessels and normally staining, regularly arranged collagen fibers. The thicker deeper layer was avascular and relatively acellular and was characterized by regularly arranged swollen and fragmented collagen fibers. Most cells (≈90%) in the external layer stained strongly positive for PCNA, indicating metabolic activation. In addition, between 5% (in recently formed blebs) and ≈2% (in established blebs) showed cytologic and/or immunohistochemical changes characteristic of apoptosis. All cells in the deeper layer, regardless of time after surgery, also demonstrated cytologic and/or immunohistochemical staining characteristic of metabolic activation and/or apoptosis. In addition, the deeper layer contained a large number of minute membrane-bound vesicles (presumed death messengers). 9  
Further histologic and immunohistochemical examination of 19 noninflamed eyes that had been treated with Molteno implants 11 days to 20 years previously showed that all but the earliest specimen capsules had two layers: a moderately cellular outer layer of normally stained collagen and an inner avascular hypocellular layer of altered collagen. Capsules contained metabolically active fibroblasts and macrophages, showing swelling, vacuolation, and apoptosis with localized loss of extracellular matrix in the inner layers of older capsules. Type I collagen was present in trace amounts. Collagens types III and VI and fibronectin were present in high concentration in the capsules. Basement membrane material (collagen type IV and laminin) and thrombospondin were concentrated in the inner avascular layers. These results support previous conclusions that the normal capsule life cycle includes continual inner surface degeneration and external surface renewal. The cells and tissue matrix components of the outer capsule layer matched those involved in the initial phase of wound healing in vascular connective tissue. The tissue matrix components and widespread apoptosis found in the inner fibrodegenerative layer reflect scar tissue remodelling induced by exposure to aqueous. 10  
Ten noninflamed eyes that had been treated with Molteno implants 2 months to 22.9 years previously were examined and showed that MMP (matrix metalloproteinase)-1, -2, and -3 were present in the bleb walls of the implants. TIMP (tissue inhibitor of MMP)-2 was expressed in most bleb capsules. The observations from this study support the hypothesis that bleb capsules undergo a cycle of collagen breakdown and renewal throughout the life of the bleb, as members of the MMP family are localized in the bleb wall. 11  
These histologic, cytologic, and immunohistochemical studies of capsules demonstrated that the normal life cycle of capsules includes continual inner surface degeneration and external surface renewal. These processes involve widespread metabolic activation and/or apoptosis, and the balance between them regulates the permeability of capsules. 9  
On the basis of these clinical and histologic observations, we developed the following hypothesis:
  1.  
    Drainage of aqueous (with a relatively low oxygen tension of 30 mm Hg, compared with 40 to 90 mm Hg for interstitial tissue fluid, and a very low protein content of 6 to 15 mg/100 mL, compared with 1500 mg/100 mL for interstitial tissue fluid) 12,13 into vascular subconjunctival connective tissue dilutes interstitial tissue fluid, reducing the levels of oxygen and protein. The tissue reacts by capillary vasodilation with increased oxygen tension, leakage of protein, and activation of cells. Activated fibroblasts escape from blood vessels into the tissue and actively migrate against the flow of aqueous while synthesizing collagen to produce a barrier to the passage of aqueous. This process continues for as long as cells in close proximity to patent capillaries (<50 μm) are exposed to aqueous. 14
  2.  
    Once sufficient fibrosis has occurred to resist the passage of aqueous, the IOP rises and exceeds the postcapillary venule pressure (15–25 mm Hg) in the deeper part of the bleb capsule. This zone (>50 μm from the nearest patent blood vessel) becomes avascular and changes the tissue environment, as aqueous displaces the interstitial tissue fluid. Under these conditions, cells deplete nutrients and are exposed to proapoptotic factors that include low protein concentration and hypoxia which in turn activates the intrinsic (mitochondrial) pathway of apoptosis. 15
  3.  
    The effects of apoptosis include breakdown of the deeper layers of the collagen barrier and the formation and transport of minute membrane-bound vesicles expressing Fas ligand with the flow of aqueous to the fibroproliferative outer portion of the capsule, where they activate the extrinsic pathway of apoptosis to destroy activated cells and inhibit the fibroproliferative response. The two responses balance each other after ≈6 weeks, as cells continue to migrate into the capsule where they are activated and synthesize small amounts of collagen in the outer layers before apoptosing and breaking down collagen in the inner layer for the rest of the patient's life (Figs. 1, 2).
Figure 1.
 
The authors' hypothesis regarding cellular activity in established capsules around Molteno implants: There is ongoing migration of cells from superficial blood vessels into the outer layers of the capsule. There, they become metabolically active and synthesize collagen before encountering high concentrations of aqueous in the inner layers of the capsule which induce apoptosis with release of enzymes and Fas ligand. These enzymes break down collagen, while membrane-bound vesicles expressing Fas ligand are carried by aqueous flow to the outer layers where they induce apoptosis to produce a long-term balance between synthesis and breakdown of collagen. 9,10
Figure 1.
 
The authors' hypothesis regarding cellular activity in established capsules around Molteno implants: There is ongoing migration of cells from superficial blood vessels into the outer layers of the capsule. There, they become metabolically active and synthesize collagen before encountering high concentrations of aqueous in the inner layers of the capsule which induce apoptosis with release of enzymes and Fas ligand. These enzymes break down collagen, while membrane-bound vesicles expressing Fas ligand are carried by aqueous flow to the outer layers where they induce apoptosis to produce a long-term balance between synthesis and breakdown of collagen. 9,10
Figure 2.
 
Vertical section of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs. 3, 6, 7, and 9 10 1112) showing Tenon's tissue (a) with blood vessels; an outer fibroproliferative layer of capsule (b) with normal-staining collagen and elongated cells; an inner fibrodegenerative layer of capsule (c) of poorly staining, swollen, and fragmented collagen containing irregularly sized cells and cell fragments; an inner surface of capsule (d) with swollen, irregularly flattened cells; and a cavity (e). All figures are identically oriented and labeled. Hematoxylin and eosin; original magnification, × 100.
Figure 2.
 
Vertical section of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs. 3, 6, 7, and 9 10 1112) showing Tenon's tissue (a) with blood vessels; an outer fibroproliferative layer of capsule (b) with normal-staining collagen and elongated cells; an inner fibrodegenerative layer of capsule (c) of poorly staining, swollen, and fragmented collagen containing irregularly sized cells and cell fragments; an inner surface of capsule (d) with swollen, irregularly flattened cells; and a cavity (e). All figures are identically oriented and labeled. Hematoxylin and eosin; original magnification, × 100.
This communication reports the findings of transmission and scanning electron microscopic examination of capsules surrounding Molteno implants, including types of cells with their patterns of apoptosis and distribution of normal and altered extracellular matrix components, and correlates these findings with the light microscopic cytologic and immunohistochemical findings from the same capsules. 
Methods
Ocular Specimens
Thirty-one specimens of bleb capsules and overlying connective tissue were examined by electron microscopy. All eyes had had a Molteno implant inserted from 0.3 to 14.9 years previously, with IOP control of between 5 and 21 mm Hg at final follow-up in all but two cases. Details of the ocular specimens are shown in Table 1
Table 1.
 
Clinical Features of Capsule Specimens
Table 1.
 
Clinical Features of Capsule Specimens
Case Type of Glaucoma Age at Operation (y), Sex Type Of Molteno Implant Vicryl Tie AIFS Final IOP (mm Hg) Hypotensive Medication at Final Follow-up Age Of Capsule (y) Specimen Obtained Specimen immunohistochemically Stained
1 PXG 81, M 2 Plate Yes No 14 0.3 Postmortem No
2 Neovascular 37, M 1 Plate* No Yes 14 0.3 Enucleation Yes
3 Traumatic 76, F 1 Plate No No 18 Adrenaline Timalol 0.4 Enucleation Yes
4 Neovascular 75, M 1 Plate No No 16 Acetazolamide Timolol 0.5 Enucleation Yes
5 Buphthalmos 0.01, F 1 Plate No No 15 0.6 Enucleation No
6 Neovascular 64, M 1 Plate No No 11 1.3 Enucleation Yes
7 Buphthalmos 28, F 2 Plate No No 6 1.5 Enucleation No
8 Traumatic 57, M 1 Plate No No 20 1.7 Postmortem No
9 Neovascular 81, F 1 Plate No No 5 Acetazolamide Timolol 2.5 Enucleation Yes
10 Uveitic 36, F 2 Plate Yes Yes 15 Adrenaline 2.5 Enucleation Yes
11 POAG 95, M Small Molteno3 Yes No 10 Timolol 2.5 Postmortem Yes
12 PXG 73, M 2 Plate No No 15 2.8 Postmortem Yes
13 Juvenile 17, M 2 Plate Yes No 6 3.3 Enucleation Yes
14 Neovascular 70, M 1 Plate No No 28 3.8 Enucleation Yes
15 ACG 65, F 2 Plate No No 12 Timolol 3.8 Enucleation Yes
16 PXG 72, M 2 Plate Yes No 8 4.4 Postmortem Yes
17 Neovascular 83, F 2 Plate No No 16 4.5 Postmortem No
18 Neovascular 75, M 1 Plate No Yes 17 Acetazolamide 4.9 Postmortem Yes
19 Uveitis 20, F 2 Plate No Yes 21 Acetazolamide 5.5 At subsequent surgery No
20 ICE 55, M 2 Plate No Yes 10 7.3 At subsequent surgery No
21 PXG 82, M 2 Plate Yes No 8 7.4 Postmortem No
22 PXG 68, M 2 Plate Yes No 18 7.5 Postmortem No
23 Neovascular 75, M 1 Plate No Yes 17 8.9 Postmortem No
24 Traumatic 24, M 2 Plate No No 25 9.2 Enucleation No
25 Neovascular 76, M 1 Plate No No 10 10.4 Postmortem No
26 PXG 86, F 2 Plate Yes No 9 10.7 Postmortem No
27 POAG 93, F 2 Plate No No 13 13.4 Postmortem No
28 Traumatic 63, M 2 Plate Yes No 6 Acetazolamide 13.7 Postmortem No
29 PXG 74, F 2 Plate Yes No 9 13.7 Postmortem No
30 Buphthalmos 23, F 2 Plate No No 15 14.0 Enucleation Yes
31 PXG 73, F 2 Plate Yes No 15 14.9 Postmortem Yes
Informed consent for donation of eyes or capsule tissue for research purposes was obtained from patients before their operation or death. This study adhered to the tenets of the Declaration of Helsinki. 
Surgical Technique
The surgical techniques for Molteno implant insertion with immediate and delayed aqueous drainage have been described. 16 20  
Collection of Specimens
Sixteen eyes were enucleated 1 to 4 hours after death, 13 painful blind eyes were enucleated from living patients, and 2 specimens of capsule tissue were obtained during subsequent surgical intervention. To facilitate antigen retrieval in subsequent immunohistochemical staining, 15 eyes enucleated from living patients or postmortem were injected with formol saline (a 10% solution of 37% formaldehyde in phosphate buffer in saline) using a 30-gauge needle inserted across the limbus to distend the capsules and were then placed in formol saline for 3 hours and microwaved for 20 minutes at 50°C. The lateral half of each capsule and adjacent tissue was excised and cut into small blocks that were placed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). After removal of the episcleral plate of the implant, the remaining capsule and globe were placed in 70% alcohol to harden before standard paraffin processing for light microscopy. The remaining 14 globes from both groups had 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), injected directly into the blebs before placing them in a large volume of the same fixative which was maintained at 72 hours at 4°C before further processing. Capsule tissue removed during surgery was placed immediately in a large volume of fixative. 
Preparation for Scanning Electron Microscopy
After fixing in 2.5% glutaraldehyde, the specimens were washed three times in cacodylate buffer, postfixed with the secondary fixative (1% osmium tetroxide) for 2 hours, and then washed three times in buffer as before. The samples were then dehydrated in a graded series of ethanol from 25% to 100% and were then critical point dried (Bal-Tec CPD-030; Bal-Tec AG, Balzers, Liechtenstein). Samples were mounted on aluminum stubs and coated with 10 nm of gold palladium with a Peltier-cooled, high-resolution sputter coater (model K575X; EM Technologies, Ltd., Kent, UK). 
Preparation for Transmission Electron Microscopy
After preliminary fixation, specimens then passed through the following program at 20°C: (1) 0.1 M cacodylate, 5 minutes; (2) 70% ethanol, 10 minutes; (3) 95% ethanol, 10 minutes; (4) 95% ethanol, 10 minutes; (5) 100% ethanol, 10 minutes; (6) 100% ethanol, 10 minutes; (7) 100% ethanol, 10 minutes; (8) propylene oxide, 10 minutes; (9) propylene oxide, 10 minutes; (10) PO:agar resin 2:1, 20 minutes; (11) PO:agar resin 1:1, 40 minutes; (12) PO:agar resin 1:2, 1 hour; (13) agar resin, 1 hour; (14) agar resin, 4 hours; (15) agar resin, 4 hours; and (16) agar resin, 6 hours. 
Results
Scanning Electron Microscopy
All capsules showed a similar structure, with the outer one quarter to one third of the capsules consisting of regularly layered collagen fibers of normal appearance (Fig. 3). The remaining inner two thirds to three quarters of capsules showed progressive changes with increasing time after surgery. These changes included loosening, fragmentation, and disintegration of collagen fibrils, which developed a frosted appearance, and localized swelling of individual fibrils at ≈6 months after surgery. The inner surface initially showed a layer of amorphous material with flattened cells resembling fibroblasts. With increasing time after surgery, the amorphous material developed a granular and pitted appearance, with localized areas of loss, before finally disappearing by ≈12 months after surgery. The overall number of cells decreased, and an increasing proportion developed surface blebbing and disintegrated, releasing numerous minute membrane-bound vesicles characteristic of apoptosis. These changes were accompanied by loosening of collagen fibrils in the deeper layers, which developed interconnected cavities followed by the appearance of loose networks of elastin fibrils exposed on the inner surface of capsules by the disintegration of surrounding collagen (Figs. 4, 5). 
Figure 3.
 
Scanning electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, came case as in Figs. 2, 6, 7, and 9 10 1112) showing apparently normal collagen fibers with a cell on the lower right outer surface.
Figure 3.
 
Scanning electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, came case as in Figs. 2, 6, 7, and 9 10 1112) showing apparently normal collagen fibers with a cell on the lower right outer surface.
Figure 4.
 
Scanning electron microscopic image showing inner surface of a 0.4-year-old capsule (case 3 in Table 1) showing loosening and disintegration of collagen and flattened cells with blebbing. Note the collagen fibrils partly covering some of the cells and the free membrane-bound vesicles.
Figure 4.
 
Scanning electron microscopic image showing inner surface of a 0.4-year-old capsule (case 3 in Table 1) showing loosening and disintegration of collagen and flattened cells with blebbing. Note the collagen fibrils partly covering some of the cells and the free membrane-bound vesicles.
Figure 5.
 
Scanning electron microscopic image of inner surface of an 8.9-year-old bleb (case 23 in Table 1) showing extensive lysis of collagen fibers and many apoptotic cells showing surface blebbing.
Figure 5.
 
Scanning electron microscopic image of inner surface of an 8.9-year-old bleb (case 23 in Table 1) showing extensive lysis of collagen fibers and many apoptotic cells showing surface blebbing.
Transmission Electron Microscopy
Vertical sections through capsules demonstrated a continuous transition from normal subconjunctival connective tissue containing collagen and elastin fibrils, together with capillary blood vessels to the outer layer of the capsule with regularly arranged dense collagenous fibers (Figs. 6, 7). Blood vessels on the outer capsule surface showed a normal structure of endothelial cells with tight junctions, and occasional cells close to blood vessels showed marked degeneration of organelles with preservation of nuclear and cytoplasmic membranes characteristic of apoptosis (Fig. 8). The outer capsule contained tissue histiocytes, fibroblasts, and myofibroblasts in approximately equal numbers. Cells contained numerous mitochondria with regularly arranged cristae, moderately prominent rough endoplasmic reticulum, and normal cell membrane in close contact with regularly arranged collagen fibrils, suggesting active synthesis of collagen (Fig. 9). 
Figure 6.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs. 2, 3, 7, and 9 10 1112) showing a portion of a myofibroblast in (a) Tenon's tissue, actin fibrils in the cytoplasm; darkly staining, normal mitochondria; and synthesis of protocollagen fibrils.
Figure 6.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs. 2, 3, 7, and 9 10 1112) showing a portion of a myofibroblast in (a) Tenon's tissue, actin fibrils in the cytoplasm; darkly staining, normal mitochondria; and synthesis of protocollagen fibrils.
Figure 7.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs 2, 3, 6, 9 10 1112) showing a tissue histiocyte in (a) Tenon's tissue, with a normal-staining nucleus, nucleoli, mitochondria, and secretory vacuoles.
Figure 7.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs 2, 3, 6, 9 10 1112) showing a tissue histiocyte in (a) Tenon's tissue, with a normal-staining nucleus, nucleoli, mitochondria, and secretory vacuoles.
Figure 8.
 
Transmission electron microscopic image of a 2.5-year-old-capsule (case 11 in Table 1) showing apoptosis of a cell next to a blood vessel in (a) the outer surface of the capsule.
Figure 8.
 
Transmission electron microscopic image of a 2.5-year-old-capsule (case 11 in Table 1) showing apoptosis of a cell next to a blood vessel in (a) the outer surface of the capsule.
Figure 9.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, and 10 1112) showing a fibroblast in (c) the outer fibroproliferative layer of the capsule with active rough endoplasmic reticulum; normal, dark-staining mitochondria; and collagen fibers directly attached to the cell surface.
Figure 9.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, and 10 1112) showing a fibroblast in (c) the outer fibroproliferative layer of the capsule with active rough endoplasmic reticulum; normal, dark-staining mitochondria; and collagen fibers directly attached to the cell surface.
Cells in the inner capsule layer showed features which became more marked on approaching the inner surface. There were two characteristic patterns of cellular behavior: the first involved swelling and disintegration with breakdown of collagen (in 90%–95% of cells). Early changes in cells included swelling of mitochondria and rough endoplasmic reticulum, together with blebbing of the cell surface with loosening and irregular orientation of adjacent collagen fibers (Fig. 10). These changes progressed to marked changes of overall swelling, vacuolation, distortion, blebbing with swelling, and disorganization of cytoplasmic organelles, ending in fragmentation of cell bodies and release of membrane-bound vesicles, suggesting a combination of osmotic swelling and apoptosis (Fig. 11). These changes were accompanied by marked disorientation and lysis of adjacent collagen fibrils. The second characteristic pattern involved a minority of cells in this layer (≈5%) that showed the classic ultrastructural features of early apoptosis—namely, nuclear chromatin condensation with marked condensation of cytoplasm accompanied by preservation of intact cytoplasmic organelles (Fig. 12). 
Figure 10.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, 9, 11, and 12) showing a darkly staining remnant of a myofibroblast in (c) the inner layer. Note extensive disorganization and lysis of adjacent collagen fibrils.
Figure 10.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, 9, 11, and 12) showing a darkly staining remnant of a myofibroblast in (c) the inner layer. Note extensive disorganization and lysis of adjacent collagen fibrils.
Figure 11.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, 9, 10, and 12) showing disruption of an apoptotic cell and remnants of collagen fibrils on (d) the inner surface of capsule.
Figure 11.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, 9, 10, and 12) showing disruption of an apoptotic cell and remnants of collagen fibrils on (d) the inner surface of capsule.
Figure 12.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, and 9 1011) showing an apoptotic myofibroblast and histiocyte on (d) the inner surface of the capsule. Note condensation of the cytoplasm with preservation of intact cellular organelles typical of the intermediate stages of apoptosis on the inner surface of the capsule. Compare this to similar cells from the outer layer in Figures 7 and 8.
Figure 12.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, and 9 1011) showing an apoptotic myofibroblast and histiocyte on (d) the inner surface of the capsule. Note condensation of the cytoplasm with preservation of intact cellular organelles typical of the intermediate stages of apoptosis on the inner surface of the capsule. Compare this to similar cells from the outer layer in Figures 7 and 8.
A few (≈5%) collagen fibrils in Tenon's tissue showed the typical 640-nm cross striations characteristic of collagen 1; however, cross striations were not observed in the collagen of the capsules. 
Discussion
The findings of the present study provide additional information to support the authors' hypothesis that there is a cycle of collagen synthesis and breakdown in bleb walls involving three interrelated cellular processes. These are:
  1.  
    The formation of new collagen by fibroblasts in the outer wall of capsules.
  2.  
    The breakdown of collagen by apoptotic fibroblasts in the inner wall of capsules.
  3.  
    The formation of membrane-bound vesicles by apoptotic fibroblasts in the inner layers of the capsules. These vesicles are then carried by aqueous to the outer layers of the capsule where they induce apoptosis in activated cells and suppress collagen synthesis. 21,22
Collagen Synthesis
The transmission electron microscopic observations demonstrating active collagen synthesis by fibroblasts and myofibroblasts in the outer layers of capsules were supported by light microscopy findings of strongly staining fibroblast-like cell nuclei surrounded by regularly arranged, normally staining, strongly birefringent collagen fibers. Immunohistochemical stains identified fibroblasts, myofibroblasts, bone marrow–derived angiogenic cells and macrophages; and most (≈90%) stained strongly positive for proliferating cell nuclear antigen (PCNA), indicating metabolic activity. 8 10 Collagen stains demonstrated the presence of significant amounts of collagen-3 and -6 and only trace amounts of collagen-1. 10  
Collagen Breakdown
Both scanning and transmission electron microscopy demonstrated collagen breakdown around apoptotic cells in the inner layer, and the breakdown was also visible on light microscopy. The initial changes consisted of decreased affinity for tissue stains, followed by swelling and fragmentation of individual fibrils and loss of birefringence as all traces of collagen disappeared. The majority of the cells (≈90%) became swollen, vacuolated and stained weakly before becoming apoptotic and disintegrating into clusters of minute membrane-bound vesicles. A minority of cells retained their normal dimensions and affinity for stains before becoming apoptotic with chromatin condensation blebbing of cell membranes and apoptotic bodies. Apoptotic cells produced visible alteration of ground substance and collagen in many cases. This appeared on polarized light microscopy as a clear zone of decreased refractive index around altered cells. Immunohistochemical staining demonstrated that most cells in the inner capsule were metabolically active, of the same types, and surrounded by the same collagens as those of the outer capsule. Most of the cells in which morphology suggested apoptosis (≈20%) stained positive for both caspase-3 and TUNEL. 9,10  
Immunohistochemical studies of the patterns of expression of MMPs and TIMPs show abundant expression of MMP-1, -2, and -3, expression of TIMP-2 and minimal expression of TIMP-1 and -3 in the inner layer of capsules. This pattern of expression of MMPs and TIMPs has been observed in the sclera from patients with scleritis and other pathologic processes characterized by collagen breakdown. 11  
Membrane-Bound Vesicles
The membrane-bound vesicles produced by apoptotic cells in the present study were visible on light microscopy as numerous variably basophilic vesicles ranging from 0.4 μm (the limit of optical resolution) to ≈4.0 μm diameter. This range corresponded to that observed by electron microscopy. The distribution of these vesicles with a large number found in the inner layers and a progressively smaller number in the outer layers of capsules was consistent with that found with light microscopy. 9 Immunohistochemical staining shows that a proportion of these membrane-bound vesicles stained positive for markers of apoptosis (PCNA and TUNEL). 10 Immunohistochemical staining for the expression of factors possibly associated with the extrinsic apoptotic pathway has shown a high concentration of TIMP-2 in the innermost layers of capsules, suggesting that in addition to its role in activating MMP-2, it may be involved in triggering apoptosis in the capsule. 11  
Conclusions
The electron microscopic observations from this study were consistent with findings of previous light microscopic studies of bleb capsules that have shown a complex cycle of simultaneous cell activation and apoptosis. The first, an inflammatory fibroproliferative component, resembles the initial stages of the wound-healing response characteristic of higher animals with a high-pressure blood circulation. The second, an avascular noninflammatory apoptotic fibrodegenerative response, resembles the predominant tissue defense response of primitive animals that lack an efficient blood circulation. 13 This tissue response has been shown to regulate embryonic development in all animals and exert an anti-inflammatory action in most tissues of mammals. 23 Additional information regarding the interactions between these processes in glaucoma drainage capsules from future, more detailed immunohistochemical study of capsules offers hope that improved methods of minimizing the fibroproliferative and enhancing the apoptotic fibrodegenerative responses of connective tissue to the aqueous will be developed in the foreseeable future. 22,24  
Footnotes
 Supported by the Healthcare Otago Charitable Trust, Dunedin, New Zealand (TB).
Footnotes
 Disclosure: A.G. Dempster, None; A.C.B. Molteno, Molteno Ophthalmic, Ltd. (I), P; T.H. Bevin, None; A.M. Thompson, None
The authors thank the patients and their families who kindly donated the eyes and tissue. 
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Figure 1.
 
The authors' hypothesis regarding cellular activity in established capsules around Molteno implants: There is ongoing migration of cells from superficial blood vessels into the outer layers of the capsule. There, they become metabolically active and synthesize collagen before encountering high concentrations of aqueous in the inner layers of the capsule which induce apoptosis with release of enzymes and Fas ligand. These enzymes break down collagen, while membrane-bound vesicles expressing Fas ligand are carried by aqueous flow to the outer layers where they induce apoptosis to produce a long-term balance between synthesis and breakdown of collagen. 9,10
Figure 1.
 
The authors' hypothesis regarding cellular activity in established capsules around Molteno implants: There is ongoing migration of cells from superficial blood vessels into the outer layers of the capsule. There, they become metabolically active and synthesize collagen before encountering high concentrations of aqueous in the inner layers of the capsule which induce apoptosis with release of enzymes and Fas ligand. These enzymes break down collagen, while membrane-bound vesicles expressing Fas ligand are carried by aqueous flow to the outer layers where they induce apoptosis to produce a long-term balance between synthesis and breakdown of collagen. 9,10
Figure 2.
 
Vertical section of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs. 3, 6, 7, and 9 10 1112) showing Tenon's tissue (a) with blood vessels; an outer fibroproliferative layer of capsule (b) with normal-staining collagen and elongated cells; an inner fibrodegenerative layer of capsule (c) of poorly staining, swollen, and fragmented collagen containing irregularly sized cells and cell fragments; an inner surface of capsule (d) with swollen, irregularly flattened cells; and a cavity (e). All figures are identically oriented and labeled. Hematoxylin and eosin; original magnification, × 100.
Figure 2.
 
Vertical section of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs. 3, 6, 7, and 9 10 1112) showing Tenon's tissue (a) with blood vessels; an outer fibroproliferative layer of capsule (b) with normal-staining collagen and elongated cells; an inner fibrodegenerative layer of capsule (c) of poorly staining, swollen, and fragmented collagen containing irregularly sized cells and cell fragments; an inner surface of capsule (d) with swollen, irregularly flattened cells; and a cavity (e). All figures are identically oriented and labeled. Hematoxylin and eosin; original magnification, × 100.
Figure 3.
 
Scanning electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, came case as in Figs. 2, 6, 7, and 9 10 1112) showing apparently normal collagen fibers with a cell on the lower right outer surface.
Figure 3.
 
Scanning electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, came case as in Figs. 2, 6, 7, and 9 10 1112) showing apparently normal collagen fibers with a cell on the lower right outer surface.
Figure 4.
 
Scanning electron microscopic image showing inner surface of a 0.4-year-old capsule (case 3 in Table 1) showing loosening and disintegration of collagen and flattened cells with blebbing. Note the collagen fibrils partly covering some of the cells and the free membrane-bound vesicles.
Figure 4.
 
Scanning electron microscopic image showing inner surface of a 0.4-year-old capsule (case 3 in Table 1) showing loosening and disintegration of collagen and flattened cells with blebbing. Note the collagen fibrils partly covering some of the cells and the free membrane-bound vesicles.
Figure 5.
 
Scanning electron microscopic image of inner surface of an 8.9-year-old bleb (case 23 in Table 1) showing extensive lysis of collagen fibers and many apoptotic cells showing surface blebbing.
Figure 5.
 
Scanning electron microscopic image of inner surface of an 8.9-year-old bleb (case 23 in Table 1) showing extensive lysis of collagen fibers and many apoptotic cells showing surface blebbing.
Figure 6.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs. 2, 3, 7, and 9 10 1112) showing a portion of a myofibroblast in (a) Tenon's tissue, actin fibrils in the cytoplasm; darkly staining, normal mitochondria; and synthesis of protocollagen fibrils.
Figure 6.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs. 2, 3, 7, and 9 10 1112) showing a portion of a myofibroblast in (a) Tenon's tissue, actin fibrils in the cytoplasm; darkly staining, normal mitochondria; and synthesis of protocollagen fibrils.
Figure 7.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs 2, 3, 6, 9 10 1112) showing a tissue histiocyte in (a) Tenon's tissue, with a normal-staining nucleus, nucleoli, mitochondria, and secretory vacuoles.
Figure 7.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as in Figs 2, 3, 6, 9 10 1112) showing a tissue histiocyte in (a) Tenon's tissue, with a normal-staining nucleus, nucleoli, mitochondria, and secretory vacuoles.
Figure 8.
 
Transmission electron microscopic image of a 2.5-year-old-capsule (case 11 in Table 1) showing apoptosis of a cell next to a blood vessel in (a) the outer surface of the capsule.
Figure 8.
 
Transmission electron microscopic image of a 2.5-year-old-capsule (case 11 in Table 1) showing apoptosis of a cell next to a blood vessel in (a) the outer surface of the capsule.
Figure 9.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, and 10 1112) showing a fibroblast in (c) the outer fibroproliferative layer of the capsule with active rough endoplasmic reticulum; normal, dark-staining mitochondria; and collagen fibers directly attached to the cell surface.
Figure 9.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, and 10 1112) showing a fibroblast in (c) the outer fibroproliferative layer of the capsule with active rough endoplasmic reticulum; normal, dark-staining mitochondria; and collagen fibers directly attached to the cell surface.
Figure 10.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, 9, 11, and 12) showing a darkly staining remnant of a myofibroblast in (c) the inner layer. Note extensive disorganization and lysis of adjacent collagen fibrils.
Figure 10.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, 9, 11, and 12) showing a darkly staining remnant of a myofibroblast in (c) the inner layer. Note extensive disorganization and lysis of adjacent collagen fibrils.
Figure 11.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, 9, 10, and 12) showing disruption of an apoptotic cell and remnants of collagen fibrils on (d) the inner surface of capsule.
Figure 11.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, 9, 10, and 12) showing disruption of an apoptotic cell and remnants of collagen fibrils on (d) the inner surface of capsule.
Figure 12.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, and 9 1011) showing an apoptotic myofibroblast and histiocyte on (d) the inner surface of the capsule. Note condensation of the cytoplasm with preservation of intact cellular organelles typical of the intermediate stages of apoptosis on the inner surface of the capsule. Compare this to similar cells from the outer layer in Figures 7 and 8.
Figure 12.
 
Transmission electron microscopic image of a 7.5-year-old capsule (case 22 in Table 1, same case as Figs. 2, 3, 6, 7, and 9 1011) showing an apoptotic myofibroblast and histiocyte on (d) the inner surface of the capsule. Note condensation of the cytoplasm with preservation of intact cellular organelles typical of the intermediate stages of apoptosis on the inner surface of the capsule. Compare this to similar cells from the outer layer in Figures 7 and 8.
Table 1.
 
Clinical Features of Capsule Specimens
Table 1.
 
Clinical Features of Capsule Specimens
Case Type of Glaucoma Age at Operation (y), Sex Type Of Molteno Implant Vicryl Tie AIFS Final IOP (mm Hg) Hypotensive Medication at Final Follow-up Age Of Capsule (y) Specimen Obtained Specimen immunohistochemically Stained
1 PXG 81, M 2 Plate Yes No 14 0.3 Postmortem No
2 Neovascular 37, M 1 Plate* No Yes 14 0.3 Enucleation Yes
3 Traumatic 76, F 1 Plate No No 18 Adrenaline Timalol 0.4 Enucleation Yes
4 Neovascular 75, M 1 Plate No No 16 Acetazolamide Timolol 0.5 Enucleation Yes
5 Buphthalmos 0.01, F 1 Plate No No 15 0.6 Enucleation No
6 Neovascular 64, M 1 Plate No No 11 1.3 Enucleation Yes
7 Buphthalmos 28, F 2 Plate No No 6 1.5 Enucleation No
8 Traumatic 57, M 1 Plate No No 20 1.7 Postmortem No
9 Neovascular 81, F 1 Plate No No 5 Acetazolamide Timolol 2.5 Enucleation Yes
10 Uveitic 36, F 2 Plate Yes Yes 15 Adrenaline 2.5 Enucleation Yes
11 POAG 95, M Small Molteno3 Yes No 10 Timolol 2.5 Postmortem Yes
12 PXG 73, M 2 Plate No No 15 2.8 Postmortem Yes
13 Juvenile 17, M 2 Plate Yes No 6 3.3 Enucleation Yes
14 Neovascular 70, M 1 Plate No No 28 3.8 Enucleation Yes
15 ACG 65, F 2 Plate No No 12 Timolol 3.8 Enucleation Yes
16 PXG 72, M 2 Plate Yes No 8 4.4 Postmortem Yes
17 Neovascular 83, F 2 Plate No No 16 4.5 Postmortem No
18 Neovascular 75, M 1 Plate No Yes 17 Acetazolamide 4.9 Postmortem Yes
19 Uveitis 20, F 2 Plate No Yes 21 Acetazolamide 5.5 At subsequent surgery No
20 ICE 55, M 2 Plate No Yes 10 7.3 At subsequent surgery No
21 PXG 82, M 2 Plate Yes No 8 7.4 Postmortem No
22 PXG 68, M 2 Plate Yes No 18 7.5 Postmortem No
23 Neovascular 75, M 1 Plate No Yes 17 8.9 Postmortem No
24 Traumatic 24, M 2 Plate No No 25 9.2 Enucleation No
25 Neovascular 76, M 1 Plate No No 10 10.4 Postmortem No
26 PXG 86, F 2 Plate Yes No 9 10.7 Postmortem No
27 POAG 93, F 2 Plate No No 13 13.4 Postmortem No
28 Traumatic 63, M 2 Plate Yes No 6 Acetazolamide 13.7 Postmortem No
29 PXG 74, F 2 Plate Yes No 9 13.7 Postmortem No
30 Buphthalmos 23, F 2 Plate No No 15 14.0 Enucleation Yes
31 PXG 73, F 2 Plate Yes No 15 14.9 Postmortem Yes
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