Tissue plasminogen activator reduces the elevated intraocular pressure induced by prednisolone in sheep.

We have previously shown that tissue plasminogen activator (tPA) injected in the vitreous of sheep, reduced or prevented the elevation of the intraocular pressure (IOP) normally produced by the instillation of 1% prednisolone. We now report the effect of tPA when injected into the anterior chamber (AC) in amounts of 0.01, 0.001 and 0.0001 µg diluted in a volume of 50 µL. Lyophilized tPA, obtained as Actilyse 50 mg from Boehringer Ingelheim containing arginine was utilized. The Actilyse was diluted in balanced salt solution to obtain the desired amount of tPA in 50 µL. An identical solution containing only arginine was prepared to inject into the contralateral eye as a control. Six sheep of the Corriedale breed were selected. At the beginning of the study all eyes received instillation of 1% prednisolone 3 times/day for 10 days to elevate their IOP from 10 mm Hg to about 23 mm Hg. Then, 0.0001 µg was injected into one of the eyes and its effect was followed for up to 55:00 h while the instillation of prednisolone continued in both eyes. The same protocol was implemented for the 0.001 and 0.01 µg amounts after extended washout and IOP was over 22 mm Hg. The injection of 0.0001 µg into the AC had no effect on an IOP of 23.0 mm Hg at 6:00 and 30:00 h after injection. 0.001 µg of tPA reduced IOP from 23.1 to 18.6 mm Hg at 6:00 h but IOP recovered to 22.3 mm Hg at 30:00 h. Injection of 0.01 µg produced a marked and prolonged reduction of IOP. From a baseline of 23.0, IOP was reduced to 14.0, 14.7, 21.2, and 20.9 mm Hg at 5.0, 23.0, 27.0 and 45.5 h, respectively. The 0.423 µg of arginine, which is associated with 0.01 µg tPA, was injected alone and had no effect. Recombinant human tPA injected in the AC is effective in reversing steroid-induced IOP elevation in sheep. The reduction of IOP elevation may be the result of an effect on extra-cellular matrix turnover in the TM. These findings suggest that tPA may by useful as a therapeutic agent in steroid-induced glaucomas.

Sildenafil stimulates aqueous humor turnover in rabbits.

Sildenafil citrate increases ocular blood flow and accelerates the rate of anterior chamber refilling after paracentesis. The latter effect could have resulted from a reduction in outflow facility or from an increase in aqueous humor (AH) production. In this study, we used scanning ocular fluorophotometry to examine the effects of sildenafil on AH turnover, and thus, AH production in eyes of live normal rabbits. For this, the rate of aqueous humor flow (AHF) was quantified with a commercially available fluorophotometer that measured the rate of fluorescein clearance from the anterior segment, which predominantly occurs via the trabecular meshwork. After ≈2 h of control scans to determine the baseline rate of AHF, the rabbits were fed 33 mg of sildenafil and allowed ≈45 min for the drug to enter the systemic circulation. Thereafter, fluorescence scans were retaken for an additional 90-120 min. Sildenafil ingestion increased AHF by about 36%, from 2.31 µL/min to 3.14 µL/min (P < 0.001, as two-tailed paired data, n = 20 eyes). This observation indicates that sildenafil citrate, which is a phosphodiesterase type-5 inhibitor currently marketed as a vasodilator (e.g., Viagra, Revatio), stimulates AHF in rabbits. Our results seem consistent with reports indicating that the drug dilates intraocular arteries and augments intraocular vascular flow. These physiological responses to the agent apparently led to increased fluid entry into the anterior chamber. As such, the drug might have utility in patients with ocular hypotony resulting from insufficient AH formation.

Interaction between mechanical and osmotic forces in the isolated rabbit lens.

Based on our previous work showing that cow and rabbit lenses isolated with their accommodation anatomical components intact change volume during simulated accommodation in vitro, and that hyposmolality and hyperosmolality also produce volume changes, we tested the idea that exerting these forces simultaneously may add or counteract each other. Further, we attempted to find a point at which osmotic and mechanical forces may cancel each other. Using previously described methodology, we found that combined stretching and anisotonic conditions applied to a lens always produced less of a volume change than that observed on its paired lens from the fellow eye that was only subjected to anisotonic conditions. Our results suggest that a stretching force that increases the equatorial diameter by 0.4% and reduces the lens volume by 1.8% could be canceled by a hyposmotic force of about -20 to -30 mOsM. Counter-intuitively, lenses that were subjected to stretching and hyperosmolality had less volume decrease than their paired lenses only exposed to hypertonicity. This latter observation is likely due to the prevention by the mechanical stretching forces of the shortening of the equatorial diameter, which normally occurs in hypertonic media.

Effect of sildenafil citrate on intraocular pressure and blood pressure in human volunteers.

Anecdotal reports have suggested that the vasodilator, sildenafil citrate, which evokes its effect via a select inhibition of PDE5, has the potential to increase intraocular pressure (IOP) in some individuals. An ocular hypertensive effect by sildenafil was also recently described in a sheep animal model. In contrast, clinical studies have not found a direct association between sildenafil ingestion (commonly consumed as Viagra) and changes in IOP. However, some such studies also reported no effects of sildenafil on systemic blood pressure (BP) at the time of the IOP determination. Given this surprising result, our purpose was to repeat a study in human volunteers in the city of Corrientes, Argentina to corroborate the effects of sildenafil on human IOP and systemic BP. For the present study, 9 healthy volunteers (male and female, 18-74 years old) were selected as subjects after ophthalmic and cardiovascular evaluation indicated that they exhibited normal parameters for their age. In a masked, placebo-controlled study, the subjects ingested 100 mg sildenafil citrate (provided as Vorst from Laboratorios Bernabo, Argentina) in one session, and a placebo on a second separate occasion. IOP was measured with a Goldman applanation tonometer by an ophthalmologist, and BP by a second physician, neither of whom witnessed the tablet ingestion by the volunteers, nor provided with information on the nature of the test compounds. A third individual administered the tablets. The average baseline IOP of this group of 9 was 13.1 ± 0.6 mm Hg. Subsequent to sildenafil ingestion, IOP increased by 26% to 16.5 ± 0.8 mm Hg 60 min later (P < 0.005, as paired data), and returned to control values within 2 h. Both systolic and diastolic BP were significantly reduced by sildenafil ingestion. At the point of maximal systemic hypotension (90 min), the systolic and diastolic pressures declined by 15% and 13%, respectively. No significant changes in IOP or BP were recorded after ingestion of the placebo. Our results suggest that sildenafil can elicit a transient IOP increase that may be of importance to patients chronically treated with PDE5 inhibitors for various vascular diseases (e.g., pulmonary hypertension). We discuss possible mechanisms by which PDE5 inhibition might lead to a rise in IOP.

Changes in rabbit and cow lens shape and volume upon imposition of anisotonic conditions.

In vivo, mammalian lenses have the capacity to effect fully reversible changes in shape, and possibly volume, during the accommodation process. Isolated lenses also change shape by readily swelling or shrinking when placed in anisotonic media. However, the manner by which the lens changes its shape when its volume is changed osmotically is not firmly established. Putatively, the lens could swell or shrink evenly in all directions, or manifest distinctive swelling and/or shrinking patterns when exposed to anisotonic media. The present study measured physical changes in lenses consistent with the latter alternative using methods we developed for determining rapid changes in lens shape and volume. It was found in isolated rabbit and cow lenses that the length of the axis between the anterior and posterior poles (A-P length) primarily increases under hypotonic conditions (-40 to -100 mOsM), with smaller, or no changes, in equatorial diameter (ED). Hypertonic conditions (+50 to +100 mOsM) on rabbit lenses elicited a predominant reduction in ED, while the A-P length was only marginally reduced. Hypertonic solutions of +150 mOsM were required to obtain similar changes in cow lens shape. The ratio of the A-P length to the ED was taken as a measure of "circularity". This ratio increased gradually in rabbit and cow lenses bathed in hypotonic solutions because of the increase in the A-P length. The calculated lens volume increased in tandem with the increase in "circularity". Lens circularity also increased under hypertonic conditions due to the decrease in ED, but this increase in circularity during shrinkage was not as pronounced as that which occurred during swelling. As such, the lens has a tendency upon swelling to change its shape by approaching the structure of a globular spheroid (as occurs during accommodation for near focusing), but lens shrinkage does not result in a flatter lens with a reduced A-P length as occurs during dis-accommodation for distance focusing. Moreover, osmotically evoked shape changes appear irreversible, in contrast to the mechanically elicited shape changes of accommodation.

Fluid transport phenomena in ocular epithelia.

This article discusses three largely unrecognized aspects related to fluid movement in ocular tissues; namely, (a) the dynamic changes in water permeability observed in corneal and conjunctival epithelia under anisotonic conditions, (b) the indications that the fluid transport rate exhibited by the ciliary epithelium is insufficient to explain aqueous humor production, and (c) the evidence for fluid movement into and out of the lens during accommodation. We have studied each of these subjects in recent years and present an evaluation of our data within the context of the results of others who have also worked on electrolyte and fluid transport in ocular tissues. We propose that (1) the corneal and conjunctival epithelia, with apical aspects naturally exposed to variable tonicities, are capable of regulating their water permeabilities as part of the cell-volume regulatory process, (2) fluid may directly enter the anterior chamber of the eye across the anterior surface of the iris, thereby representing an additional entry pathway for aqueous humor production, and (3) changes in lens volume occur during accommodation, and such changes are best explained by a net influx and efflux of fluid.

Fluid transport across the isolated porcine ciliary epithelium.

PURPOSE: To quantify spontaneous fluid transport across the isolated porcine ciliary epithelium and determine its sensitivity to the electrolyte transport inhibitors ouabain and bumetanide, as well as bath Cl(-) and HCO(3)(-) levels. METHODS: A complete annulus of ciliary body was mounted in a custom-designed chamber appropriate for quantifying net fluid movement, as well as the transepithelial potential difference (PD) across the in vitro ciliary epithelium. RESULTS: A spontaneous and stable fluid flow (FF) in the blood-to-aqueous direction was measured over a 4-hour period. This flux solely reflected the secretory activity of the isolated ciliary epithelium (CE), given the absence of externally applied osmotic or pressure gradients. In contrast to FF, the PD declined during the 4 hours in vitro, suggesting that the integrity of the tight junctions may have been compromised during this time so that an increased movement of counter ions via the paracellular pathway could have shunted the PD, while at the same time transcellular fluid transport remained unaffected. The FF in the blood-to-aqueous direction (2.3 +/- 0.2 muL/hr; n = 7) was eliminated by a unilateral reduction in the bath Cl(-) levels on the blood side of the preparation and restored on reintroducing the anion to the bathing medium. This linkage between FF and blood side [Cl(-)] is consistent with the existence of a net Cl(-) flux across the porcine CE in the same direction as the fluid transport. Addition of bumetanide to the blood-side bath inhibited FF by approximately 40%, whereas the removal of CO(2)/HCO(3)(-) from the blood-side bathing solution elicited a approximately 50% reduction in FF. Ouabain inhibited the FF from either side of the preparation, although the effects were more rapid when the glycoside was applied to the blood side of the tissue. Overall, these findings indicate the dependence of FF on active ionic transport by the isolated CE. CONCLUSIONS: Isolated porcine ciliary epithelial preparations transport fluid in the blood-to-aqueous direction, indicating that measurements of volumetric fluid flow across this preparation may serve as a suitable model for future studies directed toward the pharmacological control of secretion.

Spontaneous fluid transport across isolated rabbit and bovine ciliary body preparations.

PURPOSE: To quantify spontaneous fluid transport across the isolated ciliary bodies of rabbit and bovine and to determine their osmotic permeabilities. METHODS: A complete annulus of ciliary body was mounted in a custom-designed chamber appropriate for detecting net fluid movement across the in vitro preparation. RESULTS: A net fluid flow in the blood-to-aqueous direction was measured. It was generally observed that tissue freshness is a critical parameter for detection of such flow. The spontaneous, baseline fluid transport rate lasted, on average, approximately 4 hours. This flow solely reflects the secretory activity of the isolated ciliary epithelium, since the in vitro arrangement precludes contributions from ultrafiltration. Both the isolated rabbit and bovine ciliary body epithelia transported fluid in the absence of an external osmotic or pressure gradient. After corrections for area and possible collapse of the processes, a total flux rate of approximately 23 microL/hour or 13% of the in vivo flow in rabbit was estimated. This value agrees with predictions of ionic fluxes and short-circuit current measurements, which are also obtained in vitro. The fluid flow is bicarbonate dependent in rabbit and chloride dependent in bovine, consistent with ionic transport mechanisms described in these species. Ouabain inhibited the fluid flow across both species, indicating dependence on active ionic transport. Irrespective of the spontaneous fluid transport, a flow elicited by an osmotic gradient allowed for a calculation of the osmotic permeability coefficient (P(f); approximately 10(-3) cm/s) in line with reports in other epithelia. In addition, mannitol permeability (5.6 x 10(-6) cm/sec) was similar to that measured in "tight" epithelia, as determined by measurements of radiolabeled fluxes of the sugar across rabbit ciliary bodies mounted in the chambers used for the present fluid transport study. CONCLUSIONS: This work demonstrates that isolated ciliary epithelial preparations transport fluid in the blood-to-aqueous direction. The present observations suggest that mounting arrangements for measuring volumetric fluid flow across the ciliary epithelium is suitable for future studies directed toward the pharmacological control of secretion.

Chloride channel gene expression in the rabbit cornea.

PURPOSE: The maintenance of stromal hydration by the corneal endothelium relies on active transendothelial anion transport, with bicarbonate and chloride the major anions carrying the current. However, the ion transport pathways that operate to maintain stromal hydration have yet to be fully elucidated. METHODS: We used RT-PCR to identify the gene expression profile of members of the ClC family of chloride channels in freshly isolated samples of rabbit corneal endothelium, stroma, and epithelium. The expression of a separate group of genes was also examined to confirm the purity of the sample collection protocol. The expression of the ClC-2 and ClC-3 channel protein in the cornea was also evaluated by light and electron microscopic immunolabelling. RESULTS: The mRNA for ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 were expressed in both the corneal epithelium and endothelium, and in the stroma. The mRNA for the skeletal muscle specific channel ClC-1 and the kidney specific chloride channel ClC-Ka were not detectable. ClC-4 mRNA was not detected in any rabbit tissue examined. The expression pattern of the mRNAs for collagens V, VI, VII, and VIII demonstrated the absence of contamination in epithelial and endothelial samples. ClC-2 and ClC-3 immunolabelling confirmed the presence of these proteins in corneal endothelium, stroma, and epithelium. CONCLUSIONS: Together with cystic fibrosis transmembrane conductance regulator (CFTR) and calcium activated chloride channel-1 (CLCA1), these results bring the number of chloride channel genes known to be expressed in the corneal endothelium and epithelium to seven. These channels are likely to be important for the maintenance of corneal transparency.

Steroid-induced ocular hypertension in normal cattle.

OBJECTIVES: To determine whether the bovine eye develops elevation of intraocular pressure (IOP) in response to topical corticosteroid use and to develop a reliable model of steroid-induced elevation of IOP in an animal. METHODS: Intraocular pressure was monitored by Perkins applanation tonometry in a group of 12 cows receiving topically administered prednisolone acetate in 1 eye 3 times a day for a period of 49 days after the establishment of baseline IOP values. Perkins readings were converted to IOP in mm Hg using calibration curves derived from in vitro cannulation manometric experiments and validated with in vivo manometric measurements. Intraocular pressure was also monitored for 50 days after the discontinuation of corticosteroid therapy. RESULTS: Intraocular pressure began to increase after 3 weeks of treatment in 100% of the cow eyes receiving corticosteroid and reached a peak 1 week later. Peak interocular IOP differences between the corticosteroid-treated eye and the fellow control eye reached up to 15 mm Hg and began to decline after the discontinuation of treatment but remained significantly elevated for a period of 3 more weeks. CONCLUSIONS: Bovine eyes exhibit a robust steroid-induced ocular hypertensive response, with 100% occurrence in this trial. The IOP elevation caused by corticosteroid slowly subsides after discontinuation of treatment. Clinical Relevance The mechanisms of steroid-induced glaucoma may be related to those involved in primary open-angle glaucoma and could provide the clues to elucidate the pathogenesis of the latter. The high prevalence of corticosteroid-induced elevation of IOP in the cow and the large amount of tissue available will permit studies on the mechanism of this phenomenon not previously possible.

Presence of CFTR in the conjunctival epithelium.

PURPOSE: To determine the presence of the cystic fibrosis transmembrane conductance regulator (CFTR) in the conjunctiva and examine the possibility of its regional expression in rabbit, rat and porcine conjunctival epithelia given distinct differences in morphological appearance between the bulbar and palpebral epithelia. METHODS: Two specific anti-CFTR antibodies, against different epitopes in the R domain of the CFTR molecule were used in immunofluorescent labeling of frozen fixed sections isolated from the bulbar and palpebral regions of fresh rabbit, porcine and rat conjunctivae. CFTR expression was also determined in the rabbit conjunctival epithelium using RT-PCR methods. RESULTS: CFTR immunoreactivity in the conjunctival epithelium exhibits polarized expression and is associated with the apical domain of conjunctival epithelial cells. An identical pattern of staining obtained in porcine cryosections using either of the anti-human CFTR antibodies confirmed the specificity of the positive apical staining. RT-PCR analysis produced bands at the predicted size for CFTR mRNA transcripts in both bulbar and palpebral portions of the rabbit conjunctival epithelium. CONCLUSIONS: Apical localization of CFTR in the conjunctival epithelium is consistent with the function of this protein as a chloride channel or as a regulator of channel activity. The identification of CFTR in both bulbar and palpebral portions of the conjunctiva provides evidence that the mechanisms for Cl secretion reside throughout the conjunctiva. This finding suggests that manipulations of the CFTR Cl channel could affect transepithelial Cl transport rates and water movement into the tear film.

Beta-adrenergic inhibition of rabbit lens anterior-surface K(+) conductance.

PURPOSE: To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical parameters and examine the distribution of beta adrenoceptors about the epithelial surface. METHODS: The electrophysiological experiments encompassed the isolation of lenses within a vertically arranged, Ussing-type chamber under short-circuit conditions, an approach that allowed for measurements of short-circuit current (I(sc)) across, in separate experiments, discrete surface regions. Epithelial beta receptors were localized by immunofluorescent labeling of lens cryosections primarily exposed to a polyclonal antibody against human beta( 2)-adrenoceptors. Reverse transcription - polymerase chain reaction (RT-PCR) was used to generate cDNA (using specific primers based upon the sequence of the previously cloned human beta(2) receptor) from rabbit lens RNA extracted from mechanically sequestered anterior and equatorial epithelial cells. RESULTS: Asymmetrical I(sc) reductions with increases in translens resistance were elicited with epinephrine, isoproterenol, terbutaline, forskolin, and a lipid-permeable cAMP analogue. Electrical changes were recorded across the anterior aspect and not observed when the above compounds were applied to solutions bathing the equatorial and posterior surfaces. Immunohistochemical observations indicated the expression of beta receptors from the anterior epithelium to the equatorial region. RT-PCR yielded cDNA of expected basepair length for the apparent fragment of the beta(2)-adrenoceptor, which exhibited a sequence homology 90% identical with its human equivalent in both the anterior and equatorial epithelia. CONCLUSIONS: The cAMP-sensitive conductance(s) appear limited to the anterior epithelium and undetectable equatorially. The asymmetrical I(sc) responses do not seem to arise from a spatial heterogeneity in epithelial receptor expression.

Characterization of serotonergic receptors in rabbit, porcine and human conjunctivae.

PURPOSE: To characterize the serotonin (5-HT) receptors linked to the modulation of adenylyl cyclase activity in rabbit, porcine and human conjunctivae. METHODS: Serotonin receptor-subtype expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) and receptor subtype-specific polyclonal antibodies for the immunofluorescent labeling of conjunctival cryosections. In addition, measurements of the effects of serotonergics on the short-circuit current (I(sc)) across rabbit and porcine conjunctivae were contrasted. RESULTS: RT-PCR assays indicated the expression of 5-HT(1B ) and 5-HT(1D) receptors, subtypes negatively coupled to adenylyl cyclase, in the rabbit conjunctiva. This approach also suggested the co-expression of 5-HT(1B), 5-HT(1D), 5-HT(1F), 5-HT(4) and 5-HT(7) mRNA's in the porcine conjunctiva, and 5-HT( 1D), 5-HT(1F) and 5-HT(7) in the human conjunctiva. Since the 5-HT(4) and 5-HT(7) receptors are positively linked to adenylyl cyclase, these results implied that the porcine and human tissues exhibited subtypes both positively and negatively linked to the enzyme. However, immunohistochemical observations, using currently available antibodies solely localized the 5-HT(7) moiety in the porcine and human epithelia, suggested that the 1B/1D forms may be minor elements. Consistent with this prospect, 5-HT was a stimulant of the transepithelial I(sc) across the porcine conjunctiva, an opposite response from earlier findings that demonstrated inhibitory effects by 5-HT on the rabbit I(sc), which are now explained by the localization of the 1B/1D receptors in the rabbit stratified epithelium. CONCLUSIONS: The 5-HT receptors expressed by mammalian conjunctivae are not identical. In terms of 5-HT receptor expression, the porcine tissue may be a more appropriate model for human, than is the rabbit, in that 5-HT may serve as a secretagogue in the human epithelium.

Fluid circulation determined in the isolated bovine lens.

PURPOSE: In 1997, a theoretical model was developed that predicted the existence of an internal, Na(+)-driven fluid circulation from the poles to the equator of the lens. In the present work, we demonstrate with a novel system that fluid movement can be measured across the polar and equatorial surface areas of isolated cow lenses. We have also determined the effects of ouabain and reduced bath [Na(+)]. METHODS: Lenses were isolated in a chamber with three compartments separated by two thin O-rings. Each compartment, anterior (A), equatorial (E), and posterior (P), was connected to a vertical capillary graduated in 0.25 µL. Capillary levels were read every 15 minutes. The protocols consisted of 2 hours in either open circuit or short circuit. The effects of ouabain and low-Na(+) solutions were determined under open circuit. RESULTS: In 21 experiments, the E capillary increased at a mean rate of 0.060 µL/min while the A and P levels decreased at rates of 0.044 and 0.037 µL/min, respectively, closely accounting for the increase in E. The first-hour flows under short circuit were approximately 40% larger than those in open-circuit conditions. The first-hour flows were always larger than those during the second hour. Preincubation of lenses with either ouabain or low-[Na(+)] solutions resulted in reduced rates of fluid transport. When KCl was used to replace NaCl, a transitory stimulation of fluid transport occurred. CONCLUSIONS: These experiments support that a fluid circulation consistent with the 1997 model is physiologically active.

Sildenafil accelerates anterior chamber refilling after paracentesis in sheep and rabbits.

PURPOSE: Sildenafil increases ocular blood flow. Thus, the authors investigated if it also increases anterior chamber (AC) refilling after paracentesis. METHODS: Corriedale sheep and albino rabbits were used as animal models. Intraocular pressure (IOP) was measured, paracentesis performed on one eye, and AC refilling followed by observation using oblique illumination. IOP measurements continued as the AC formed. After IOP stabilization, sildenafil (100 mg) was orally administered. Forty to 60 minutes later, AH was withdrawn from the contralateral eye. The point at which IOP recovered was used to determine refilling time. Paracentesis volumes were either 60, 120, or 300 µL in sheep, and 50 or 100 µL in rabbits. RESULTS: IOP recovered in approximately 49, 56, and 50 minutes after the 60, 120, and 300 µL withdrawals in sheep. The refilling times of the contralateral eye after sildenafil ingestion were approximately 19, 26, and 37 minutes for the respective AH withdrawals. With rabbits, IOP recovered in approximately 13 minutes after the 50 and 100 µL AH withdrawals. After sildenafil, the IOP recovery times of the fellow eye were approximately 6 minutes. AH refilling rates were estimated by dividing the paracentesis volume by IOP recovery time. After sildenafil, such rates were larger than the AH formation rate attributed to secretion by the ciliary epithelium. CONCLUSIONS: Sildenafil accelerates the rate of AC refilling and might have beneficial utility as an agent enhancing fluid entry into the AC of patients who experienced AH loss during eye surgery, as well as in some cases of ocular hypotony.

An hypothesis on pressure transmission from anterior chamber to optic nerve.

Few studies have characterized how pressure in the anterior chamber (AC) of the eye is transmitted via the vitreous to the vitreous-ganglion cell interface. We are aware of only one study that simultaneously measured the pressures in the AC and vitreous humor; and of only one study that simultaneously measured the pressures in the AC and the suprachoroidal space (SCS). The pressure in the AC is defined as the intraocular pressure (IOP), which when elevated beyond statistically normal limits is a recognized risk factor for glaucoma, a malady best described as an optic neuropathy with degeneration and eventual death of the retinal ganglion cells (GC's) and highly characteristic changes in the optic nerve head (ONH). Most investigators currently believe that the prevalent risk factor for GC apoptosis is ocular hypertension, but no one has demonstrated how an increase in IOP in the AC is transmitted to the GC's. In patients with primary open angle glaucoma, the pressure in the AC increases due to an increase in the resistance of the trabecular meshwork (TM) outflow pathway. We questioned how such increased pressure in the AC would be transmitted to the GC to produce the changes in the ONH seen in glaucoma. Based on our preliminary data and purview of the literature, we hypothesize that a pressure increase originating in the AC is likely transmitted via both the SCS and the vitreous, with transmission via the former pathway probably most efficient in affecting the GC. Independently of the mechanism that produces GC apoptosis, the ones that are first affected, as repeatedly shown by visual field tests, are the most peripheral ones; i.e., those whose axons are the most external as they form the ONH and enter the lamina cribrosa. There are no published reports explaining this peculiarity. The dogma is that the pressure transmitted via the vitreous is higher at the periphery because it is transmitted across a shorter distance, since the vitreous acts as a buffer that absorbs part of the pressure being transmitted. We propose that IOP is not only transmitted via the vitreous but also via the SCS. Increases in IOP could be efficiently applied via the SCS to the most external axons of the ONH as they leave the eye. Our hypothesis can also explain low-tension glaucoma in which the most peripheral GC's are also affected first, because pressure is transmitted without decay due to a reduced uveoscleral (UVS) flow.

Gene expression changes in steroid-induced IOP elevation in bovine trabecular meshwork.

PURPOSE: To determine whether gene expression changes occur in the trabecular meshwork (TM) of cow eyes with steroid-induced intraocular pressure (IOP) elevation. METHODS: Adult female Braford cows (n = 4) were subjected to uniocular prednisolone acetate treatment for 6 weeks. IOP was monitored with an applanation tonometer. At the conclusion of the experiment, animals were euthanized, eyes were enucleated, and the TM was dissected and stored in an aqueous nontoxic tissue storage reagent. RNA was extracted and subjected to microarray analysis using commercial oligonucleotide bovine arrays. Some of the genes differentially expressed between control and experimental eyes were confirmed by quantitative RT-PCR and some of the respective proteins were studied by immunoblotting. RESULTS: IOP began to increase after 3 weeks of treatment, reaching a peak 2 weeks later. IOP differences between corticosteroid-treated and fellow control eyes were 6 ± 1 mm Hg (mean ± SD) at the conclusion of the study. Microarray analysis revealed that expression of 258 genes was upregulated, whereas expression of 187 genes was downregulated in the TM of eyes with steroid-induced IOP elevation. Genes identified to be differentially expressed include genes coding for cytoskeletal proteins, enzymes, growth and transcription factors, as well as extracellular matrix proteins and immune response proteins. A number of relevant gene networks were detected by bioinformatic analysis. CONCLUSIONS: Steroid-induced IOP elevation alters gene expression in the bovine TM. Identification of genes with changing expression in this model of open-angle glaucoma may help elucidate the primary changes occurring at the molecular level in this condition.

Effects of sildenafil and tadalafil on intraocular pressure in sheep: implications for aqueous humor dynamics.

PURPOSE: To determine the effects of vasodilators on intraocular pressure (IOP) and the protein content of sheep aqueous humor (AH), because the vasodilators may increase fluid leakage from the fenestrated capillaries of the ciliary body to the extracellular tissue and directly to the anterior chamber (AC) via the iris, and some senior patients (older than 70) treated with sildenafil have exhibited elevated IOP. METHODS: Experiments were performed on domestic sheep residing on a ranch in Argentina. These docile and compliant animals readily swallowed tablets of sildenafil (50 and 100 mg) and tadalafil (20 mg). IOP was monitored by Perkins applanation tonometry in 21 normal sheep receiving orally administered drugs. In addition, paracentesis was performed on six sheep to quantify changes in AH protein levels. RESULTS: Ingestion of both sildenafil and tadalafil increased sheep IOP from normal levels of approximately 9 to 11 mm Hg within 1 hour. The IOP elevation was approximately 1.6-fold with both doses of sildenafil. IOP returned to control values within 4 hours. With the longer-lasting vasodilator tadalafil, IOP remained 1.6- to 1.9-fold higher than normal for at least 48 hours and returned to control levels within 4 days. The AH protein content was approximately 39% higher in sheep given 100 mg sildenafil. CONCLUSIONS: These data are consistent with a vasodilator-evoked increase in plasma-like fluid in the AC, which likely accounts for the IOP elevation. The results are discussed with a model for AH dynamics that may be of importance to senior individuals treated for vascular diseases with these compounds.

Treatment of sheep steroid-induced ocular hypertension with a glucocorticoid-inducible MMP1 gene therapy virus.

PURPOSE: To investigate whether intracameral injection of the adenovirus vector AdhGRE.MMP1 would reduce or prevent elevated intraocular pressure (IOP) induced by corticosteroids in living animals. METHODS: Glucocorticoid-inducible adenovirus vectors carrying wild-type or mutant forms of human metalloproteinase 1 (MMP1 and mutMMP1) cDNAs were generated. An adenovirus carrying no gene (Ad5.CMV.Null) was used as an additional control. Sheep were injected intracamerally with 30 microL of each vector, either previously or after the induction of increased IOP with topical prednisolone or sub-Tenon triamcinolone under various protocols. IOP was measured with a Perkins tonometer. Inflammation was monitored by visual inspection. RESULTS: In eyes in which IOP was already elevated to 24 to 30 mm Hg, injection of AdhGRE.MMP1 reduced IOP by 70% in 24 hours and to 10 to 13 mm Hg in 48 hours. In eyes with normal IOP (9-11 mm Hg), preinjection of the virus protected against the increase in IOP normally produced by the corticosteroid. IOP remained at a level of approximately 12 mm Hg for 5 days despite the continuous application of the corticosteroid. Injections of the control viruses had no hypotensive effects. There were no signs of ocular inflammation or discomfort to the animals. CONCLUSIONS: A single dose of a gene therapy vector carrying an inducible metalloproteinase human gene can both protect against the IOP increase produced by corticosteroid instillation in the sheep model and quickly reverse the IOP increase previously elicited by the corticosteroid. These results are a first step toward a treatment of steroid-glaucoma with inducible overexpression of extracellular matrix modulator genes.

Suppression of corticosteroid-induced ocular hypertension in sheep by anecortave.

OBJECTIVE: To confirm the ocular hypotensive effects of anecortave acetate on an ovine model for steroid-induced ocular hypertension. Eyes of normal sheep exhibit a robust steroid-induced ocular hypertensive response. Recent observations in an uncontrolled, interventional case series indicated that anecortave elicited hypotensive effects when administered as a sub-Tenon depot in the eyes of a small sample of patients with glaucoma. METHODS: Intraocular pressure (IOP) was monitored by Perkins applanation tonometry in 16 normal sheep receiving topically administered prednisolone acetate, 0.5%, in both eyes, 3 times daily, a protocol that doubled IOP within 12 days. Half of the sheep had received a unilateral sub-Tenon injection of anecortave in 1 eye prior to the initiation of the bilateral prednisolone instillations, while the 8 remaining sheep received the unilateral anecortave sub-Tenon depot after the IOP was maximally elevated by the prednisolone instillations. RESULTS: In these 2 sets of experiments, the presence of the anecortave depot suppressed the steroid-induced IOP elevation and reverted the elevated IOP to baseline levels. Measurements of aqueous outflow facility indicated that eyes treated with prednisolone plus anecortave exhibited a 5.8-fold higher outflow facility than the fellow eyes solely exposed to prednisolone, indicating that anecortave prevented the increase in outflow resistance produced by the corticosteroid. CONCLUSION: Elucidation of the mechanisms of action of anecortave in animal models may prove relevant to the design of novel interventions for the management of primary open-angle glaucoma.