The canine eye: the vessels of aqueous drainage.

Lumenal castings of canine vessels of aqueous drainage, prepared by microvascular injection of room-temperature vulcanizing silicone or Batson's methacrylate corrosion compound No. 17, were viewed with a stereo dissecting microscope and by scanning electron microscopy. The pathways for aqueous drainage in the dog appear to extend radially from the interstices of the filtering meshwork, through the deep intrascleral venous plexus, to the midscleral venous circle of Hovius, which also serves choroidal venous drainage. Venous blood mixed with aqueous humor may egress the globe from the venous plexus of Hovius via the anterior ciliary vein or posterior vortex veins. The canine pathways of aqueous drainage are distinguished from those in the primate by the absence of Schlemm's canal and the intrascleral mergence of drainage routes for aqueous humor and uveal venous blood.

The canine eye: lens depression and aqueous outflow.

The effects on facility of outflow of lens depression (controlled mechanical retrodisplacement of the crystalline lens to simulate cyclotonia in excised eye preparations), prolonged perfusion, and intracameral hyaluronidase were evaluated in 16 pairs of freshly excised canine eyes. Lens depression increased facility of aqueous outflow by an amount greater than could be attributed to mechanical or temporal disruption of the hyaluronidase-sensitive outflow barrier and appears to act, at least in part, on the hyaluronidase-resistant portion of the outflow barrier. These data suggest that in addition to accelerating the washout effect, mechanical tension on the iridocorneal angle in the dog increases facility of aqueous outflow in a manner similar to the direct trabecular component of cyclotonic-induced facility increases observed in primate eyes.

Anatomic correlates of changing aqueous outflow facility in excised human eyes.

Five pairs of excised human eyes were examined for anatomic correlates of changes in aqueous outflow facility that result from changing the intraocular pressure (IOP) and from placing mechanical tension on the irido-corneal angle (lens depression). An increase in IOP from 0 to 40 mm Hg tended inconsistently to compress trabecular meshwork as a whole and to distend the juxtacanalicular tissue. The most constant and significant effect of increasing IOP, however, was to compress Schlemm's canal, thereby progressively diminishing its volume to its virtual collapse at 40 mm Hg IOP. Lens depression increased Schlemm's canal volume and partially prevented its collapse at high levels of IOP. The mean frequency of endothelial vacuoles was remarkably constant at IOPs from 2.5 to 40.0 mm Hg, with and without lens depression, but was significantly lower when the IOP was reduced to 0 mm Hg. When correlated with existing physiologic data, these anatomic findings suggest that Schlemm's canal collapse, but not vacuole frequency, is an important contributor to pressure-dependent changes in aqueous outflow facility.

Trabeculotomy in the immature, enucleated human eye.

Greater than 50 per cent of resistance to aqueous outflow was eliminated by internal trabeculotomy covering 30 degrees of the limbal circumference in enucleated, postmorten infant eyes subjected to constant-pressure acqueous perfusion. The proportion of total outflow resistance eliminated after limited trabeculotomy was greater in infant eyes than that observed in eyes of older children andthan that which others have observed in adult eyes. These data suggest that in the infant eye aqueous humor may flow circumferentially in Schlemm's canal but that the capacity for circumferential flow diminishes with increasing age.

The canine eye: in vitro dissolution of the barriers to aqueous outflow.

Facility of aqueous outflow and time-dependent changes in its hyaluronidase-sensitive and hyaluronidase-resistant components were evaluated in freshly excised canine eyes by constant pressure quantitative aqueous perfusion. Mean baseline facility of outflow was 0.24 microliter/min/mm Hg. With prolonged perfusion at constant intraocular pressure, facility of outflow was observed to increase almost linearly for at least 3 hr and continued to increase for up to 10 hr, reaching a maximum several times the initially measured facility. Perfusion with pooled dog aqueous humor did not prevent the time-dependent increase in measured facility. Rapid exchange of anterior chamber contents with perfusion solution alone produced an immediate threefold increase in facility, again followed by a gradual time-dependent facility increase. Rapid exchange of anterior chamber contents with hyaluronidase produced an immediate fivefold increase in facility with stabilization of measured facility over 3 hr and subsequent perfusion. The time-dependent changes in measured facility of outflow or "washout phenomenon" appeared to result from the gradual dissolution of the hyaluronidase-sensitive component of the barriers to aqueous outflow in the canine eye.

The canine eye: in vitro studies of the intraocular pressure and facility of aqueous outflow.

Possible interdependence of facility of aqueous outflow and intraocular pressure was examined by quantitative aqueous perfusion at four levels of intraocular pressure in 69 canine eyes, freshly excised post-mortem. Statistically similar, time-dependent increases in facility of outflow occurred over steps of increasing and decreasing, and at constant intraocular pressue. Pretreatment with intracameral hyaluronidase diminished the rate of facility increase, reducing it to 20% or less in one third of the eyes so perfused. Observed increases in canine facility of outflow with changing intraocular pressure appeared to result primarily from "washout" of the hyaluronidase-sensitive component of the barriers to aqueous outflow and not from direct effects of the intraocular pressure.

The canine eye: pectinate ligaments and aqueous outflow resistance.

The contribution of the pectinate ligaments to total aqueous outflow resistance was studied in 30 excised canine eye pairs by means of constant-pressure aqueous perfusion. Incision of these ligaments over one and two quadrants of the angle circumference increased the facility of aqueous outflow significantly more than could be attributed to normal "washout" alone. Neither mechanical nor enzymatic disruption of angle glycosaminoglycans could fully account for this phenomenon. The pectinate ligaments may therefore contribute to the canine aqueous outflow barrier by compartmentalizing the glycosaminoglycans in the spaces of Fontana. In addition, these ligaments, analogous to iris processes in the human, prevent widening of the canine angle and hold the filtration structures in a relatively compressed state, a situation that can be reversed by ligament incision.

Regional microvascular anatomy of the rabbit ciliary body.

Vascular luminal castings of rabbit eyes were microdissected and studied with the scanning electron microscope to delineate the anatomy of the ciliary body microvasculature. We found regional microvascular differences corresponding to well known gross topographical and ultrastructural differences that may indicate regional functional specialization. The major arterial circle of the iris, derived solely from the long posterior ciliary arteries, supplies the ciliary body via two types of arterioles: anterior and posterior. Spiral iridial process capillaries arise from the anterior (iris) arterioles, are radially arranged along the back of the iris and drain directly into the iris veins. Arterioles from the posterior (ciliary) arterioles enter the head of the process, supplying its tortuous capillaries, some of which drain back into the iris veins. Other capillaries turn posteriorly to form relatively straight, parallel capillaries within the process leaf that drain into the choroidal system via marginal process veins. More posterior arterioles supply capillaries to the base of major processes, to the interprocess ciliary web, and to minor processes. The presence of a dual arteriolar supply to the ciliary processes has also been found in primates and suggests that the rabbit may represent a suitable animal model for the study of factors governing regional ciliary process perfusion.

Comparative microvascular anatomy of mammalian ciliary processes.

Methylmethacrylate lumenal castings of the ciliary body microvasculature were prepared from eight mammalian species and studied with the scanning electron microscope. In all of these species, the ciliary body is supplied by the major arterial circle, which originates solely from the long posterior ciliary arteries without contribution from the anterior ciliary circulation. In contrast to primates and rabbits, the ciliary processes of the eight species we studied are supplied by only one type of arteriole, which travels posteriorly from the major arterial circle to the iris root, where it gives rise to ciliary process arterioles. Using precise microdissection techniques, we found marked interspecies variations in ciliary process angioarchitecture among the mammalian eyes examined. Rodents (rat and guinea pig) demonstrated several interesting similarities to primates, with extensive interprocess connections and irregularly dilated, concentrically parallel capillaries traveling posteriorly to empty into the choroidal veins. In addition, rat ciliary process arterioles displayed marked focal constrictions suggestive of precapillary "sphincter" agonal activity. The carnivore ciliary process (cat and dog) is supplied by a single arteriole traveling posteriorly throughout its length and sending capillary arcades to its margin from where they drain outward into venous sinuses at the base of the process. Ungulate processes (sheep, goat, pig and cow) receive blood from multiple arterioles that occupy the process core along with veins that empty into the choroidal circulation. These vessels serve delicate capillaries lying on the sides, margin and head of each process. The anatomic variations described here should be considered in the design and interpretation of physiologic and immunohistochemical studies of ciliary body vascular perfusion in non-primate animal models.

Human trabecular meshwork organ culture: morphology and glycosaminoglycan synthesis.

Human corneoscleral explants were maintained for several weeks in defined, serum-free media. Trabecular cell vitality, as judged by vital stain exclusion, is high for at least one month. Trabecular ultrastructure, as compared to that of fresh eyes, first shows minor cellular and extracellular matrix degradation after 3 weeks in culture. The biosynthetic profiles of trabecular glycosaminoglycans (GAGs) change significantly by 3 weeks in culture. Eyes that are stored at 5 degrees C for up to 48 hr postmortem exhibit changes in trabecular ultrastructure and in GAG profiles; both characteristics return to normal by 7 days in culture. The incorporation pattern of 35S-sulfate and 3H-glucosamine into the GAGs of the trabecular meshwork (TM) is distinct from corneal or scleral incorporation. The relative incorporation of 3H-glucosamine into trabecular GAGs, as determined by sequential enzymatic degradation, is: 22.3% hyaluronic acid (HA), 27.9% chondroitin sulfate (CS), 21.3% dermatan sulfate (DS), 5.9% keratan sulfate (KS), 17.7% heparan sulfate (HS) and 4.9% unidentified material. The relative incorporation of 35S-sulfate into trabecular GAGs is: 0% HA, 32.9% CS, 34.8% DS, 7.7% KS, 13.8% HS and 11.1% into unidentified material. This profile is in good agreement with the profile that was previously obtained for human and nonhuman primate meshworks prior to culture. We conclude that corneoscleral explant organ culture is a useful tool for extracellular matrix studies within a time window from 7 to at least 14 days in culture.

Optic nerve head microvasculature of the rabbit eye.

Vascular luminal castings of rabbit eyes were microdissected and studied with scanning electron microscopy to elucidate the three-dimensional angioarchitecture of the optic nerve head. Using sequential microdissection, an incomplete arterial circle was identified as terminal branches of two to three short posterior ciliary arteries around the optic nerve head. Several recurrent branches from the arterial circle form a pial arterial network. This pial system supplies the optic nerve head microvasculature and receives numerous venules from them. The only large vessel to enter the optic nerve is a central retinal artery that has few branches within the optic nerve and provides several branches at the surface of the optic disc. Moderately numerous vessels connect the retinal and ciliary vascular layers within the optic nerve head. Few arterioles to the optic nerve head arise from the choroid; however, there are a small number of capillary and numerous venous connections between them. These results indicate that the principal blood supply of the rabbit optic nerve head is derived from the short posterior ciliary arteries by the arterial circle. The retinal arteries contribute to the surface vasculature of the optic nerve head. The pial system also plays a significant role in both supply and drainage of the rabbit optic nerve head.

An endothelin-1 induced model of optic nerve ischemia in the rabbit.

PURPOSE: To evaluate blood flow reduction and topographic optic nerve changes after the local administration of endothelin-1 in vivo, delivered to the perineural region of the anterior optic nerve in the rabbit. METHODS: Endothelin-1 (five rabbits) in a dosage of 0.1 microgram/day or balanced salt solution (two rabbits) was delivered to the perineural region of the anterior optic nerve with osmotically driven minipumps. Optic nerve blood flow was determined by the colored microspheres technique after 14 days of local endothelin-1 or balanced salt solution administration to the microvasculature of the optic nerve. In addition, optic nerve blood flow was determined in two rabbits that had no minipump implants. The morphologic changes induced by reduction of blood flow were assessed in five additional rabbits implanted with osmotically driven minipumps containing endothelin-1 (0.1 microgram/day). These rabbits were observed for 8 weeks, and the morphologic optic nerve changes were monitored with a confocal scanning laser ophthalmoscope. RESULTS: Independent of intraocular pressure, endothelin-1 induced a decrease in blood flow of approximately 38% in the experimental eye, compared to the decrease induced by balanced salt solution or to the decrease in rabbits without minipumps (analysis of covariance, P = 0.0092). Multivariate statistical analysis showed a significant change in topometric parameters (cup area, cup depth, rim volume) obtained with a confocal scanning laser ophthalmoscope, indicating an increase in optic nerve cupping and a decrease of the perineural rim volume in the experimental eyes (P = 0.017). CONCLUSIONS: The current results suggest that morphologic optic nerve alterations can be induced experimentally in the rabbit model after ischemia produced by the local administration of endothelin-1 to the perineural region of the anterior optic nerve.

Expression of matrix metalloproteinases and inhibitor by human trabecular meshwork.

Extracellular matrix (ECM) turnover and remodeling are initiated, at least in part, by the regulated secretion of members of a family of matrix metalloproteinases. Human and bovine trabecular mesh-work in culture secrete interstitial collagenase, both the 72- and the 92-kD forms of type IV collagenase (gelatinases) and stromelysin, and the tissue inhibitor of metalloproteinases (TIMP). These proteinases and TIMP were identified by immunoblotting western transfers from sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using several specific antiprotein and antipeptide polyclonal antibodies. Gelatinase and stromelysin enzymatic activities were also analyzed by substrate SDS-PAGE, in which proteinase substrates were polymerized into the gels before electrophoresis to allow subsequent activity assays. These matrix metalloproteinases and TIMP are secreted at low basal levels into trabecular culture medium; their secretion levels are increased several-fold by treatment of the cultures with the phorbol mitogen. 12-O-tetradecanoylphorbol-13-acetate (TPA). Characteristics of the trabecular matrix metalloproteinases and TIMP are similar to those secreted by numerous other tissues, including the retinal pigment epithelium. These proteinases may serve an important role in the maintenance and regulation of the trabecular extracellular matrix and, subsequently, of the aqueous humor outflow pathway in normal and glaucomatous eyes.

Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye.

The glycosaminoglycans (GAGs) extractable from the trabecular meshworks (TM) of human and non-human primate eye have been analyzed by sequential enzymatic degradation and cellulose acetate electrophoresis. For comparison, similar extracts of the cornea, sclera, iris, and ciliary body have also been analyzed. The distribution of glycosaminoglycans in human and in cynomolgus monkey TMs are similar, although not identical. The human TM contains hyaluronic acid (HA), chondroitin-4-sulfate and/or 6-sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), and an unidentified band of Alcian Blue staining material, which is resistant to the enzymes that we used. Based upon quantitation of the Alcian Blue staining intensities of extracted GAGs, which have been corrected by a relative dye-binding factor, the GAGs of the human TM include: 29.0% HA, 14.1% CS, 21.5% DS, 20.3% KS, and 15.0% HS. The cynomolgus monkey trabecular GAGs include: 12.8% HA, 14.3% CS, 15.2% DS, 42.1% KS, and 15.6% HS.

Laser trabeculoplasty induces stromelysin expression by trabecular juxtacanalicular cells.

PURPOSE: The mechanism by which laser trabeculoplasty reduces elevated intraocular pressure in primary open-angle glaucoma has been established. To test the hypothesis that trabecular extracellular matrix turnover is involved, stromelysin expression after laser treatment of anterior segment organ cultures was evaluated. METHODS: Argon laser trabeculoplasty, using typical clinical treatment parameters, was applied to anterior segment organ cultures. Stromelysin levels and activity were then evaluated at various times by immunoblots of Western transfers and by zymography. Stromelysin mRNA levels were evaluated by dot blot and by reverse transcription, followed by polymerase chain reaction amplification. Stromelysin protein was localized by immunohistochemistry, and image analysis was used for quantitation. Stromelysin mRNA was localized by in situ hybridization. RESULTS: Trabecular stromelysin protein, activity, and mRNA levels were detectably elevated by 8 hours and were several-fold higher by 24 hours after treatment. Stromelysin immunostaining was elevated dramatically in the juxtacanalicular and insert regions of the meshwork, but only modestly in other regions. Stromelysin mRNA increases also were localized primarily to these regions. The juxtacanalicular stromelysin immunostaining increase was sustained for at least 1 week, whereas the insert levels declined somewhat after day 2. CONCLUSIONS: A stromelysin increase, localized primarily to the juxtacanalicular region of the meshwork, the putative site of the aqueous humor outflow resistance, should degrade trabecular proteoglycans, the putative outflow resistance source, and allow their uptake and further degradation by the juxtacanalicular cells. If diminished juxtacanalicular extracellular matrix turnover is responsible for the glaucomatous reduction in aqueous humor outflow, an increase in stromelysin in this specific area of the meshwork should ameliorate the problem. Thus, the observations support the working hypothesis and may explain the efficacy of this treatment for glaucoma.

Betaxolol and timolol. A comparison of efficacy and side effects.

Betaxolol hydrochloride, 0.5%, and timolol maleate, 0.5%, were compared in a 26-week randomized, double-masked study involving 46 patients with primary open-angle glaucoma. The two drugs were comparable with regard to both intraocular pressure-lowering efficacy and side effects. Since betaxolol is a selective beta 1-adrenergic antagonist, it may be advantageous for patients in whom beta 2-blockade could be harmful.

Exercise training reduces intraocular pressure among subjects suspected of having glaucoma.

The effect of exercise conditioning on elevated intraocular pressure has not been previously described among sedentary individuals. We prospectively observed intraocular pressure for nine sedentary subjects suspected of having glaucoma before and after 3 months of aerobic exercise training. Mean (+/- SEM) aerobic capacity, as assessed by maximal oxygen uptake, increased 6.3 +/- 1.6 mL.kg-1.min-1 (30%) (P less than .02). Mean intraocular pressure decreased 4.6 +/- 0.4 mmHg (20%) (P less than .001) at the end of the conditioning period. With cessation of exercise and subsequent detraining, intraocular pressure returned to elevated preconditioning levels by 3 weeks. Regular aerobic exercise is associated with a reduction in elevated intraocular pressure and may represent an effective nonpharmacologic intervention for patients suspected of having glaucoma.

Trabecular cell division after argon laser trabeculoplasty.

The usefulness of argon laser trabeculoplasty (ALT) in the treatment of some patients with glaucoma has been established; yet, the mechanism of ALT is not understood. The hypothesis that the mechanism involves trabecular cell division was examined. Autoradiographic assessment of tritiated thymidine incorporation into trabecular cell DNA in a human corneoscleral explant organ culture system demonstrated a basal level of DNA replication that increased by 180% in the two days immediately after ALT. When labeling was initiated five days after ALT, DNA replication declined to 70% of the control level. The autoradiographic findings were confirmed using biochemical evaluation of tritiated thymidine incorporation into acid-precipitable macromolecules (DNA) in excised trabecular meshwork. One biologic response of the trabecular meshwork after laser trabeculoplasty is a change in the level of ongoing trabecular cell division.

Sources of variability of topometric data with a scanning laser ophthalmoscope.

OBJECTIVE: To determine in a two-part study whether misalignment between the patient and the laser scanner is a major source of variability with the Heidelberg Retina Tomograph (Heidelberg [Germany] Engineering). METHODS: Three topographic images of the right optic nerve were acquired with the Heidelberg Retina Tomograph in eight patients with glaucoma. The correlations between average cup volume, variability of cup volume estimates, and direction of imaging were evaluated. Furthermore, the correlations between average rim volume, variability of rim volume estimates, and direction of imaging were evaluated. Next, the optic nerve cup volume and rim volume estimates of a rabbit's left eye were compared between three series of five topographic images acquired from three slightly different directions. RESULTS: Average cup volume, variability of cup volume estimates, and variability in the direction of imaging correlated significantly among the patients with glaucoma (multiple R2 = .95; P < .001). Average rim volume, variability of rim volume estimates, variability in the direction of imaging, and variability in the mean height of the contour line also correlated significantly (multiple R2 = .88; P = .03). In the rabbit eye, the cup volume and the rim volume differed significantly among the three image series (analysis of variance, P < .001 and P = .04, respectively). CONCLUSION: Misalignment between the patient and the laser scanner may account for significant variability with the Heidelberg Retina Tomograph.

The efficacy of apraclonidine as an adjunct to timolol therapy. Apraclonidine Adjunctive Therapy Study Group.

OBJECTIVE: To compare the intraocular pressure (IOP) lowering efficacy of 0.5% and 1.0% apraclonidine hydrochloride when used adjunctively with 0.5% timolol maleate in 129 patients. DESIGN: A multicenter, randomized, double-masked clinical trial. Adult patients of either sex diagnosed as having either open-angle glaucoma or ocular hypertension were enrolled in the study. Patients using only 0.5% timolol maleate twice daily for at least 4 weeks and who had 8 AM IOPs of at least 22 mm Hg and no greater than 30 mm Hg 12 hours after dosing were eligible for the study. After 8 AM baseline IOPs were obtained while patients were taking timolol only, they were then randomized to receive either 0.5% or 1.0% apraclonidine twice daily in addition to their timolol. Intraocular pressures were measured at 8 AM (before morning dosing) and at 11 AM (3 hours after dosing) on days 14 and 90 and at 8 AM only on day 45. RESULTS: Both concentrations of apraclonidine produced significant IOP reductions from baseline at all visits (P < .001). At 8 AM, after the nighttime dose, the additional mean IOP reduction from the timolol baseline ranged from 2.5 to 3.3 mm Hg (10.3% to 13.6% reduction, respectively). At 11 AM, 3 hours after the morning dose, the additional IOP reduction from the timolol baseline ranged from 4.7 to 5.2 mm Hg (20.0% to 21.7%, respectively). No difference in IOP reduction was observed between the 0.5% and 1.0% apraclonidine concentrations and no loss of IOP efficacy was observed for either concentration for the duration of the study. Sensitivity to 0.5% and 1.0% apraclonidine was observed in nine (13.8%) and 13 (20.3%) patients, respectively. Overall, therapy was discontinued owing to ocular or nonocular side effects with 0.5% and 1.0% apraclonidine in 14 (21.5%) and 16 (25%) patients, respectively. CONCLUSIONS: We believe that 0.5% apraclonidine is equally effective as 1.0% apraclonidine when used twice daily as the first adjunctive drug to timolol. The drug effect is maintained for at least 90 days.