Viscocanalostomy in Rhesus Monkeys - JAMA … in Rhesus Monkeys ... Embryology, University of ......

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Viscocanalostomy in Rhesus MonkeysErnst R. Tamm, MD; Roberto G. Carassa, MD; Daniel M. Albert, MD;B’Ann T. Gabelt, MS; Sarit Patel, MD; Carol A. Rasmussen, BA; Paul L. Kaufman, MD

Objective: To examine structural changes and aque-ous humor outflow after viscocanalostomy in live nor-mal monkey eyes.

Methods: Viscocanalostomy surgery was performed in1 eye of each of 4 rhesus monkeys. Outflow facility wasdetermined before and after surgery. All eyes were fixedand examined by light and/or electron microscopy 36 or63 days postoperatively.

Results: Schlemm canal was replaced by scar tissue at thesurgical site. The juxtacanalicular zone contained homo-geneous material, probably high-molecular-weight 1.4% so-dium hyaluronate. The sclera external to Schlemm canalwas overhydrated, and remains of a scleral lake were pres-ent in 1 animal. Multiple defects were present in the en-dothelial lining of Schlemm canal inner and outer wall. Finefibrillar material and sheath-derived plaque material partlybridged the defects. Along the inner wall, aggregations ofthrombocytes covered some defects in the endothelial lin-ing of the canal. At 90° to 180° from the surgical site, smalland fewer breaks in the inner wall were seen. Postsurgery

outflow facility (n=2) was approximately 30% higher inthe treated eye than in the contralateral control, correctedbilaterally for presurgery baseline.

Conclusions: The most likely explanations for the in-crease in outflow facility in monkeys after viscocanalos-tomy are focal disruptions of the inner wall endothe-lium of Schlemm canal and disorganization of thejuxtacanalicular zone, resulting in direct communica-tion of juxtacanalicular zone extracellular spaces with thelumen of Schlemm canal. The continuous presence of so-dium hyaluronate might prevent repair of these defectsby interfering with thrombocyte function.

ClinicalRelevance: Innonhumanprimates,viscocanalos-tomy appears to decrease outflow resistance through per-sisting focal disruption of the inner wall endothelium andopening of the juxtacanalicular or cribriform region of thetrabecularmeshwork, thetissuemostaffectedbypathologicchanges in primary open-angle glaucoma in humans.

Arch Ophthalmol . 2004;122:1826-1838

A NTERIOR CHAMBER DRAIN-age surgery is an essentialtool in glaucoma manage-ment. Nonpenetratingtechniques are being re-

fined to reduce the postoperative compli-cations associated with more traditionaldrainage procedures. Viscocanalostomyand a related procedure called deep scle-rectomy have garnered much attention re-cently. Although more difficult and per-haps slightly less effective at loweringintraocular pressure (IOP) than standardtrabeculectomy,1 they lessen the risk ofsome of the postoperative problems of tra-beculectomy and thus seem attractive.

Stegmann et al2 recognized the need fora technique that would be successful inareas with a high risk of postoperative in-fection. They proposed the viscocanalos-tomy procedure, based on Krasnov’s3 andZimmerman and coworkers’4 work onnonpenetrating “trabeculectomy.” Ide-ally, there is no obvious or intentional in-vasion of the anterior chamber and no iri-

dectomy. These patients typically havequiet eyes postoperatively, do not be-come hypotonous, and, in 1- to 5-year fol-low-up, have good IOP control, albeit notas low as with trabeculectomy.2,5-7 They of-ten have no clinically visible filtering bleb,or they may have a small, relatively flatbleb, quite different from results with tra-beculectomy. Antimetabolite is not used.

Critical components of the surgical pro-cedure are creation of superficial and deepscleral flaps, unroofing of Schlemm canaland Descemet membrane followed by can-nulation of the cut ends of Schlemm ca-nal and intracanalicular injection ofhigh-molecular-weight 1.4% sodium hy-aluronate (Healon GV; Pfizer Inc, NewYork, NY), and excision of the deep scleralflap. The hypothesis was that aqueous hu-mor leaves the anterior chamber by per-colating through Schlemm canal endothe-

For editorial commentsee page 1868

Author Affiliations:Department of Anatomy,Molecular Anatomy, andEmbryology, University ofErlangen-Nürnberg, Germany(Dr Tamm); Department ofOphthalmology and VisualSciences, University HospitalS. Raffaele, Milan, Italy(Dr Carassa); and Departmentof Ophthalmology and VisualSciences, University ofWisconsin–Madison, Madison,Wis (Drs Albert, Patel, andKaufman and Mss Gabelt andRasmussen).Financial Disclosure: None.

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lium and Descemet membrane into the hollowed-out“lake,” and then enters the widened cut ends of Schlemmcanal.

As pointed out by Johnson and Johnson,8,9 it is un-clear how this mechanism would be effective in lower-ing outflow resistance, as the current concept of aque-ous outflow dynamics implies that most of the resistanceto outflow of aqueous humor is internal to Schlemm ca-nal endothelium.10 Indeed, more recent histopathologi-cal studies on human and monkey cadaver eyes showedthat the dilation of Schlemm canal with viscoelastic agents(1.4% sodium hyaluronate or 2.3% sodium hyaluronate[Healon 5; Pfizer Inc]) caused disruptions in the endo-thelial lining of Schlemm canal,11,12 an effect that couldsignificantly reduce outflow resistance in the trabecularmeshwork (TM), but would imply that viscocanalos-tomy is a penetrating rather than a “nonpenetrating” sur-gical technique. To obtain more data on the functionaland structural effects of viscocanalostomy in the livingeye, we performed viscocanalostomy in the eyes of rhe-sus monkeys and studied structure and function of theoutflow pathways 1 to 2 months after surgery.

METHODS

Four normal adult male and female rhesus monkeys (Macaca mu-latta), aged 19 to 20 years, were studied. Baseline slitlamp bio-microscopy and IOP measurements using a minified Gold-mann applanation tonometer13 were performed. Baseline perfusionoutflow facility and tonographic outflow facility were deter-mined on 2 animals (monkeys AI09 and AI10). Perfusion out-flow facility was determined by 2-level constant-pressure per-fusion of the anterior chamber with the use of Bárány solution,14

correcting for internal apparatus resistance.15 The eyes were can-nulated with a branched needle, with one branch connected toa pressure transducer and the other branch to a reservoir inflowline. Tonographic outflow facility was determined with an elec-tronic Schiøtz tonographer (model 720 D; Berkeley Bioengineer-ing, San Leandro, Calif). Applanation tonometry was done be-fore tonography. If IOP readings were less than 20 mm Hg, the5.5-g weight was used; between 20 and 29 mm Hg, the 7.5-gweight was used. Tonography was done for 4 minutes on eacheye. Facility was calculated by means of tables and the Frieden-wald nomogram, according to methods described in Becker-Shaffer’s Diagnosis and Therapy of the Glaucomas.16 All animalshad IOP measured weekly with the Goldmann tonometer. Vis-cocanalostomy surgery (see description in the “Surgical Tech-nique” section) was performed after perfusion-induced inflam-mation had subsided (28 days). Two monkeys (81051 and AI45)were used as practice surgery animals. These animals were notoriginally intended to be part of the study and so did not havebaseline facility measurements performed. They were to be usedto work out any technique differences or problems we might en-counter in adapting a human procedure to monkeys. The sur-gery went well, and these animals were included in subsequenttesting. In all, surgery was done in 1 eye of each of 4 animals.None of the animals received a sham procedure. One practiceanimal (AI45) had a structurally abnormal control eye unsuit-able for physiological testing. The monkeys were divided into 2groups, each with 1 normal and 1 practice animal. Group 1 hadperfusion and tonographic outflow facility determinations andgroup 2 had tonographic outflow facility determination 33 to 42days after surgery. The animals were divided into 2 groups topermit different structural approaches and physiology proto-cols for maximum information.

TISSUE FIXATION

Group 1

After perfusion inflammation had subsided (21 to 28 days af-ter perfusion, 63 days after surgery), both anterior chambersof each monkey were exchanged with cationic 5-nm and non-cationized 10-nm gold solution as tracers to delineate flow path-ways and to label extracellular matrix, at an IOP of approxi-mately 15 mm Hg, and then perfused at 25 mm Hg with Itosolution from an elevated reservoir. Under deep general anes-thesia with intravenous pentobarbital sodium, 15 mg/kg, theseanimals were then perfused through the heart with phosphate-buffered saline, 0.1 mol/L (pH 7.4), followed by Ito solution.The eyes were enucleated, windows were cut in the cornea andsclera, and the eyes were placed in the same fixative and sentto Germany for electron microscopy.

On arrival, the eyes were placed in cacodylate buffer (pH7.4) for 24 hours to wash out fixative. Each eye was bisectedand the anterior halves were cut into quadrants by meridionalsectioning. Each quadrant was further dissected into wedge-shaped specimens 1.0 to 1.2 mm wide that contained TM, cili-ary muscle, iris, and adjacent cornea and sclera. In the supe-rior quadrant, the distance of each specimen to the 12-o’clocklimbus, which had been marked by a suture during enucle-ation of the eye, was identified and documented. All wedgeswere dehydrated in ascending concentrations of alcohol andembedded in epoxy resin according to standard protocols. One-micrometer semithin sections were cut from each specimen ofthe superior quadrant (6-8 per monkey) and from at least 3 speci-mens of the other 3 quadrants. All semithin sections were stainedwith toluidine blue O and examined by light microscopy. Sub-sequently, ultrathin sections were cut from each specimen thathad been investigated by light microscopy and stained with leadcitrate and uranyl acetate. Both semithin and ultrathin sec-tions were assigned a unique identifying number and were ex-amined by a masked observer (E.R.T.).

Group 2

Tonographic outflow facility determination at day 35 after vis-cocanalostomy was followed by perfusion through the heart withphosphate-buffered neutral formalin on day 36. The eyes wereenucleated and placed in 10% formalin for processing and analy-sis by one of us (D.M.A.). The eyes were histologically pro-cessed, embedded in paraffin, and serially sectioned at 5 µm.Every 10th section was stained with hematoxylin-eosin and cov-erslipped. The eye sections were assigned a unique identifyingnumber and a masked observer examined the hematoxylin-eosin–stained sections under a microscope. The eyes were ex-amined and several histologic features were recorded: (1) thepatency and integrity of Schlemm canal, (2) evidence of breaksin the TM, (3) the presence of scleral lakes, (4) the presenceand degree of inflammation, (5) the presence and degree of fi-brosis, and (6) any other unusual histologic ocular features.Emphasis was placed on the area of, and adjacent to, the sur-gical site, as well as the area 180° from the surgical site. Thesurgically treated eyes from group 2 monkeys were comparedwith the contralateral control eye from one of the animals.

Anesthesia and Antibiotics

Intramuscular ketamine hydrochloride anesthesia (10 mg/kg,supplemented every 20-30 minutes as needed with 5 mg/kg)was used for all procedures. In addition, animals received in-travenous pentobarbital sodium (10-15 mg/kg) for perfusionoutflow facility, or acepromazine maleate (0.2-0.5 mg/kg in-




tramuscularly) for tonographic outflow facility. For perfu-sion, animals were maintained under ketamine anesthesia un-til slitlamp biomicroscopy and IOP measurement wereperformed, intravenous lines were in place, and the perfusionapparatus was calibrated. At that point they were given pen-tobarbital and no further ketamine. For the viscocanalostomysurgery, animals were intubated and maintained under isoflu-rane inhalation anesthesia. All were maintained with intrave-nous fluids (5% dextrose–lactated Ringer solution) at a rate of10 mL/kg per hour. Animals were given subtenon gentamicinsulfate and methylprednisolone acetate (20 mg) postopera-tively. Penicillin G procaine and penicillin G benzathine, 50000U/kg, were given intramuscularly for 5 days and methylpred-nisolone acetate, 1 mg/kg, was given intramuscularly for 4 weeks,with the dose tapered down during the last week.

All experiments were performed in accordance with the As-sociation for Research in Vision and Ophthalmology State-ment on the Use of Animals in Ophthalmic and Vision Re-search.

SURGICAL TECHNIQUE

Phase 1: Preparation of the Surgical Field

After a bridle suture was passed under the superior rectus muscle,a fornix-based conjunctival flap was prepared. To avoid dam-age to Schlemm canal, collector channels, and the sclera itself,hemostasis was maintained by repeated irrigation with orni-pressin solution, 5 IU/mL (Por 8; Sandoz, Basel, Switzerland),so as to use as little thermal coagulation as possible.

Phase 2: Preparation of the Intrascleral Chamber

With a 20-gauge diamond knife, a 5�5-mm limbus-based para-bolic incision was made in the sclera, and a 200-µm superfi-cial flap was dissected with a single-use bevel-up spatula (bothfrom Grieshaber, Schaffhausen, Switzerland). By the same tech-

nique, an inner concentric 4�4-mm limbus-based scleral flapwas sculpted beneath the previous one, keeping the surface ofthe cut so close to the choroid as to have a dark reflex. Whenthe cut was advanced limbally, Schlemm canal was deroofedand the 2 openings of the canal remained patent at the edgesof the cut.

Phase 3: Injection of Viscoelastic in Schlemm Canal

The ostia of Schlemm canal were then cannulated with a 190-µm-diameter blunt cannula (Grieshaber) and filled with high-molecular-weight 1.4% sodium hyaluronate. To limit damageto the Schlemm canal walls, the injection of sodium hyaluro-nate was started while the ostia were approached, the cannulainsertion did not exceed 1 mm, and a small amount of sodiumhyaluronate was slowly injected on each side 3 to 4 times. Ap-proximately 275 µL of sodium hyaluronate was injected. Mostof it did not go into Schlemm canal because of high reflux.

Phase 4: Realization of WindowOverlying Anterior Chamber

By gently pulling the inner scleral flap upward and delicatelydepressing the floor of the canal and Descemet membrane withthe tip of a cotton swab, the membrane itself was cleaved an-teriorly from the cornea for approximately 1 mm (Figure 1),and aqueous was seen to percolate through this window andto enter the lake. As soon as the window was completed, theinner scleral flap was excised by means of Vannas scissors.

Phase 5: Sealing of the Lake

The ostia of Schlemm canal were cannulated again and so-dium hyaluronate was gently injected 2 to 3 times in each op-ening. Finally, the outer flap was tightly sutured with seven 10-0nylon stitches, and sodium hyaluronate was injected under-neath the flap to temporarily fill the intrascleral lake and pre-vent it from collapsing and scarring in the early postoperativeperiod. Finally, 2 wing 8-0 silk sutures were passed to hold theconjunctiva in place.

SODIUM HYALURONATE

In a separate experiment, approximately 25 to 30 µL of sodiumhyaluronate was injected into the anterior chamber of 1 eye ofan adult female cynomolgus monkey (Macaca fascicularis). Ap-proximately 25 to 30 µL of aqueous humor was then removedand the IOP was checked to ensure that it was not elevated. Slit-lamp examination was performed 24 hours later to assess theamount of inflammation, which was minimal. Fixation for thisanimal was similar to that of the animals in group 1. Under deepgeneral anesthesia with intravenous pentobarbital sodium, 15 mg/kg, the animal was perfused through the heart with phosphate-buffered saline, 0.1 mol/L (pH 7.4), followed by Ito solution. Theeyes were enucleated, windows were cut in the cornea and sclera,and the eyes were placed in the same fixative and sent to Ger-many for electron microscopy. Wedge-shaped specimens fromeach quadrant of the anterior eye were processed for light andelectron microscopy as described in “Tissue Fixation, Group 1.”

RESULTS

OUTFLOW FACILITY

One animal had preoperative and postoperative perfu-sion outflow facility determinations (monkey AI10). Post-

Figure 1. Phase 4 of the viscocanalostomy procedure, showing realization ofthe window overlying the anterior chamber. See the “Methods” section fordetails.




operative facility (35 days after surgery) increased by 50%in the treated eye and 22% in the control eye of this ani-mal compared with baseline. The ratio of facility in theviscocanalostomy-treated and control eyes (V/C) changedfrom 0.78 before to 1.22 after surgery. Tonographic out-flow facility was determined before and 34 days after sur-gery on 2 animals (monkeys AI09 and AI10). Facility in-creased by 68.5% ± 0.5% in the viscocanalostomy-treated eyes and 42.5%±0.5% in the control eyes. (Resultsare given as mean±SEM unless otherwise indicated.) TheV/C facility ratio changed from 0.66±0.10 before to1.21±0.16 after surgery. Tonographic outflow facility datawere collected 1 day before perfusion outflow facility on3 occasions and 9 days before in 1 other. One animal hadboth preoperative and postoperative testing done (AI10)and so accounted for 2 of the 4 test points. The ratios ofV/C facility were calculated for both types of facility mea-surement. The ratio of V/C facility for tonographic out-flow was 0.86±0.18. The ratio of V/C facility for perfu-sion outflow was 0.84±0.12. The ratio of tonographicfacility to perfusion facility was 1.00±0.07.

INTRAOCULAR PRESSURE

The presurgery IOP for the 4 eyes that underwent theviscocanalostomy procedure was 17.3±1.6 mm Hg. TheIOP at time of death for these eyes was 16.3±0.6 mm Hg,a nonsignificant decrease of 6%. The presurgery IOP forthe 3 fellow eyes was 17.3±1.2 mm Hg, while the IOP attime of death was 16.7±0.3 mm Hg, a nonsignificant de-crease of 4%.

ELECTRON MICROSCOPY

The eyes of 2 monkeys that underwent viscocanalostomyin 1 eye were examined at 63 days after surgery. The entirecircumference of operated-on and control eyes was inves-tigated. In the operated-on eyes of both animals, Schlemm

canal and juxtacanalicular TM had disappeared in the cen-ter of the superior quadrant, where the operation had beenperformed (Figure 2). In an area of about 2 mm in cir-cumferential width, irregular whorls of elastic and collag-enous fibers occupied the position where Schlemm canaloriginally had been located (Figure 2). At the inner aspectof the TM, uveal trabecular lamellae covered by TM cellsremained intact and the chamber angle was open.

Adjacent to the operated-on area, Schlemm canal waspresent (Figure 3A) in both operated-on eyes. In noneof the eyes was Schlemm canal abnormally dilated. Therewere, however, marked structural changes in the outer partsof the TM along a distance of about 2 to 6 mm next to theoperated-on site. In this area, trabecular lamellae had be-come replaced by a network of fibroblastlike cells that wereembedded in a loose collagenous matrix (Figure 3A). Thistissue appeared not to impede flow of aqueous humor, asgiant vacuoles were present along the inner wall of Schlemmcanal (Figure 3A). In places, the juxtacanalicular zone wasfilled with homogeneous material that was not seen in con-trol eyes. By electron microscopy, this material was elec-tron dense and had a fine granular structure (Figure 3B).The inner wall endothelial lining of Schlemm canal fre-quently showed intercellular openings approximately 100nm to 2 µm in diameter (Figure 3B and C). Through theseopenings, extensions of the fine granular material pro-truded into the lumen of Schlemm canal. Both 5- and 10-nmgold particles that were embedded in the fine granular ma-terial were seen at the outer side of the inner wall next tothe openings, as well as in the part of the material that ex-tended through the openings (Figure 3C). We hypoth-esized that the fine granular material was sodium hyal-uronate that had been injected into Schlemm canal duringsurgery and was pressed through the openings in Schlemmcanal endothelium into the juxtacanalicular region of theTM. To support this hypothesis, we performed an addi-tional experiment and injected sodium hyaluronate di-rectly into the anterior chamber of the eye of an addi-

BA

Figure 2. Chamber angle at the center of the superior quadrant (monkey Al10), where the operation was performed (semithin sections, toluidine blue O stain).Irregular whorls of elastic and collagenous fibers (arrows) mark the position of Schlemm canal before the operation. At the inner aspect of the trabecularmeshwork, uveal trabecular lamellae covered by trabecular meshwork cells are left intact. Bars indicate 42 µm (A) and 19 µm (B).




tional monkey. Twenty-four hours after injection, sodiumhyaluronate was observed by light microscopy in contactwith the surfaces of ciliary processes and iris as homoge-neous material that stained intensely with toluidineblue O (Figure 4A). By electron microscopy (Figure 4B),the same material was electron dense and finely granular,and showed essentially the same ultrastructural character-

istics as the material that was found in the juxtacanalicu-lar TM of monkeys that underwent viscocanalostomy. Simi-lar electron-dense and finely granular material was observedafter anterior chamber injection at the inner side of the pe-ripheral cornea (not shown) and at the inner surface andthe intertrabecular spaces of the uveal meshwork (Figure 4Cand D), but not in the juxtacanalicular region.

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Figure 3. Trabecular meshwork of a monkey eye 2 months after viscocanalostomy (monkey 81051), 4 to 6 mm from the site operated on. A, Trabecular lamellaehave become replaced by a network of fibroblastlike cells embedded in a loose collagenous matrix (star). Giant vacuoles are present along the inner wall ofSchlemm canal (SC), and the juxtacanalicular zone is filled with homogeneous material (arrow) (semithin section, toluidine blue O stain). B and C, Electronmicrographs of the area shown in A. B, The juxtacanalicular zone is filled with fine granular, electron-dense material (star). In areas of contact with the inner wall,the endothelial lining of Schlemm canal shows openings through which extensions of the fine granular material protrude into the lumen of Schlemm canal(arrows). C, Homogeneous fine granular material is present close to an opening between endothelial cells (E) of Schlemm canal (star). Gold particles measuring5 nm (solid arrows) and 10 nm (open arrows) that are in contact with the material are present in the lumen of Schlemm canal. Bars indicate 30 µm (A), 4.25 µm (B),and 0.135 µm (C).




In one of the viscocanalostomy-treated monkeys(AI10), at a distance of about 2 mm from the 12-o’clocklimbus, a large triangular defect was present in the scleraadjacent to the anterior portion of the ciliary muscle(Figure 5A). The defect had a circumferential exten-sion of about 1 mm. The walls of the defect had a lengthof approximately 0.5 to 0.6 mm and were not coveredby cells. We concluded that this defect resulted from theoperation and was a remaining part of the intrascleral res-ervoir, the so-called scleral lake that was created by re-moving the inner layer of the sclera. Between the sclerallake and the TM, the sclera appeared to be markedly lessdense than in other parts of the eye (Figure 5A). In theTM of this region, there were fewer trabecular lamellae,

and large intertrabecular spaces were present (Figure 5B).The lumen of Schlemm canal formed protrusions to-ward the TM (Figure 5C and D). Similar protrusions werenot observed in control eyes. On serial sections, it be-came evident that these protrusions communicated di-rectly with the intertrabecular spaces and were not Son-dermann canals, which are blind diverticula in the innerwall of Schlemm canal that are completely covered by en-dothelium. Sondermann canals are extremely rare in mon-keys (E.R.T., unpublished data, 2003), consistent withthe findings in control eyes. A similar large scleral de-fect was not present in the same area of the other oper-ated-on eye. However, in some distinct areas, larger de-fects, approximately 1 to 5 µm in length, in the endothelial

B

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TM

Figure 4. Iris (Ir), ciliary processes (CP), and trabecular meshwork (TM) of a monkey eye 24 hours after anterior chamber (AC) injection of high-molecular-weightsodium hyaluronate. A and C, The injected material is present close to the surface of the ciliary processes (A), the posterior surface of the iris (arrows, A), and theinnermost parts of the uveal trabecular meshwork (arrows, C) close to the anterior chamber (semithin sections, toluidine blue O stain). B and D, By electronmicroscopy, sodium hyaluronate next to the surface of the ciliary epithelium (CE in B) and in the trabecular meshwork (D) is of electron-dense, homogeneous, finegranular appearance (stars) and has essentially the same ultrastructural characteristics as the fine granular material observed in the trabecular meshwork ofmonkeys after viscocanalostomy (Figure 3). Bars indicate 40 µm (A), 1.5 µm (B), 20 µm (C), and 0.59 µm (D).




covering of Schlemm canal were observed that were notassociated with the fine granular material nor with throm-bocytes (Figure 6). Such defects were seen along theinner wall and, more rarely, in the outer wall of Schlemmcanal in this area (Figure 6A and B). By electron micros-copy, fine fibrillar material and sheath-derived plaque ma-terial was seen to partly bridge over the defects(Figure 6C). Numerous 5- and 10-nm gold particles wereattached to this extracellular material (Figure 6D). In aregion 2 to 4 mm nasal from the 12-o’clock limbus andin an area where defects in the outer wall were fre-quently observed, the extracellular material between theouter wall of Schlemm canal and the outer side of thesclera and cornea was markedly less dense than in thecontrol eye or in other regions of this eye (Figure 7).Thus, at the limbus, a sharp boundary was formed at theanterior end of the TM that separated corneal stroma ofnormal extracellular matrix density localized anteriorlyto the TM from stroma of considerably less density thatwas localized opposite to Schlemm canal and TM. By elec-tron microscopy of this area of diminished extracellular

matrix density, large electron-empty spaces were seen be-tween cells that showed typical ultrastructural charac-teristics of fibroblasts. The amount of collagenous fibersappeared to be greatly reduced.

At the nasal, temporal, and inferior sides of both eyes,approximately 90° to 180° from the center of the surgi-cal site and beyond the extent of the Schlemm canal can-nulation, obvious scarlike changes in the TM were notobserved. Still, the normal contour of Schlemm canal hadchanged and often showed irregularly branched cul-de-sac–like protrusions that reached toward the inner partsof the TM (Figure 8). Such protrusions were not ob-served in control eyes. The inner wall in this area formednumerous giant vacuoles, and the juxtacanalicular areawas again partly filled with homogeneous material. Alongthe inner wall, aggregations of thrombocytes were fre-quently observed that appeared to cover defects in theendothelial lining of the canal. By electron microscopy,individual thrombocytes were found at the outer side ofthe inner wall in close association with intercellular junc-tions of the endothelium, some of which appeared open

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Figure 5. Chamber angle of a monkey eye (monkey AI10) treated by viscocanalostomy about 2 mm from the 12-o’clock limbus where the surgery was performed2 months earlier (semithin sections, toluidine blue O stain). A, A large triangular defect (SL), probably representing a remaining part of the scleral lake createdduring surgery, is present in the sclera adjacent to the anterior portion of the ciliary muscle (CM). The walls of the defect are approximately 0.5 to 0.6 mm longand are not covered by cells. In a distinct area (solid arrows) between defect and trabecular meshwork (open arrow), the sclera appears to be markedly less densethan in other parts of the eye. AC indicates anterior chamber. B, In the trabecular meshwork of this region, the number of trabecular lamellae is reduced and largeintertrabecular spaces are present (open arrows). SC indicates Schlemm canal. C and D, Serial sections of the trabecular meshwork, same region as in A and B.The lumen of Schlemm canal forms protrusions toward the trabecular meshwork (solid arrows) that communicate directly with the intertrabecular spaces. Barsindicate 90 µm (A), 22.5 µm (B), and 14.8 µm (C and D).




(ie, the cells were separated), others closed (ie, the cellswere not separated). In addition, aggregates of throm-

bocytes were observed (Figure 9). In these areas, cy-toplasmic protrusions of individual thrombocytes were

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Figure 6. Trabecular meshwork of a viscocanalostomy-treated monkey eye (monkey 81051) about 4 mm from the 12-o’clock limbus where the surgery wasperformed 2 months earlier. A and B, Larger defects, about 1 to 5 µm long, are present in the inner and outer wall of Schlemm canal (SC, solid arrows) (semithinsection, toluidine blue O stain). C and D, By electron microscopy, fine fibrillar material and sheath-derived plaque material (solid arrows) are seen that partlybridge the defects between neighboring endothelial cells (open arrows). Inset in D, Numerous 5-nm (triple arrow) and 10-nm (double arrows) gold particles areattached to this extracellular material. Bars indicate 40 µm (A), 13.8 µm (B), 1 µm (C), and 0.46 µm (D).




seen that filled the gaps between adjacent inner wall cells.Gold particles had become attached to extracellular fi-bers close to the openings, indicating that this region wasused for flow of aqueous. In addition, gold particles wereseen in intracellular vesicles in neighboring Schlemm ca-nal endothelial cells next to the openings. In contrast,no gold particles were observed in association with theextracellular matrix in immediately adjacent areas thatwere separated from the lumen of Schlemm canal by anintact endothelial layer. More rarely observed were con-tracted thrombocytes that closed openings of the innerwall, or larger thrombotic plaques that consisted of nu-merous aggregated thrombocytes and fibrinlike mate-rial between them (Figure 9D and E).

Discontinuities in the periphery of Descemet mem-brane were observed at the base of the opercular area,an extension of Descemet covering the anterior TM, whichis not present in humans (Figure 10). Between the endsof the discontinuous Descemet membrane, cells or smalleropen spaces up to 5 µm in width were present. In otherareas, Descemet membrane was interrupted by a smallslit only. These discontinuities are normal for monkeysand were present in both control and experimental eyesand distributed equally around the circumference. No evi-dence of iatrogenic disruptions of Descemet membranewas observed in any eye.

LIGHT MICROSCOPY

The viscocanalostomy-treated eyes of monkeys AI09 andAI45 were examined at 36 days after surgery, along withthe contralateral control eye of monkey AI09. In all vis-cocanalostomy-treated eyes, the sclera of the surgical areawas mildly edematous and contained at least minimal fi-brosis of the scleral flap interface. Schlemm canal was notdilated in any eyes. The lamella of the corneoscleral anduveal TM in viscocanalostomy-treated eyes showed breaksthat were not observed in control eyes. There were mi-croscopic breaks in both the inner and outer wall ofSchlemm canal in both viscocanalostomy-treated eyes.In these 2 eyes, there were also breaks in the inner andouter walls of Schlemm canal 180° from the surgical site.A full-thickness discontinuity in Descemet membrane ofthe peripheral cornea was seen in 1 animal (AI09).

COMMENT

Baseline physiological testing showed small differences inoutflow facility between the eyes for each animal; the eyewith lower facility was selected for viscocanalostomy sur-gery. As with clinical studies,1,2,6,7,17 there was a marked de-crease in IOP after surgery. Still, in our monkeys this effectwas transient and recovery to near baseline occurred within30 days. Outflow facility, determined after surgery whenthe eyes were quiet, increased to a greater extent, relativeto baseline values, in the eyes undergoing viscocanalos-tomy. By this time IOP had recovered to baseline levels.We assume that larger decreases in IOP as reported for hu-man patients with glaucoma and larger increases in facil-ity were not observed because preoperative IOP and base-line facility in our monkeys were within the reference range.In addition, the higher-than-physiologic pressure gradi-

B

A

SC

Figure 7. Trabecular meshwork, Schlemm canal (SC), and adjacent corneaand sclera in a region 2 to 4 mm nasal from the 12-o’clock limbus whereviscocanalostomy was performed 2 months earlier (A), and the contralateralcontrol eye (B) (monkey 81051) (semithin sections, toluidine blue O stain).The extracellular material between the outer wall of Schlemm canal and theouter side of the sclera-cornea is markedly less dense in A than in B(asterisks). In A, a sharp boundary is formed at the anterior end of thetrabecular meshwork (arrows) that separates corneal stroma of normalextracellular matrix density, which lies anteriorly from the trabecularmeshwork, from an extracellular matrix of considerably less density, which liesopposite to Schlemm canal and trabecular meshwork. Bars indicate 33 µm.

SC

Figure 8. Outer trabecular meshwork and Schlemm canal (SC) of a monkeyeye (monkey 81051) 2 months after viscocanalostomy, approximately 90°from the center of the surgical site and beyond the extent of the Schlemmcanal cannulation (semithin section, toluidine blue O stain). Schlemm canalforms cul-de-sac–like protrusions that reach toward the inner parts of thetrabecular meshwork. The inner wall of Schlemm canal forms giant vacuolesand the juxtacanalicular area is filled with homogeneous material (asterisk).Along the inner wall endothelium, aggregations of thrombocytes are presentthat appear to cover defects in the endothelial lining of the canal. Arrowsindicate aggregations of thrombocytes; bar, 8.6 µm.




ent or flow rate during external perfusion or tonographymight contribute to the increase in outflow facility. Dur-ing perfusion from an open reservoir, IOP can be up to 12mm Hg higher than spontaneous IOP. The resultant higherpressure gradient and flow rate across the TM could fur-ther loosen already weakened (by the injection and reten-

tion of viscoelastic material) cell adhesions to each otherand to their extracellular matrix in the inner wall and jux-tacanalicular zone, relaxing the meshwork so as to facili-tate flow through it,18,19 as well as driving fluid through theinner wall breaks in Schlemm canal. Under this scenario,the surgical procedure might be more clearly effective func-

B

D E

A

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SCSC

SCSC

Figure 9. Electron micrographs of monkey eyes treated by viscocanalostomy 2 months previously (monkey 81051). A, Thrombocytes (solid arrows) are found atthe outer side of the inner wall of Schlemm canal (SC) in close association with intercellular junctions of the endothelium, some of which appear open (ie, the cellsare separated; open arrows). B, Aggregated thrombocytes (arrows) adhere to the inner wall of Schlemm canal in the region of a larger gap between adjacent innerwall cells. C, Higher magnification of B. Thrombocyte processes fill the gap between adjacent endothelial cells (solid arrow). Gold particles measuring 5 and 10 nmattach to extracellular fibers close to the opening and are seen in intracellular vesicles in neighboring Schlemm canal endothelial cells (open arrows).D, A contracted thrombocyte (arrow) fills an opening in the inner wall. E, A thrombotic plaque (arrow) consisting of numerous aggregated thrombocytes andfibrinlike material between them closes an opening in the inner wall. Bars indicate 1.7 µm (A), 1.15 µm (B and D), 0.42 µm (C), and 2.15 µm (E).




tionally in patients with glaucoma with elevated IOP, es-pecially if they are not receiving secretory suppressants.

Experimental glaucoma can be induced in monkeysby laser treatment of the chamber angle tissues.20,21 How-ever, this procedure induces scarring of TM and Schlemmcanal22,23 and would preclude cannulation and injectionof sodium hyaluronate in Schlemm canal as required foreffective viscocanalostomy.

In contrast to the modest changes in outflow facility,structural changes of the conventional outflow pathwaysthat persisted for at least 1 to 2 months postoperatively weremore pronounced. These changes included numerousbreaks in the inner and outer walls of Schlemm canal en-dothelium that were observed over large parts of the cir-cumference in all of the operated-on monkey eyes. Goldparticles with a high affinity for extracellular matrix thatwere added to the perfusion fluid were observed in areasof disruption where underlying extracellular matrix bridgedthe defects or was partly protruding into the lumen ofSchlemm canal, and were directly associated with this ma-trix. Because these particles must have been carried by aque-ous flow, we assume that aqueous humor passed throughthe breaks in the endothelial lining of Schlemm canal. Com-parable breaks were reported in a recent study on humanand monkey cadaver eyes that had undergone viscoca-nalostomy.11 In these acute experiments, the walls ofSchlemm canal became disrupted after marked dilation ofSchlemm canal due to cannulation and injection of so-

dium hyaluronate. It seems reasonable to assume that a simi-lar scenario is responsible for the structural defects inSchlemm canal endothelium in our material. Still, dila-tion of Schlemm canal after injection of sodium hyaluro-nate appears to be an acute and transient phenomenon, sincein our material 1 to 2 months after surgery, Schlemm ca-nal was of normal size or appeared narrowed, or was com-pletely obliterated close to the site of surgery. In addition,we did not observe signs of disrupted septae bridging thelumen of Schlemm canal that were seen in acute experi-ments on cadaver eyes.11

It is well established that acute disruption of the endo-thelial lining of Schlemm canal in monkey eyes, eg, by treat-ment with disodium EDTA, ethylene glycol-bis(2-aminoethylether)-N,N,N�,N�-tetraacetic acid (EGTA),24,25

or cytochalasin B,24,26 is associated with a marked increasein outflow facility. It has been calculated that the inner wallendothelium of Schlemm canal accounts for only less than10% of total outflow resistance,27 since it forms numerousmicrometer-size pores that allow relatively free passage ofaqueous humor.10 Therefore, the effects of inner wall dis-ruption on outflow facility are thought to be due to a wash-out of extracellular matrix components from the juxta-canalicular or cribriform region of the TM into the lumenof Schlemm canal. Data from microcannulation experi-ments indicate that most of the outflow resistance is lo-cated in this region, at a distance of approximately 7 to 14µm from the inner wall endothelium.28 Notwithstanding

B

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NFTM

Op

Figure 10. Discontinuities in Descemet membrane (arrows) at the opercular area of control eyes (A and B) and one eye treated by viscocanalostomy 2 monthspreviously (C; monkey AI10) (semithin sections, toluidine blue O stain). A, The discontinuities are frequently observed at the base of the opercular area (Op), anextension of Descemet membrane covering the anterior nonfiltering trabecular meshwork (NFTM), not present in humans. The frequency and structure of discontinuitiesdo not differ between control and viscocanalostomy-treated eyes. Between the ends of the discontinuous Descemet membrane, cells (A) or smaller open spaces up to5 nm in width (B) may be present. In other areas, Descemet membrane appears to be interrupted only by a small slit (arrow, C). Bars indicate 20 µm.




this evidence, there is still uncertainty as to the exact siteand nature of the resistance, with most advocating the jux-tacanalicular region but some advocating the inner wall en-dothelium.29 It seems reasonable to assume that focal dis-ruption of the inner wall endothelium and resulting washoutof extracellular material from the outer parts of the TMcaused the effects on outflow facility in our monkeys. Ourfindings are informative regardless of the resistance distri-bution between the sites in question; indeed, from the vis-cocanalostomy mechanistic standpoint, both the extracel-lular matrix washout and inner wall endothelial defects areimportant.

It is surprising that these defects remained open forat least 1 to 2 months after surgery. In the living eye, de-fects in Schlemm canal endothelium that are larger thanthe physiologic pore size of 0.25 µm to approximately 2µm are occluded by platelet aggregation, a process thatoccurs within minutes.30 This process appeared to be in-effective, or at least significantly delayed, in our mate-rial, as only some pores were observed that were com-pletely or partially occluded by thrombocytes. Moreover,it is unclear whether the thrombocytes were effective insealing the endothelial defects, as numerous gold par-ticles were often seen associated with extracellular ma-trix components close to occluded pores, but not in im-mediately adjacent areas that were separated from thelumen of Schlemm canal by an intact endothelial layer,indicating that passage of fluid through the pores did stilloccur. A likely explanation for the persistence of endo-thelial defects in Schlemm canal could be a direct or in-direct action of sodium hyaluronate on thrombocyte ag-gregation. Indeed, we observed in some of the defectshomogeneous, granular, electron-dense material that wasnot observed in control eyes. Although we do not havedirect molecular proof, we assume that this material re-flects the presence of remaining sodium hyaluronate, sincesodium hyaluronate that was directly injected into theanterior chamber of a monkey in a parallel experimentshowed similar ultrastructural characteristics. After an-terior chamber injection, sodium hyaluronate was foundin the posterior chamber, on the inner surface of the pe-ripheral cornea, and on the inner uveal parts of the TM,but not in the juxtacanalicular region. This distributionindicates that in a structurally intact TM, sodium hyal-uronate is too viscous to enter the fluid pathways of thejuxtacanalicular region, which are considerably smallerthan those of the corneoscleral and uveal TM.10 Hyal-uronan has been shown to inhibit platelet adhesion andaggregation,31 and sodium hyaluronate might have simi-lar effects on the adhesion of platelets to Schlemm canalendothelium. Identification of the molecular processesby which sodium hyaluronate might prevent healing ofSchlemm canal defects could provide important infor-mation on how to increase the effectiveness of agents thatmay be used to disrupt the endothelial lining of Schlemmcanal to therapeutically decrease outflow resistance.

Reduction of IOP in viscocanalostomy putatively re-quires aqueous humor to percolate into an intrascleral res-ervoir (the so-called scleral lake) that is created by remov-ing the inner layers of the sclera. From there it is thoughtto enter the widened cut ends of Schlemm canal and/orcut ends of collector channels. As a scleral lake was cre-

ated during viscocanalostomy of our monkeys, such a fluidpathway may have existed for a time but was absent bythe time of our investigation 1 to 2 months after surgery.At the site of surgery, Schlemm canal was obliterated andopen ends were not observed. In addition, a larger openintrascleral reservoir next to Descemet membrane orSchlemm canal endothelium did not remain in any of themonkeys. There were, however, regions of hydrated scleranext to Schlemm canal and close to the site of surgery insome of the monkeys. These regions were filled with cellsexpressing the typical structural characteristics of scleralfibroblasts and loosely arranged collagen fibers, and mightrepresent healing stages of a former scleral lake, based onsurgical anatomy and postoperative ultrasound biomi-croscopy studies in humans. In one of the monkeys, therewas an intrascleral open space at a site considerably dis-tant from Schlemm canal, which was likely a remainingpart of the scleral lake created by removing scleral tissue.Thus, scleral spaces that were generated by removing theinner scleral layers were largely occluded 1 to 2 monthsafter surgery. However, the hydrated sclera indicates thatfluid was still moving through this region, which may con-stitute a low-resistance pathway for egress of aqueoushumor from the anterior chamber, and could also be func-tionally equivalent to a scleral lake. Ultrasound biomi-croscopy data on human patients indicate that clear sclerallakes may remain open until at least 7 to 9 months aftersurgery.32 This might indicate that scarring and/or endog-enous removal of sodium hyaluronate occurs at a muchfaster rate in monkeys than in humans. Reasons for thismight be species-specific differences or the presence of moreactive tissue repair mechanisms in monkeys vs human pa-tients with glaucoma. On the basis of our data, we cannotsay whether collector channels were cut open during sur-gery and whether they remained so despite the ongoinghealing process. Theoretically, an opening of collector chan-nels could have had effects on outflow facility, as about25% of outflow resistance appears to be localized distal toSchlemm canal, probably in the aqueous veins.33 We didnot see cyclodialysis in these animals despite step serialsectioning in 2 different laboratories, in all probability ex-cluding this as a possible factor for changes in outflow re-sistance in our experiments.

We did see discontinuities in the periphery of Desce-met membrane in the opercular area (an extension ofDescemet membrane covering the anterior TM, not pres-ent in humans), but these are normal for monkeys,34 werepresent in control eyes as well, and were distributed equallyaround the circumference. These are not related to the sur-gical procedure, and, in fact, we saw no evidence of iat-rogenic disruptions of Descemet membrane in any eye. Be-cause in both humans and normal monkeys aqueous humorcan be seen percolating through Descemet membrane dur-ing viscocanalostomy surgery, this implies either that mi-croperforations occur but were missed in our examina-tion because of their infrequency, or that Descemetmembrane and the endothelium at the corneal peripheryare in fact leaky in the absence of covering stroma. Thelatter seems especially likely in the monkey, given the nor-mal discontinuities in Descemet membrane. This could bepresent but more subtle in the human. Indeed, small fis-sures that are frequently associated with Hassall-Henle warts




have been described in Descemet membrane in human pe-ripheral cornea. These fissures are predominantly local-ized on the endothelial side of Descemet membrane andmay contain processes of endothelial cells or collagenfibrils.35,36 Some of these fissures have been observed topenetrate the entire thickness of Descemet membrane tothe corneal stroma.36

In summary, the most likely explanation for the de-crease in outflow resistance in monkeys after viscoca-nalostomy is the focal disruption of the inner wall en-dothelium and the opening of the juxtacanalicular orcribriform region of the TM. We hypothesize that a simi-lar effect is the major reason for the decrease in IOP inhuman patients after this type of glaucoma surgery. Op-ening of the outer TM had only modest effects on facil-ity in normal monkeys but might be very effective in hu-man patients, where the cribriform region is the tissuethat is most affected by the pathologic changes that oc-cur during primary open-angle glaucoma.37

Submitted for Publication: November 5, 2003; final re-vision received March 18, 2004; accepted May 14, 2004.Correspondence: Paul L. Kaufman, MD, Department ofOphthalmology and Visual Sciences, University of Wis-consin, 600 Highland Ave, Madison, WI 53792-3220.Funding/Support: This study was supported by grantEY02698 from the National Eye Institute, Bethesda, Md(Dr Kaufman); grant SFB 539 from the Deutsche For-schungsgemeinschaft, Bonn, Germany (Dr Tamm); Phar-macia Corp (acquired by Pfizer Inc, New York, NY); Re-search to Prevent Blindness Inc, New York (Drs Kaufmanand Albert); and the Ocular Physiology Research and Edu-cation Foundation, Madison, Wis (Dr Kaufman).Acknowledgment: We thank Karin Göhler, MTLA, forexcellent technical assistance and Marco Gößwein for hisexcellent processing of the electron micrographs.

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