October 2012
Volume 53, Issue 11
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Glaucoma  |   October 2012
A Model to Measure Fluid Outflow in Rabbit Capsules Post Glaucoma Implant Surgery
Author Affiliations & Notes
  • Dan Q. Nguyen
    From the Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Australia; the
  • Craig M. Ross
    From the Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Australia; the
  • Yu Qin Li
    From the Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Australia; the
  • Surinder Pandav
    From the Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Australia; the
  • Bruce Gardiner
    School of Computer Science and Software Engineering, The University of Western Australia, Perth, Australia; and the
  • David Smith
    School of Computer Science and Software Engineering, The University of Western Australia, Perth, Australia; and the
  • Alicia C. How
    Singapore National Eye Centre, Singapore.
  • Jonathan G. Crowston
    From the Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Australia; the
  • Michael A. Coote
    From the Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Australia; the
  • Corresponding author: Michael A. Coote, Royal Victorian Eye and Ear Hospital, 32 Gisborne Street, East Melbourne, Victoria 3002, Australia; mcoote@bigpond.net.au  
Investigative Ophthalmology & Visual Science October 2012, Vol.53, 6914-6919. doi:10.1167/iovs.12-10438
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      Dan Q. Nguyen, Craig M. Ross, Yu Qin Li, Surinder Pandav, Bruce Gardiner, David Smith, Alicia C. How, Jonathan G. Crowston, Michael A. Coote; A Model to Measure Fluid Outflow in Rabbit Capsules Post Glaucoma Implant Surgery. Invest. Ophthalmol. Vis. Sci. 2012;53(11):6914-6919. doi: 10.1167/iovs.12-10438.

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

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Abstract

Purpose.: Prior models of glaucoma filtration surgery assess bleb morphology, which does not always reflect function. Our aim is to establish a model that directly measures tissue hydraulic conductivity of postsurgical outflow in rabbit bleb capsules following experimental glaucoma filtration surgery.

Methods.: Nine rabbits underwent insertion of a single-plate pediatric Molteno implant into the anterior chamber of their left eye. Right eyes were used as controls. The rabbits were then allocated to one of two groups. Group one had outflow measurements performed at 1 week after surgery (n = 5), and group two had measurements performed at 4 weeks (n = 4). Measurements were performed by cannulating the drainage tube ostium in situ with a needle attached to a pressure transducer and a fluid column at 15 mm Hg. The drop in the fluid column was measured every minute for 5 minutes. For the control eyes (n = 6), the anterior chamber of the unoperated fellow eye was cannulated. Animals were euthanized with the implant and its surrounding capsule dissected and fixed in 4% paraformaldehyde, and embedded in paraffin before 6-μm sections were cut for histologic staining.

Results.: By 7 days after surgery, tube outflow was 0.117 ± 0.036 μL/min/mm Hg at 15 mm Hg (mean ± SEM), whereas at 28 days, it was 0.009 ± 0.003 μL/min/mm Hg. Control eyes had an outflow of 0.136 ± 0.007 μL/min/mm Hg (P = 0.004, one-way ANOVA). Hematoxylin and eosin staining demonstrated a thinner and looser arrangement of collagenous tissue in the capsules at 1 week compared with that at 4 weeks, which had thicker and more densely arranged collagen.

Conclusions.: We describe a new model to directly measure hydraulic conductivity in a rabbit glaucoma surgery implant model. The principal physiologic endpoint of glaucoma surgery can be reliably quantified and consistently measured with this model. At 28 days post glaucoma filtration surgery, a rabbit bleb capsule has significantly reduced tissue hydraulic conductivity, in line with loss of implant outflow facility, and increased thickness and density of fibrous encapsulation.

Introduction
The role of glaucoma surgery is to decrease the intraocular pressure (IOP). This is achieved by increasing the facility of aqueous outflow from the eye. Surgically induced outflow facility is thus the key functional endpoint, but is currently not directly measured in experimental glaucoma surgery models. 
Current models are limited, focusing on endpoints such as IOP, bleb morphology, the inflammatory response, or histology. In animal models without elevated IOP, or reduced trabecular outflow, the use of IOP as an indirect measure of outflow facility is of limited value. Prior modeling suggests that when normal trabecular outflow facility is reduced, there is a nonlinear increase in IOP. 1  
The morphologic appearance of a bleb is only moderately defined and assessment is usually subjective. Although some structure function correlation exists, clinicians are aware of morphologic and functional estrangement. Bleb failure has a number of morphologic and histologic variables, including large blebs with thick, vascularized walls. 2  
Histology is valuable but direct relationships between capsule histology and function are limited. Scarring can be assessed directly by histologic markers that have previously been taken as a surrogate of surgical success. 35  
Modeling suggests that the bleb capsule is not the principal site of absorption. Aqueous must traverse the bleb into the surrounding subconjunctival tissue and be absorbed into subconjunctival capillaries. 1 The indirect effect of progressive scarring increases resistance to flow through the bleb wall, thereby reducing the ability of a bleb capsule to distribute aqueous for subconjunctival absorption; thus lowering of bleb hydraulic conductivity leads to higher bleb pressure and IOP. 1  
The hydraulic conductivity of the bleb capsule is thus a critical determinant of the success of glaucoma surgery because it defines the surgically induced outflow facility. Yet, hydraulic conductivity of the surgical outflow pathway, as the physiologic endpoint of glaucoma surgery, remains unmeasured by experimental models. We sought to address these issues with a surgical model in which the bleb size was fixed using an implant, surgical intervention was standardized, with the intention to directly measure hydraulic conductivity, the outcome of most clinical significance in glaucoma surgery. 
Methods
Experiments were performed on nine healthy female New Zealand White rabbits at the Royal Victorian Eye and Ear Hospital and Centre for Eye Research Australia. The study was approved by the Royal Victorian Eye and Ear Hospital Animal Research and Ethics Committee and procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
All animals underwent general anesthesia with intramuscular ketamine (35 mg/kg; Pfizer [Parke-Davis], Morris Plains, NJ) and xylazine (5 mg/kg; Bayer, Leverkusen, Germany). Each rabbit underwent insertion of a single-plate pediatric implant (Molteno Implant; Molteno Ophthalmic Ltd., Dunedin, New Zealand) into the superotemporal quadrant of their left eye under microscopic view and sterile conditions. 
In brief, the implant surgery involved conjunctival dissection, followed by insertion of the pediatric implant (Molteno Ophthalmic Ltd.) under the conjunctiva into the superotemporal fornix with the tube extending from the limbus into the anterior chamber via a 25-gauge incision. The implant was secured with 10/0 nylon to the sclera to allow efflux of aqueous humor into the subconjunctival space through the plate. The animals were treated with prednisolone acetate and chloramphenicol (Allergan Inc., Irvine, CA) eye drops 3 times a day for up to 10 days after the surgery. 
Outflow Measurements
The rabbits were randomized to one of two groups. Group one had outflow measurements performed 7 days after surgery (n = 5) and group two had outflow measurements performed at 28 days (n = 4). Adapting from a previously described method, 6 all measurements were made on a single manometric apparatus that consisted of a pressure transducer connected to a pressure monitor and recorder (Transpac IV; Abbott Critical Care Systems, Sligo, Ireland) (Fig. 1A). The pressure transducer was placed between the needle connected to the implant or the anterior chamber and polyethylene tubing, attached to a ruler suspended vertically on a retort stand. The needle and the pressure transducer were held in place at the same level with a micromanipulator arm. Fluid within the tubing could be refilled via a reservoir syringe that was attached to the tubing via a stopcock, which would be closed off during measurements. Prior to each experiment, the system was filled with 0.9% saline and air bubbles were flushed out. The system was calibrated with the tubing placed such that the top of the fluid column was at the same horizontal level as the rabbit's eye at 0 mm Hg. 
Figure 1. 
 
(A) Experimental apparatus for outflow measurement of implants inserted into the anterior chamber. Fluid column and pressure transducer were used to measure only the outflow provided by the seton, as indicated by red arrow. (a) Pediatric Molteno implant with tube placed in anterior chamber. (b) A 27-gauge needle inserted into the anterior chamber and cannulating the implant tube opening. (c) Three-way stopcocks. (d) Pressure transducer connected via tubing. (e) Syringe to assist with loading of system. (f) Calibrated fluid column with height at eye level. (B) Implant tube in anterior chamber cannulated. (C) Experimental apparatus for outflow measurement of control eyes. Fluid column and pressure transducer were used to measure the outflow through normal drainage pathways in the unoperated eye, as indicated by the red arrows. (a) Eye without glaucoma implant. (b) A 27-gauge needle inserted into anterior chamber. (c) Three-way stopcocks. (d) Pressure transducer connected via tubing. (e) Syringe to assist with loading of system. (f) Calibrated fluid column with height at eye level.
Figure 1. 
 
(A) Experimental apparatus for outflow measurement of implants inserted into the anterior chamber. Fluid column and pressure transducer were used to measure only the outflow provided by the seton, as indicated by red arrow. (a) Pediatric Molteno implant with tube placed in anterior chamber. (b) A 27-gauge needle inserted into the anterior chamber and cannulating the implant tube opening. (c) Three-way stopcocks. (d) Pressure transducer connected via tubing. (e) Syringe to assist with loading of system. (f) Calibrated fluid column with height at eye level. (B) Implant tube in anterior chamber cannulated. (C) Experimental apparatus for outflow measurement of control eyes. Fluid column and pressure transducer were used to measure the outflow through normal drainage pathways in the unoperated eye, as indicated by the red arrows. (a) Eye without glaucoma implant. (b) A 27-gauge needle inserted into anterior chamber. (c) Three-way stopcocks. (d) Pressure transducer connected via tubing. (e) Syringe to assist with loading of system. (f) Calibrated fluid column with height at eye level.
The drainage tube within the anterior chamber of the study eyes was cannulated ostium in situ with a 27-gauge needle (Fig. 1B), attached to a pressure transducer and fluid column at a height of 15 mm Hg. With the IOP maintained at 15 mm Hg, the drop in the fluid column was measured every minute for 5 minutes to determine the outflow through the tube into the plate during the 5 minutes. Six of the unoperated fellow eyes were used as controls (n = 6). The anterior chamber of the control eyes was cannulated with a 27-gauge needle attached to a pressure transducer and fluid column at 15 mm Hg, and similar measurements were performed on the same day as that of the other eye (Fig. 1C). 
The drop in height of the fluid column was a reflection of the tissue hydraulic permeability of the rabbit capsule that had formed around the plate of the implant of the operated eyes and a reflection of the outflow facility through the normal pathways in the control eyes. The height of the fluid column was multiplied by the area of the lumen of the tube to obtain the volume. 
Histologic Staining
Rabbits were euthanized on day 7 or day 28 following fluid column measurements for histologic examination of the capsules. The intact eyes, including implant site and the superior conjunctiva, were removed by enucleation and fixed in 4% formaldehyde overnight before eyes were dissected and embedded in paraffin. Sections were cut on a microtome at 6 μm and stained with hematoxylin and eosin (H&E) for general tissue structure and cell morphology, and Masson's trichrome staining to assess collagen deposition and remodeling before viewing under the microscope with ×10 magnification. 
Results
Fluid Column Measurements of Outflow
At 7 days after surgery, outflow through pediatric Molteno implant capsules was 0.117 ± 0.036 μL/min/mm Hg at 15 mm Hg (mean ± SEM), whereas at 28 days, it was significantly reduced at 0.009 ± 0.003 μL/min/mm Hg at 15 mm Hg. Control eyes had a mean outflow of 0.136 ± 0.007 μL/min/mm Hg, corresponding to outflow through normal rabbit trabecular and uveoscleral outflow pathways (P = 0.004, one-way ANOVA) (Fig. 2, the Table). 
Figure 2. 
 
Mean outflow measurements.
Figure 2. 
 
Mean outflow measurements.
Table. 
 
Outflow into Control Eyes and Implant Capsules at 7 and 28 Days Following Insertion of a Pediatric Molteno Implant
Table. 
 
Outflow into Control Eyes and Implant Capsules at 7 and 28 Days Following Insertion of a Pediatric Molteno Implant
Control Eyes, n = 6 7-Day-Old Capsules, n = 5 28-Day-Old Capsules, n = 4
Mean (SEM), μL/min/mm Hg measured at 15 mm Hg 0.136* (0.007) 0.117* (0.036) 0.009* (0.003)
Histology
A representative capsule at 7 days post implant surgery (Fig. 3A) demonstrated a poorly formed and thin central capsule. The capsule was composed of mainly inactive fibrocytes with scattered narrow-caliber blood vessels and a minimal amount of collagen. The capsule was permeated by an infiltrate of neutrophils, consistent with acute inflammation/healing, which was accentuated within the common capsule of the defect spaces. Adjacent conjunctiva appeared histologically normal. Masson's trichrome staining of a 7-day post implant capsule demonstrated a loose arrangement of faintly staining collagen with a rich cell population, mainly composed of fibroblasts, normal caliber blood vessels, and acute inflammatory cells. 
Figure 3. 
 
Histology of bleb capsules at 7 days. H&E (A) and Masson's trichrome (B) staining at ×10 magnification at the center of the capsule. H&E staining taken at the limbal (C) and fornix (D) borders of the capsule, at ×10 magnification.
Figure 3. 
 
Histology of bleb capsules at 7 days. H&E (A) and Masson's trichrome (B) staining at ×10 magnification at the center of the capsule. H&E staining taken at the limbal (C) and fornix (D) borders of the capsule, at ×10 magnification.
Histologic analysis of a representative 28-day postsurgery capsule (Fig. 4) demonstrated an implantation site surrounded by a well-formed thickened fibrous capsule, which was mostly collagenous consistent with scar formation. There were only scant inflammatory cells. Masson's trichrome staining demonstrated densely packed darkly stained collagen fibers with occasional elongated fibroblasts, degraded inflammatory cells, and blood vessels. 
Figure 4. 
 
Histology of bleb capsules at 28 days. H&E (A) and Masson's trichrome (B) staining at ×10 magnification at the center of the capsule. H&E staining taken at the limbal (C) and fornix (D) borders of the capsule, at ×10 magnification.
Figure 4. 
 
Histology of bleb capsules at 28 days. H&E (A) and Masson's trichrome (B) staining at ×10 magnification at the center of the capsule. H&E staining taken at the limbal (C) and fornix (D) borders of the capsule, at ×10 magnification.
In summary, 28-day-old capsules were thicker, with more densely arranged collagenous tissue, compared with those at 7 days and also control eyes (Figs. 35). 
Figure 5. 
 
Control eye: normal conjunctiva. H&E (A) and Masson's trichrome (B) staining at ×10 magnification.
Figure 5. 
 
Control eye: normal conjunctiva. H&E (A) and Masson's trichrome (B) staining at ×10 magnification.
Discussion
We describe a new model to directly measure hydraulic conductivity in a rabbit glaucoma surgery implant model. This model can be quantified reliably and consistently, and is informed by detailed hydraulic modeling. 1 It enables researchers to focus on the relationship between hydraulic conductivity, subconjunctival tissue absorptive capacity, outflow facility, and IOP with relation to histologic characteristics. 
The 28-day time point was chosen to achieve a relatively mature capsule in a New Zealand White rabbit, which exhibits a vigorous wound healing response. The comparison was made with 7-day capsules to investigate the changes in outflow through a maturing capsule. We found that 28-day postsurgery capsules in the rabbit had significantly reduced tissue hydraulic conductivity compared with 7-day postsurgery capsules. This was in line with loss of function of the implant, and on histology, increased thickness, and density of fibrous encapsulation. 
As far as we are aware, no other study has directly measured hydraulic conductivity of a bleb capsule. Prata et al. 7 measured the flow in glaucoma drainage devices in vivo 24 hours postimplantation; however, their study involved grasping the tube with forceps through a paracentral corneal incision to cannulate the tube within the anterior chamber. The effect of this cannulation technique may decompress the eye and, importantly, change the dynamic behavior of the implant and scar complex. In contrast, in our study, there is no collapse of the anterior chamber that was kept at physiologic pressures. 
Surgical outflow is a dynamic process that involves egress of aqueous from the anterior chamber through a conductive bleb, distribution through subconjunctival tissue, and absorption into the capillary network. Our model does not measure absorption. It is assumed that this will be adequate if capsular hydraulic conductivity is not significantly impaired. Existing trabecular meshwork function is also important and may be affected by surgery. This requires us to better understand how tissue homeostasis, porosity, and hydroconductivity are maintained, so that we can maintain outflow facility over the long term. Our model also investigated flow at multiple time points with sufficient time for capsules to form. 
In glaucoma research, animal studies play an important role in the development of new treatment modalities. Previous studies have shown that rabbit conjunctiva is structurally and phenotypically similar to human ocular surface epithelial tissue. An established rabbit model of glaucoma filtration, but not implant surgery, has been previously devised. 8 This with the development of antifibrosis treatments translated from rabbit models to humans 810 lends validity to working with this animal model. 
It is our belief that conjunctival fibrosis following glaucoma filtration surgery is a common final pathway of diverse, multifactorial etiology, which includes inflammation, hemodynamics, and mechanical strains such as stretch, shear stress, and static pressure. Histology is valuable, and studies by Molteno and others 1114 have demonstrated the complex cellular events that occur. However, it is not possible to adequately model the healing response around an implant except in a living eye. Tissue culture systems cannot identify vascularized tissue responses, nor can they assess function. Therefore, direct histologic relationships with bleb function, and ultimately surgically induced outflow facility, are unclear. 
A potential limitation of this study is the relatively small numbers of rabbits used. Additionally, outflow measurements in each eye were performed for only 5 minutes. A longer time period for outflow measurements may lead to reduced error. The important advantages of this model lie in that it enables us to directly measure surgical outflow facility in a glaucoma drainage device in vivo rather than be derived from indirect estimations, calculations, and equations. Our model enables hydraulic conductivity to be calculated in the normal physiologic state of an eye post device implantation. It omits the need for fluorophotometry or use of systemic tracers and fluorescent dyes, which may disrupt the normal physiology of the eye. 
Direct measurement of surgically induced outflow should improve the accuracy of experimental verification of the effect of variations in glaucoma surgical techniques, devices, or new antifibrotic agents. Factors such as fluid mechanics that may contribute to the development of progressive scarring can also be directly tested using this model, allowing flow measurements to be tested consistently and accurately. This model is cost effective and its technique and results are repeatable, thus enabling a more informed approach to animal models of glaucoma filtration surgery, and could be used to investigate IOP–conductivity relationships in the future. 
References
Gardiner BS Smith DW Coote M Crowston JG. Computational modeling of fluid flow and intra-ocular pressure following glaucoma surgery. PLoS One . 2010;5:1–11.
Azuara-Blanco A Katz LJ. Dysfunctional filtering blebs. Surv Ophthalmol . 1998;43:93–126. [CrossRef] [PubMed]
Hitchings RA Grierson I. Clinicopathological correlation in eyes with failed fistulizing surgery. Trans Ophthalmol Soc . 1983;103:84–88.
Chang L Crowston JG Cordeiro MF Akbar AN Khaw PT. The role of the immune system in conjunctival wound healing after glaucoma surgery. Surv Ophthalmol . 2000;45:49–68. [CrossRef] [PubMed]
Van Buskirk EM. Cysts of Tenon's capsule following filtration surgery. Am J Ophthalmol . 1982;94:522–527. [CrossRef] [PubMed]
Kong YX Crowston JG Vingrys AJ Trounce IA Bui VB. Functional changes in the retina during and after acute intraocular pressure elevation in mice. Invest Ophthalmol Vis Sci . 2009;50:5732–5740. [CrossRef] [PubMed]
Prata JA Jr Mermoud A LaBree L Minckler DS. In vitro and in vivo flow characteristics of glaucoma drainage implants. Ophthalmology . 1995;102:894–904. [CrossRef] [PubMed]
Cordeiro MF Constable PH Alexander RA Effect of varying the mitomycin-C treatment area in glaucoma filtration surgery in the rabbit. Invest Ophthalmol Vis Sci . 1997;38:1639–1646. [PubMed]
Cordeiro MF Gay JA Khaw PT. Human anti-transforming growth factor-beta 2 antibody: a new glaucoma anti-scarring agent. Invest Ophthalmol Vis Sci . 1999;40:2225–2234. [PubMed]
Khaw PT Doyle JW Sherwood MB Smith MF McGorray S. Effects of intraoperative 5-fluorouracil or mitomycin C on glaucoma filtration surgery in the rabbit. Ophthalmology . 1993;100:367–372. [CrossRef] [PubMed]
McCluskey P Molteno A Wakefield D Di Girolamo N. Otago Glaucoma Surgery Outcome Study: the pattern of expression of MMPs and TIMPs in bleb capsules surrounding Molteno implants. Invest Ophthalmol Vis Sci . 2009;50:2161–2164. [CrossRef] [PubMed]
Molteno AC Thompson AM Bevin TH Dempster AG. Otago Glaucoma Surgery Outcome Study: tissue matrix breakdown by apoptotic cells in capsules surrounding Molteno implants. Invest Ophthalmol Vis Sci . 2009;50:1187–1197. [CrossRef] [PubMed]
Molteno AC Suter AJ Fenwick M Bevin TH Dempster AG. Otago Glaucoma Surgery Outcome Study: cytology and immunohistochemical staining of bleb capsules around Molteno implants. Invest Ophthalmol Vis Sci . 2006;47:1975–1981. [CrossRef] [PubMed]
Lloyd MA Baerveldt G Nguyen QH Minckler DS. Long-term histologic studies of the Baerveldt implant in a rabbit model. J Glaucoma . 1996;5:334–339. [CrossRef] [PubMed]
Footnotes
 Supported in part by Operational Infrastructure funding from the Victorian Government (to The Centre for Eye Research Australia), Dorothy Adele Edols Charitable Trust, Irene Lo and Department of Pathology, St. Vincent's Hospital Melbourne, Melbourne, Australia, and National Health and Medical Research Council Grant 529918.
Footnotes
 Disclosure: D.Q. Nguyen, None; C.M. Ross, None; Y.Q. Li, None; S. Pandav, None; B. Gardiner, None; D. Smith, None; A.C. How, None; J.G. Crowston, None; M.A. Coote, None
Figure 1. 
 
(A) Experimental apparatus for outflow measurement of implants inserted into the anterior chamber. Fluid column and pressure transducer were used to measure only the outflow provided by the seton, as indicated by red arrow. (a) Pediatric Molteno implant with tube placed in anterior chamber. (b) A 27-gauge needle inserted into the anterior chamber and cannulating the implant tube opening. (c) Three-way stopcocks. (d) Pressure transducer connected via tubing. (e) Syringe to assist with loading of system. (f) Calibrated fluid column with height at eye level. (B) Implant tube in anterior chamber cannulated. (C) Experimental apparatus for outflow measurement of control eyes. Fluid column and pressure transducer were used to measure the outflow through normal drainage pathways in the unoperated eye, as indicated by the red arrows. (a) Eye without glaucoma implant. (b) A 27-gauge needle inserted into anterior chamber. (c) Three-way stopcocks. (d) Pressure transducer connected via tubing. (e) Syringe to assist with loading of system. (f) Calibrated fluid column with height at eye level.
Figure 1. 
 
(A) Experimental apparatus for outflow measurement of implants inserted into the anterior chamber. Fluid column and pressure transducer were used to measure only the outflow provided by the seton, as indicated by red arrow. (a) Pediatric Molteno implant with tube placed in anterior chamber. (b) A 27-gauge needle inserted into the anterior chamber and cannulating the implant tube opening. (c) Three-way stopcocks. (d) Pressure transducer connected via tubing. (e) Syringe to assist with loading of system. (f) Calibrated fluid column with height at eye level. (B) Implant tube in anterior chamber cannulated. (C) Experimental apparatus for outflow measurement of control eyes. Fluid column and pressure transducer were used to measure the outflow through normal drainage pathways in the unoperated eye, as indicated by the red arrows. (a) Eye without glaucoma implant. (b) A 27-gauge needle inserted into anterior chamber. (c) Three-way stopcocks. (d) Pressure transducer connected via tubing. (e) Syringe to assist with loading of system. (f) Calibrated fluid column with height at eye level.
Figure 2. 
 
Mean outflow measurements.
Figure 2. 
 
Mean outflow measurements.
Figure 3. 
 
Histology of bleb capsules at 7 days. H&E (A) and Masson's trichrome (B) staining at ×10 magnification at the center of the capsule. H&E staining taken at the limbal (C) and fornix (D) borders of the capsule, at ×10 magnification.
Figure 3. 
 
Histology of bleb capsules at 7 days. H&E (A) and Masson's trichrome (B) staining at ×10 magnification at the center of the capsule. H&E staining taken at the limbal (C) and fornix (D) borders of the capsule, at ×10 magnification.
Figure 4. 
 
Histology of bleb capsules at 28 days. H&E (A) and Masson's trichrome (B) staining at ×10 magnification at the center of the capsule. H&E staining taken at the limbal (C) and fornix (D) borders of the capsule, at ×10 magnification.
Figure 4. 
 
Histology of bleb capsules at 28 days. H&E (A) and Masson's trichrome (B) staining at ×10 magnification at the center of the capsule. H&E staining taken at the limbal (C) and fornix (D) borders of the capsule, at ×10 magnification.
Figure 5. 
 
Control eye: normal conjunctiva. H&E (A) and Masson's trichrome (B) staining at ×10 magnification.
Figure 5. 
 
Control eye: normal conjunctiva. H&E (A) and Masson's trichrome (B) staining at ×10 magnification.
Table. 
 
Outflow into Control Eyes and Implant Capsules at 7 and 28 Days Following Insertion of a Pediatric Molteno Implant
Table. 
 
Outflow into Control Eyes and Implant Capsules at 7 and 28 Days Following Insertion of a Pediatric Molteno Implant
Control Eyes, n = 6 7-Day-Old Capsules, n = 5 28-Day-Old Capsules, n = 4
Mean (SEM), μL/min/mm Hg measured at 15 mm Hg 0.136* (0.007) 0.117* (0.036) 0.009* (0.003)
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