Ascorbic acid levels in aqueous and vitreous humors of the rabbit: effects of inflammation and ceruloplasmin

A HPLC method for determination of ascorbic acid and dehydroascorbic acid in plasma and aqueous and vitreous humors of rabbits is described. Values for total ascorbic acid concentration found in this study are in agreement with those of previous investigators. Endotoxin-induced ocular inflammation caused a decrease in the concentration of ascorbic acid in the aqueous humor and an increase in the vitreous humor. The additional lack of correlation between levels of ascorbic acid in normal aqueous and vitreous humors from the same uninflamed eye indicates that the aqueous humor is not the source of vitreal ascorbic acid. The copper concentration of the aqueous humor is increased during ocular inflammation, most likely due to the influx of ceruloplasmin from plasma when the blood-aqueous barrier is disrupted. Ceruloplasmin caused a decrease in the amount of ascorbic acid in the aqueous humor in vitro and the vitreous humor in vivo. The presence of ceruloplasmin in the aqueous humor during inflammation thus may contribute to the decreased concentration of ascorbic acid in this fluid.     

Some effects of progestogens, oestrogens and androgens on the ocular tension of rabbits and owl monkeys.

Topically applied progestogens; progesterone, quingestanol acetate and norethisterone acetate, were shown to reduce the ocular tension of conscious rabbits. Quingestanol acetate and norethisterone acetate also reduced the ocular tension of conscious owl monkeys. It was found that quingestanol acetate did not increase outflow facility in anaesthetized rabbits, but it significantly reduced the clearance of [¹⁴C]inulin from the anterior chamber suggesting a reduction in aqueous flow. Androgens and oestrogens were not found to exert hypotensive effects in either species.     

The influence of topical prostaglandins on HSA-induced uveitis in the rabbit

In this study the effect of topical administration of prostaglandins (PGs) on a human serum albumin (HSA)-induced uveitis is evaluated. Topical prostaglandin E₁ (PGE,) and prostaglandin F₂ (PGF₂) partly inhibited hyperaemia and flare in the anterior chamber after the induction of immune complex uveitis. A marked increase in the cellular response was observed in the aqueous humour after topical PGE₁ and PGF₂. Topical prostaglandins may decrease endogenous prostaglandin formation and reduce the prostaglandin-mediated inflammatory symptoms; on the other hand, they also stimulate the aqueous cellular response, possibly by facilitation of leukotriene formation.These results indicate that topical prostaglandins should not be used to treat immunogenic uveitis.     

Prostaglandin and an increased sensitivity of the sympathetically denervated rabbit eye to laser-induced irritation of the iris

Application of ruby laser light to the pigmented rabbit iris elicits an acute, dose-related injury response which is characterized by a pupil constriction and a transient increase in intraocular pressure and is accompanied by an increased infiltration of blood-plasma proteins and a release of prostaglandin-like material into the aqueous humour. In unilaterally sympathetically denervated eyes the peak intraocular pressure and pupil changes and the aqueous prostaglandin levels are notably higher than in contralateral normal eyes following laser irradiation. Indomethacin, a drug which prevents prostaglandin formation, inhibited the intraocular pressure and pupil responses in both eyes but it did not reduce the magnitude of difference in sensitivity between the sympathetically denervated and control eyes. The results suggest that the increased sensitivity of the former are not due to the raised prostaglandin levels but are the result of removal of the pupil dilator and vasoconstrictive influences of sympathetic nervous activity.     

A direct correlation between the levels of ascorbic acid and H2O2 in aqueous humor

There is evidence that H2O2 present in aqueous humor arises from ascorbic acid which is also present in this fluid, but the extent to which peroxide is derived from ascorbic acid is not known. We have measured the concentrations of H2O2 and ascorbic acid normally present in the aqueous humor of various species and also under conditions in which the level of ascorbic acid in the fluid was experimentally altered. In aqueous humor of rabbit and guinea pig the concentration of ascorbic acid was 10 times higher than that present in aqueous of rat and frog. Similarly, the concentration of H2O2 was four to 10 times higher in rabbit and guinea pig aqueous compared to that in rat and frog. Consistent with the higher concentration of ascorbic acid in posterior compared to anterior aqueous humor in the rabbit, the concentration of H2O2 was also significantly higher in the posterior aqueous. When ascorbic acid in rabbit aqueous humor was elevated by intraperitoneal administration of the compound, there was a significant increase in the level of H2O2 in both anterior and posterior aqueous humor. Moreover, when the level of ascorbic acid was lowered experimentally by placing guinea pigs on an ascorbic acid deficient diet, a 10-fold decrease in the level of both ascorbic acid and H2O2 was observed in the aqueous humor. Upon returning the animals to a normal diet, the concentrations of both compounds returned to control values. The direct correlation between the concentrations of ascorbic acid and H2O2 in aqueous humor suggests that ascorbic acid is the primary source of H2O2 in this fluid.     

Effect of various drugs on pseudofacility and aqueous humor formation in the rabbit eye.

Norepinephrine and epinephrine reduce aqueous humor formation by an amount less than sufficient to reduce the intraocular pressure. Both drugs increase pseudofacility and true outflow facility which both contribute to reduce the pressure. Neither drug changes the value of x_c, the pressure index of the capillaries, which indicates that large hemodynamic changes do not occur. Phenylephrine has no effect on pseudofacility, since, although x_c is reduced, the fluid permeability is increased in a compensatory manner: an increase in true outflow facility is responsible for the small pressure fall.Pilocarpine causes a minor increase in aqueous humor formation, has no effect on pseudofacility yet lowers the intraocular pressure, albeit by only 2 mmHg. In order to lower intraocular pressure, therefore, pilocarpine must increase true outflow facility.Prostaglandin E₁ increases intraocular pressure, aqueous humor formation, x_c and pseudofacility. PGE₁ is known to induce venous congestion and this correlates with the increase in x_c since a secondary rise in capillary pressure would follow. The increase in aqueous humor formation accounts for the increase in intraocular pressure. The increase in pseudofacility accounts for about half of the increase in total outflow facility found with PG's, at least at times up to 2 hr after topical PG application.     

Longitudinal non-invasive proton NMR spectroscopy measurement of vitreous lactate in a rabbit model of ocular hypertension

To determine whether vitreous lactate concentrations are correlated with intraocular pressure (IOP) rise, retinal ganglion cell (RGC) damage, and nerve fiber layer (NFL) thickness decrease in a rabbit model of ocular hypertension. Also, to learn whether proton nuclear magnetic resonance ((1)H-NMR) spectroscopy can provide sequential, non-invasive in vivo measurements of vitreous lactate. Intra-anterior chamber injections of 20-mum latex beads were used to impede aqueous drainage in New Zealand White rabbits, causing an elevation of IOP. Group I consisted of 12 rabbits in which unilateral elevations in IOP were achieved. Group II consisted of 6 rabbits in which treatment did not cause a change in IOP. The contralateral eye served as a control in both groups. Control eyes received an equal volume injection of vehicle only. IOP was measured for two pre-treatment days and then on post-treatment days 1, 3, 5, 7, 9, 16, 23, 30, and 37. (1)H-NMR spectroscopy was used to measure changes in vitreous lactate concentrations that may be associated with the onset and progression of the pathophysiology. Post-mortem histochemical analysis at the light microscope level was used to quantify changes in the retinal NFL thickness and in the numbers of RGC, and correlate them with IOP and percent change in lactate levels. Baseline IOP in Group I control and treated eyes were 12.0+/-1.9 and 12.5+/-1.3 mmHg, respectively. Between days 5 and 9 post-treatment, the IOP in Group I treated eyes (n=12) rose to 23.9+/-4.2 mmHg. IOP in the control eyes remained unchanged (12.0+/-1.6). Vitreous lactate levels in Group I treated eyes increased by 100%, from pre-treatment values. Levels in control eyes remained unchanged. In Group I, IOP and percent change in lactate concentration in treated eyes were closely correlated throughout the study period (r=0.95; p< or =0.05). Group II control and treated eyes showed no significant changes in either IOP or lactate. Group I treated eyes had a reduced NFL thickness (65+/- 4 microm; n=5) at the temporal medullary ray (MR) compared with control eyes (45+/-6 microm). A smaller reduction was found in the nasal MR areas, where thickness was 53+/-3 microm in treated eyes and 66+/-4 microm in control eyes. RGC numbers also were decreased in the treated eyes (241,222+/-10,920 cells) vs. 322,311+/-8726 cells in control eyes. TdT-mediated dUTP nick-end labeling (TUNEL) indicated that RGC loss in the treated eyes was most likely due to apoptosis. In vivo changes in lactate can be monitored non-invasively over time using (1)H-NMR spectroscopy. Vitreous lactate concentrations increased and returned to baseline concurrently with IOP. The brief elevation in IOP produced a reduction in both the RGC cell numbers and in the thickness of the NFL.     

Efficacy and safety of indomethacin 0.1% eye drops compared with ketorolac 0.5% eye drops in the management of ocular inflammation after cataract surgery

Purpose: To determine whether indomethacin 0.1% eye drops are at least as effective as ketorolac 0.5% eye drops in treating ocular inflammation following cataract surgery. Methods: Prospective, multicenter, investigator‐masked, parallel‐group, randomized, active‐controlled clinical trial. Cataract patients were randomized in a 1:1 ratio to receive indomethacin or ketorolac administered QID for 3 weeks beginning 1 day before surgery. The primary end‐point was aqueous flare measured by laser flare meter at postoperative Days 1 and 7. Secondary end‐points included retinal thickness, slit lamp and funduscopic examinations and postsurgical pain ratings. Safety and tolerability were also assessed. Results: A total of 86 patients were included in the per protocol population (n = 43 per treatment group). Indomethacin was found non‐inferior to ketorolac for comparison of aqueous flare at postoperative Days 1 and 7 (Day 1: 95% CI: ?2.37, 5.50; non‐inferiority upper margin, 15 ph/ms and Day 7: 95% CI: ?7.83, ?0.94; non‐inferiority upper margin, 8 ph/ms) and statistically better than ketorolac at Day 7 (p = 0.013). There were no significant between‐group differences in aqueous flare and change from baseline in retinal thickness at postoperative Days 30 and 90. Indomethacin showed a higher subjective tolerance rating than ketorolac at postoperative Days 7 and 30 (p ≤ 0.044). Conclusion: Indomethacin 0.1% was at least as effective as ketorolac 0.5% at Day 1 and more effective than ketorolac 0.5% at Day 7 in treating ocular inflammation after uncomplicated cataract surgery. Indomethacin was better tolerated than ketorolac. There were no clinically meaningful safety concerns with either treatment.     

Inhibition of the acute ocular responses to nitrogen mustard by colchicine

Colchicine, a naturally occurring alkaloid, inhibits both fast and slow axoplasmic transport by prevention of microtubule assembly. We have examined the influence of colchicine on the neurogenically mediated acute inflammatory responses to topically administered nitrogen mustard and formaldehyde on the albino rabbit eye. Topical administration of colchicine (10, 20 or 40 micrograms) once per day for 15 days inhibited the ocular hypertensive response, the leakage of protein into the anterior chamber, and the pupillary constriction observed following topical administration of nitrogen mustard. Colchicine pretreatment also inhibited the ocular hypertensive response to topically administered formaldehyde. Pretreatment of the eye with colchicine (20 micrograms day-1) for a period of 5 days proved to be ineffective in suppressing the ocular hypertensive effect of nitrogen mustard, while more prolonged pretreatment (10 or 15 days) significantly abrogated the characteristic injury responses. Topical administration of colchicine (20 and 40 micrograms) caused dilation of conjunctival and limbal vessels, but these vascular responses subsided as treatment continued. These preliminary experiments suggest that the inhibition of the ocular responses to nitrogen mustard by chronic colchicine pretreatment might be the result of modification of synthesis or release of sensory mediator(s).     

A comparison of the miotic and inflammatory effects of biologically active polypeptides and prostaglandin E2 on the rabbit eye

The ocular effects of some biologically active peptides were studied and compared to those of prostaglandin E₂ (PGE₂) to determine whether the responses of the eye to trauma, characterized by increased intraocular pressure (IOP), the development of anterior chamber flare and partial miosis, might be mediated by such peptides. One to 2 hr after intravitreal injection of 10 μg of PGE₂ into rabbit eyes, ocular hypertension, flare and iridial hyperemia, but only minimal miosis were observed. Maximum miosis developed within 2–3 hr after intravitreal injection of 1·0–100 μg of substance P (SP), SP-octapeptide (SP-8), coherin or eledoisin-related peptide (EDR), while 10–100 μg of vasoactive intestinal polypeptide (VIP), somatostatin or bradykinin (BK) yielded only submaximal miosis and angiotensin II, α-MSH and poly-dl-alanine had little or no miotic effect. None of these peptides caused iridial hyperemia or a cellular invasion of the anterior chamber and only high doses (100 μg) of VIP or BK caused significant increases in the protein concentration of the aqueous humor. Miotic doses of SP, SP-8 or EDR caused a significant increase in IOP in some, but not all, experiments. Thus, PGE₂ can be regarded as a mediator of the ocular irritative response although it may not account for the miosis that is associated with chemical or surgical trauma. However, this autacoid should not be regarded as the mediator of ocular inflammation since it does not elicit a cellular response. In contrast, some polypeptides, particularly SP, SP-8 and EDR are strong miotics and, at least under some circumstances, can act as effective ocular hypertensives, but these peptides do not reproduce any other signs of ocular irritation or inflammation. It is therefore concluded that none of the peptides studied here could, by itself, be the sole mediator of the initial ocular irritative response although some of them may account for the miosis and contribute to the ocular hypertension associated with this response. A combination of some of these peptides together with PGE₂ and/or other PGs may account for all aspects of the ocular irritative response and for most aspects of the ocular inflammatory response.     

Identification of lymphatics in the ciliary body of the human eye: A novel “uveolymphatic” outflow pathway

Impaired aqueous humor flow from the eye may lead to elevated intraocular pressure and glaucoma. Drainage of aqueous fluid from the eye occurs through established routes that include conventional outflow via the trabecular meshwork, and an unconventional or uveoscleral outflow pathway involving the ciliary body. Based on the assumption that the eye lacks a lymphatic circulation, the possible role of lymphatics in the less well defined uveoscleral pathway has been largely ignored. Advances in lymphatic research have identified specific lymphatic markers such as podoplanin, a transmembrane mucin-type glycoprotein, and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Lymphatic channels were identified in the human ciliary body using immunofluorescence with D2-40 antibody for podoplanin, and LYVE-1 antibody. In keeping with the criteria for lymphatic vessels in conjunctiva used as positive control, D2-40 and LYVE-1-positive lymphatic channels in the ciliary body had a distinct lumen, were negative for blood vessel endothelial cell marker CD34, and were surrounded by either discontinuous or no collagen IV-positive basement membrane. Cryo-immunogold electron microscopy confirmed the presence D2-40-immunoreactivity in lymphatic endothelium in the human ciliary body. Fluorescent nanospheres injected into the anterior chamber of the sheep eye were detected in LYVE-1-positive channels of the ciliary body 15, 30, and 45 min following injection. Four hours following intracameral injection, Iodine-125 radio-labeled human serum albumin injected into the sheep eye (n = 5) was drained preferentially into cervical, retropharyngeal, submandibular and preauricular lymph nodes in the head and neck region compared to reference popliteal lymph nodes (P < 0.05). These findings collectively indicate the presence of distinct lymphatic channels in the human ciliary body, and that fluid and solutes flow at least partially through this system. The discovery of a uveolymphatic pathway in the eye is novel and highly relevant to studies of glaucoma and other eye diseases.     

Species differences in the responses of the eye to irritation and trauma: a hypothesis of divergence in ocular defense mechanisms, and the choice of experimental animals for eye research

Information published during the past century, especially the last decade, has identified pronounced species differences, not only in the morphological organization of ocular structures, but also in the functional responses of the eyes of different mammals to experimental and surgical procedures, as well as to drugs and autacoids. For the most part, these differences have been regarded as peculiarities or weakness rather than as fundamental evolutionary adaptations optimally suited to the environment and behavior of each species. This paper proposes a working hypothesis of evolutionary divergence in ocular defense mechanisms, based on some of the known morphological and functional differences among mammals, and discusses the implications of these differences with regard to the choice of appropriate animals for use as models in different areas of ophthalmic research.     

Homeostasis of the retinal micro-environment: I. Magnesium,potassium and calcium distributions in the avian eye

The concentrations of Mg²⁺, K⁺ and Ca²⁺ in the intraocular fluids (IOFs) and blood plasma of chickens and pigeons were determined by atomic absorption spectrophotometry. The Mg²⁺ concentration in the IOFs of both species was greatest in the liquid vitreous adjacent to the retina followed by aqueous > blood plasma > plasma dialysate. In contrast the concentration of K⁺ in the IOFs of both chickens and pigeons was greater in the aqueous than in the liquid vitreous. The concentrations of Ca²⁺ in all IOF compartments of chicken eyes were virtually identical and were lower than that of blood plasma. The concentrations of Mg²⁺ in the IOFs of the chicken, especially in the liquid vitreous, was remarkably stable; experimentally lowering or raising the plasma Mg²⁺ concentration over a relatively wide range had little or no effect on the Mg²⁺ concentration in the IOFs of these animals. We can conclude that a high Mg²⁺ and low K⁺ concentration in the extracellular fluids of the retina is maintained in the avian eye, as in the mammalian eye, by active transport processes across the blood-retinal barrier systems. Because the avian retina is completely avascular, the site of these homeostatic transport processes must be the epithelium of the retinal choroid and/or the pecten. These findings support the concept that the contribution of the vitreous to the homeostasis of the retinal micro-environment is inversely related in vertebrates to the degree of retinal vascularization.     

The response of the isolated iris sphincter muscle of various mammalian species to substance P

Contractile responses of the isolated iris sphincter muscle from the rabbit, cow, pig, cat, dog, baboon and man to substance P were compared under isotonic conditions using carbachol as a reference standard. Iris preparations from the rabbit, pig and cow showed strong and consistent responses to substance P whilst those from the cat, dog, baboon and human eyes did not contract.     

Exogenous Na-hyaluronate in the anterior chamber of the owl monkey and its effect on the intraocular pressure

Exogenous, ultrapure (sterile, pyrogen-free), non-inflammatory fraction of Na-hyaluronate (NIF-NaHA) was introduced into the anterior chamber of owl monkeys (Aotus trivirgatus), replacing approximately 48% or 77% of the aqueous humor and creating post-injection intraocular pressures (IOPs) below normal (5-10 mmHg) or above normal (40-60 mmHg), respectively. Five different molecular weight samples (MW 1.7, 3.4, 3.7, 4.5 and 4.9 X 10(6)) were used. All solutions contained 1% NIF-NaHA and, because of the varying molecular weights, the viscosities of the solutions ranged between 10 000 and 930 000 cSt. The IOP and the rate of export of the exogenous NIF-NaHA from the anterior chamber were measured. All solutions caused an increase in the IOP, and the maximum level occurred at 4 hr after injection. In all cases, the IOP returned to normal 24 hr after injection. The highest and most persistent increase in IOP was observed after the injection of the solution with the lowest viscosity (10 000 cSt). The smallest increases in IOP over the post-operative value were observed after replacement of the aqueous humor using those samples with viscosities of 10 0000 to 300 000 cSt. The turnover (export rate) of injected NIF-NaHA depends for the most part on the viscosity of the injected solution. With increasing viscosity the rate constant, and therefore the half-life, of the injected NIF-NaHA decreases. The volume fraction of the viscous solution replacing the aqueous humor is also a determining factor in establishing the turnover rate. The molecular weight of the injected NIF-NaHA did not change during that time (48 hr) in which a sufficient amount of sample for analysis could be obtained. No evidence was found for the presence of any kind of hyaluronic acid-degrading agent in the anterior chamber.