Intracameral and Lenticular Penetration of Locally Applied Deuterium-Labeled Vitamin E in Rats.

Purpose: To confirm the intraocular dynamics of 1% deuterium (D) labeled alpha-tocopheral acetate (VEA) solution.Methods: The concentrations of D(3)-VEA and D(3)-alpha-tocopherol (VE) derived from D(3)-VEA in the aqueous humor and lens were measured after instilling 1% D(3)-VEA continuously into the cul-de-sac of rat eyes for one and three weeks. D(3)-VEA and D(3)-VE concentrations were determined by gas chromatograph/mass spectrometry.Results: D(3)-VEA and D(3)-VE concentrations in the aqueous humor after one and three weeks of continuous administration were 93.1 and 498.9, and 9.4 and 21.5 ng/mL, respectively. The concentrations in the lens were 15.0 and 6.1, and 9.8 and 4.8 ng/g, respectively.Conclusion: The penetration of VEA into the aqueous humor and lens by eyedrop application was confirmed.

Intracameral and lenticular penetration of locally applied stable isotope-labeled vitamin E.

PURPOSE: To confirm the aqueous humor and lens dynamics of 1% deuterium-labeled alpha-tocopherol acetate (D(3)-VEA) solution. METHODS: The concentrations of D(3)-VEA and D(3)-alpha-tocopherol (D(3)-VE), derived from D(3)-VEA, in the aqueous humor and lens were measured after continuous instillation of 1% D(3)-VEA into the cul-de-sac of rat eyes for 1 or 3 weeks. D(3)-VEA and D(3)-VE concentrations were determined by gas chromatography/mass selective detector. RESULTS: In the aqueous humor, the concentrations of D(3)-VEA and D(3)-VE were, respectively, 93.1 and 9.4 ng/mL after 1 week, and, respectively, 498.9 and 21.5 ng/mL after 3 weeks. In the lens, they were, respectively, 15.0 and 9.8 ng/g after 1 week, and 6.1 and 4.8 ng/g after 3 weeks. CONCLUSION: The penetration levels of alpha-tocopherol acetate by eyedrop application were confirmed in the aqueous humor and lens.

[Intracameral and lenticular penetration of locally applied deuterium-labeled vitamin E in rats].

PURPOSE: To confirm the intraocular dynamics of 1% deuterium (D)-labeled alpha-tocopherol acetate (VEA) solution. METHODS: The concentrations of D3-VEA and D3-alpha-tocopherol (VE) derived from D3-VEA in the aqueous humor and lens were measured after instilling 1% D3-VEA continuously into the cul-de-sac of rat eyes for one and three weeks. D3-VEA and D3-VE concentrations were determined by gas chromatograph/mass spectrometry. RESULTS: D3-VEA and D3-VE concentrations in the aqueous humor after one and three weeks of continuous administration were 93.1 and 498.9, and 9.4 and 21.5 ng/ml, respectively. The concentrations in the lens were 15.0 and 6.1, and 9.8 and 4.8 ng/g, respectively. CONCLUSION: The penetration of VEA into the aqueous humor and lens by eyedrop application was confirmed.

Effect of anti-inflammatory agents on corneal wound-healing process after surface excimer laser keratectomy.

PURPOSE: To investigate the effect of anti-inflammatory agents on conjunctival inflammation and corneal haze formation after excimer laser keratectomy. SETTING: Department of Ophthalmology, University of Tokyo Faculty of Medicine, Tokyo, Japan. METHODS: After excimer laser keratectomy was performed in 21 rabbits (42 eyes), saline, betamethasone 0.1%, or diclofenac 0.1% was topically applied 6 times a day for 4 weeks and then 3 times a day for 8 weeks. The degree of conjunctival inflammation was determined 1, 2, 3, and 7 days after the keratectomy. The degree of corneal haze was quantitatively measured using a digital analyzer before and once a week after the keratectomy. The expression of type IV collagen in the corneas at baseline and 4 and 12 weeks after the keratectomy was examined immunohistochemically. RESULTS: Compared with saline, betamethasone and diclofenac significantly decreased early-phase conjunctival inflammation. Betamethasone significantly inhibited corneal haze formation compared with saline at 3 to 5 and 8 to 12 weeks. Diclofenac did not inhibit corneal haze formation significantly. Although betamethasone tended to be more effective in inhibiting corneal haze formation and deposition of type IV collagen than diclofenac, there was no statistical difference between the 2 anti-inflammatory agents. CONCLUSION: Topically applied betamethasone effectively suppressed corneal haze formation after excimer laser keratectomy. Diclofenac was not statistically effective in inhibiting corneal haze formation.

Tachykinins as enhancers of prostaglandin E2-induced intraocular inflammation.

The effects of tachykinins on prostaglandin E2 (PGE2)-induced intraocular inflammation were investigated. PGE2 (0.01% or 0.1%) instillation induced iridal hyperemia and protein leakage into the aqueous humor in rabbits, but caused minimal miosis. Intravitreally injected substance P (SP) or neurokinin A (NKA), on the other hand, did not induce protein leakage into the aqueous humor in normal rabbits, but they (SP 10 micrograms/eye or NKA 50 micrograms/eye) did induce long-lasting miosis. The miotic activity of SP was about fivefold stronger than that of NKA. Intravitreally injected SP (10 micrograms/eye) but not NKA (50 micrograms/eye) increased PGE2 concentration in the aqueous humor in normal rabbits. In addition, SP (10 micrograms/eye) or NKA (50 micrograms/eye) markedly enhanced protein leakage into the aqueous humor induced by PGE2 instillation. Pretreatment with indomethacin partially blocked the enhancing effect of SP on protein leakage, while it did not block that of NKA. These results suggest that SP or NKA may enhance intraocular inflammation in vivo. However, the mechanisms of these effects of SP and NKA may be different. The enhancing effect of SP in eye inflammation may be partially due to an increased turnover of arachidonic acid into PGE2 caused by activation of the enzyme cyclooxygenase.

The role of platelet-activating factor in cell infiltration in endotoxin-induced uveitis in guinea pigs.

PURPOSE: We investigated the role of platelet-activating factor (PAF) in cell infiltration in endotoxin-induced uveitis (EIU) in guinea pigs. METHODS: To elicit EIU, lipopolysaccharide (LPS) was injected into the anterior chamber of the eye. Cell numbers in the aqueous humor after LPS injection were determined by flow cytometry. PAF and prostaglandin E2 (PGE2) production after LPS injection were also examined. RESULTS: Intracameral injection of LPS induced cell infiltration into the anterior chamber, and platelet-activating factor (PAF) was detected in the aqueous humor. In addition, topical apafant (PAF antagonist) partially inhibited cell infiltration. Intracameral injection of PAF scarcely induced cell infiltration but the reaction with EIU was accelerated by intracameral injection of a small amount of PAF. CONCLUSIONS: These results suggest that PAF has weak direct activity on cell infiltration in intraocular inflammation but enhances intraocular inflammation.

Lactoferrin suppresses loss of corneal epithelial integrity in a rabbit short-term dry eye model.

Human tear fluid contains lactoferrin at the highest concentration. In patients with dry eye such as Sjogren's syndrome, the concentration of lactoferrin in the tears is approximately half the normal value. The present study utilizes a short-term rabbit dry eye model to evaluate if lactoferrin containing eye drops can reverse any of the damage produced by blockage of blinking with an ocular speculum. Damage was evaluated based on the extent of methylene blue staining in histological sections. After 3 h of desiccation, the amount of extractable dye recovered following sacrifice increased by more than 4-fold in the vehicle-treated eyes. However, in those rabbits treated with 1% lactoferrin, dye recovery was only 40% of the value in the vehicle-treated eyes. Between 1-3 h and over a concentration range from 0.01 to 1% lactoferrin, the decreases in staining were both time and concentration dependent. Alternatively, if 1% lactoferrin was applied during the desiccation period, there was partial restoration of corneal epithelial integrity. These results suggest that lactoferrin may be of therapeutic value in decreasing the loss of corneal epithelial integrity in dry eye.

Effects of the immunosuppressant cyclosporin A on neurotransmitter release from peripheral non-adrenergic, non-cholinergic nerves.

This study was undertaken to determine the effect of the immunosuppressant cyclosporin A on neurotransmitter release from non-adrenergic, non-cholinergic nerves (tachykininergic nerves) in the rabbit iris sphincter muscle. Cumulative application of cyclosporin A (0.1 to 10 microM) caused a slow onset of contraction in a concentration-dependent manner. Both FK888 (1 microM) and capsaicin (10 microM), a substance P receptor antagonist and a substance P-depleting agent, respectively, inhibited the contractile effect of cyclosporin A, whereas atropine (1 microM) had no effect. Both cyclosporin A and capsaicin (10 microM) stimulated the release of substance P-like immunoreactivity in the iris. Neither the sodium channel blocker tetrodotoxin (1 microM), the N-type voltage-dependent Ca2+ channel blocker omega-conotoxin GVIA (1 microM) nor the P-type channel blocker omega-agatoxin IVA (0.2 microM) affected cyclosporin A (1 microM)-induced contraction. In contrast, the L-type Ca2+ channel blocker nicardipine (10 microM) inhibited this contractile effect. These results suggest that cyclosporin A stimulates substance P-like tachykinin release by activating L-type voltage-dependent Ca2+ channels, resulting in contraction of the rabbit iris sphincter muscle.

FK506-binding proteins regulate smooth muscle contractility by altering neurotransmitter release.

To determine the roles of FK506-binding proteins, receptors for the immunosuppressant FK506, in tachykinin release, we examined the effects of the FK506 derivative ascomycin, [3S-[3R[E(1S,3S,4S)],4S,5R,8S,9E,12R,14R,15S,16R,18S,1 9S,26aR]]-8-ethyl- 5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro- 5,19-dihydroxy- 3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16- dimethoxy- 4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotr icosine -1,7,20,21(4H,23H)-tetrone, on the contractility of the rabbit iris sphincter muscle. Ascomycin (10(-7) to 10(-5) M) caused concentration-dependent contractions, which were greatly attenuated by preexposure to rapamycin (10(-5) M), a FK506 receptor antagonist. Similarly, this contractile effect was abolished by preexposure to FK888 (10(-6) M), a tachykinin receptor antagonist, and to capsaicin (10(-5) M), a tachykinin-depleting agent. L-type voltage-dependent Ca2+ channel blockers, nicardipine (10(-5) M) and verapamil (5 x 10(-5) M), inhibited the ascomycin-induced contraction, but the N-type channel blocker omega-conotoxin (10(-6) M) did not. These results suggest that ascomycin stimulates tachykinin release by its binding to FK506-binding proteins and the subsequent activation of L-type Ca2+ channels. Thus, FK506-binding proteins may regulate muscle contractility by altering transmitter release from peripheral tachykininergic nerves.

16 beta-Methyl-17 alpha,21-diesterified glucocorticoids as partial agonists of glucocorticoid in rat endotoxin-induced inflammation.

OBJECTIVE AND DESIGN: We investigated the anti-inflammatory effects of 16 beta-methyl-17 alpha,21-diesterified glucocorticoids which are well known as potent topical glucocorticoids in man on endotoxin-induced uveitis (EIU) in rats. MATERIAL: Female Lewis rats were used. TREATMENT: Glucocorticoids were instilled (0.01%-1.0%) or subcutaneously injected (0.1-10 mg/kg) to rats. METHODS: To elicit EIU, LPS (500 micrograms/kg) was injected into the footpad of rats. Twelve hours after LPS injection, cell number in aqueous humor was counted by flow cytometry. Endotoxin-induced in vivo tumor necrosis factor-alpha (TNF-alpha) production was also examined. RESULTS: 16 beta-methyl-17 alpha,21-diesterified glucocorticoids showed no effects or some enhancement of cell infiltration into the aqueous humor in EIU by topical instillation. Systemic injection of these glucocorticoids showed only weak inhibition of cell infiltration and TNF-alpha production. On the other hand, betamethasone phosphate strongly inhibited the cell infiltration and TNF-alpha production. Combined systemic injection of 16 beta-methyl-17 alpha,21-diesterified glucocorticoids and betamethasone phosphate reduced the inhibitory effects of the latter. CONCLUSIONS: These results suggest that 16 beta-methyl-17 alpha,21-diesterified glucocorticoids might act as partial agonists of glucocorticoid in rats.

Up-regulation of glial fibrillary acidic protein in the retina of primate eyes with experimental glaucoma.

OBJECTIVE: To identify molecular mechanisms of retinal responses to intraocular pressure elevation in primate experimental glaucoma. METHODS: An experimental glaucoma model was created by repeated laser trabeculophotocoagulation. After the preparation of complementary DNAs from extracted total RNAs in the retinas, polymerase chain reaction (PCR) experiments were performed for the following screening target genes: beta-tubulin beta 2 and beta 5 and glial fibrillary acidic protein (GFAP). To investigate the amplified sequences derived from the PCR experiments, sequencing, subcloning, and Southern blot analysis of PCR products were performed. In addition, an immunohistochemical analysis was performed in an attempt to show the distribution of the target gene products in the retinas. RESULTS: A series of PCR experiments suggested up-regulation of gene expression for GFAP but not for beta-tubulins. Sequencing of the PCR products and results of the Southern blot analysis showed that the amplified sequences were derived mainly from the target gene, GFAP, and that increased expression of GFAP was found despite the severity of glaucoma. Immunohistochemical studies also demonstrated increased expression of GFAP proteins in Müller cells and astrocytes in the retinas of primate eyes with experimental glaucoma. CONCLUSIONS: Our study showed up-regulation of GFAP at gene and protein levels, which suggests that glial components in the retina may contribute to the pathologic processes induced by elevated intraocular pressure.

Involvement of both L- and N-type voltage-dependent Ca2+ channels in KCl- and veratridine-evoked transmitter release from non-adrenergic, non-cholinergic nerves in the rabbit iris sphincter muscle.

To determine which types of voltage-dependent Ca2+ channels mediate tachykinin release in the isolated rabbit iris sphincter muscle, we examined the effects of several Ca2+ channel modulators on contractions induced by either an elevation of the extracellular KCl concentration or application of the Na+ channel activator veratridine. Contractions caused by either 45.9 mM KCl or veratridine (10 microM) were inhibited by spantide (10 microM), a tachykinin receptor antagonist, and capsaicin (10 microM), a tachykinin-depleting agent, but were not changed by atropine. Nicardipine, an L-type Ca2+ channel blocker, inhibited contractions induced by KCl and veratridine in a concentration-dependent manner. omega-Conotoxin GVIA (1 microM), an N-type Ca2+ channel blocker, inhibited only contractions induced by lower concentrations of KCl, both when applied alone and when combined with nicardipine. Bay K 8644, an L-type Ca2+ channel activator, caused a spantide- and nicardipine-sensitive contraction in muscles partially depolarized with 15.9 mM KCl, and enhanced contractions induced by 15.9 mM KCl and veratridine (2 microM). omega-Agatoxin IVA (0.3 microM), a P-type voltage-dependent Ca2+ channel blocker, did not affect contractions induced by either KCl or veratridine. Contractions induced by exogenous substance P were not modified by any of the Ca2+ channel blockers or by Bay K 8644. These results suggest that, in the rabbit iris sphincter muscle. L- and N-type voltage-dependent Ca2+ channels are involved in neurotransmitter release from tachykininergic nerves elicited by high KCl and by veratridine.

Detection of cell adhesion molecules in lens epithelial cells of human cataracts.

PURPOSE: To detect the expression of cell adhesion molecules (CAMs), including beta integrins, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1, leukocyte-endothelial cell adhesion molecule-2, E-cadherin, and CD44, in cultured lens epithelial cells (LECs) of human cataracts. To show that LECs attach to cells or extracellular matrix components by the detected adhesion molecules. METHODS: A circular section of the anterior capsule with attached LECs obtained by anterior capsulotomy during cataract surgery was cultured in a well of an eight-chamber slide. The LEC immediately after surgery and the cell outgrowth beyond the capsular margin at 2 weeks of culture were observed after the culture was stained immunohistochemically. Functional assays of LEC growth on collagen- or laminin-coated plates were performed in the presence and absence of the antibody blocking the detected adhesion molecules. RESULTS: beta 1 integrin, ICAM-1, and CD44 were detected in both the original specimens and the cultured cells. When the antihuman anti-beta 1 integrin monoclonal antibody (mAb), anti-ICAM-1 mAb, or anti-CD44 mAb was added at 10 micrograms/ml to the incubation medium, LEC migration and proliferation were inhibited significantly on the collagen- or laminin-coated plates. When the mAb blocking these three CAMs were added each at 1 microgram/ml, LEC proliferation also were inhibited. CONCLUSIONS: beta 1 integrin, ICAM-1 and CD44 are all involved in LEC attachment and growth on collagen and laminin in vitro. It can be assumed that these CAMs are involved in adhesion of LECs to extracellular matrix components of the lens capsule. Understanding the characteristics of the adhesion molecules in LEC may lead to the development of a new approach to inhibit secondary cataract formation.

The effects of betamethasone derivatives on endotoxin-induced uveitis in rats.

The anti-inflammatory effects of betamethasone and its derivatives (betamethasone 21-phosphate, betamethasone 21-acetate, betamethasone 17-valerate, clobetasol 17-propionate and betamethasone dipropionate) on endotoxin-induced uveitis (EIU) in rats was investigated. Among the compounds examined, betamethasone, betamethasone 21-phosphate, betamethasone 21-acetate and clobetasol 17-propionate strongly inhibited cell infiltration into the aqueous humor in the EIU by instillation into the eye in a dose-dependent manner (0.01-1.0%) and by systemic administration (1 mg kg-1). On the other hand, betamethasone 17-valerate and betamethasone dipropionate showed only weak inhibitory effects or enhancement of cell infiltration by instillation into the eye (0.01-1.0%) and by systemic administration (1 mg kg-1). Combined systemic administration of betamethasone dipropionate and betamethasone reduced the inhibitory effect of betamethasone on cell infiltration and gene expression of IL-1 beta. In an in vitro interleukin-8 (IL-8) release assay, betamethasone showed stronger inhibition of the IL-8 release from rat peritoneal exudate cells (PEC) than betamethasone dipropionate, and simultaneous addition of betamethasone dipropionate with betamethasone reduced the inhibitory effect of the latter. These results suggest that the betamethasone derivative betamethasone dipropionate might behave as an anti-glucocorticoid in rats.

The effects of betamethasone derivatives on endotoxin-induced uveitis in guinea pigs.

OBJECTIVE AND DESIGN: We reported previously that the betamethasone derivative betamethasone dipropionate behaves as an anti-glucocorticoid in rat endotoxin-induced uveitis (EIU). In the present study, we produced EIU in guinea pigs and investigated the effects of betamethasone dipropionate on the EIU. MATERIAL: Male Hartley guinea pigs were used. TREATMENT: Glucocorticoids were instilled into the eye. METHOD: To elicit EIU, lipopolysaccharide (LPS) was injected into the anterior chamber of the eye. Cell numbers in the aqueous humor after LPS injection were determined by flow cytometry. Prostaglandin E2 (PGE2) production after LPS injection into the anterior chamber was also examined. RESULTS: Intracameral injection of LPS (1 microgram/eye) induced cell infiltration into the anterior chamber and PGE2 production. Betamethasone dipropionate inhibited cell infiltration and PGE2 production more strongly than betamethasone. These results suggest that betamethasone dipropionate is a potent glucocorticoid in guinea pigs. CONCLUSIONS: Structure-activity relationships of glucocorticoids in the guinea pig EIL model may differ from those in the rat EIU model.

Endothelin-1 mediated Ca2+ influx does not occur through L-type voltage-dependent Ca2+ channels in cultured bovine trabecular meshwork cells.

We evaluated whether endothelin-1-induced changes in cytosolic Ca2+ concentration ([Ca2+]i) involve Ca2+ influx through L-type membrane voltage-dependent Ca2+ channels in cultured bovine trabecular meshwork (TM) cells. The cells were loaded with the fluorescent Ca2+ indicator fura-2 and cell-associated fluorescence was measured with a digital video-imaging analyzer. Application of carbachol (10(-3) M) and norepinephrine (10(-5) M) increased [Ca2+]i only transiently. Endothelin-1 (10(-9) M to 10(-7) M) also increased [Ca2+]i in a concentration-dependent manner, and its effects were larger than those of carbachol and norepinephrine. Unlike carbachol or norepinephrine, endothelin-1 evoked a peak transient effect followed by a sustained elevated level of [Ca2+]i. The only level of the sustained elevated component was dependent on extracellular Ca2+. However, pretreatment with diltiazem (10(-5) M and 10(-4) M), an L-type Ca2+ channel blocker, did not affect either component of the endothelin-1-induced increase in [Ca2+]i. These results suggest that in cultured bovine TM cells endothelin-1 receptors are coupled to Ca2+ signaling mechanisms. However, the extracellular Ca(2+)-dependent increase in [Ca2+]i may not involve Ca2+ influx through L-type Ca2+ channels.

Effects of the cytokines on the proliferation of and collagen synthesis by human cataract lens epithelial cells.

AIMS: To assess the effects of the cytokines, interleukin-1 (IL-1), IL-1 receptor antagonist (IL-1ra), transforming growth factor-beta 2 (TGF-beta 2) and basic fibroblast growth factor (b-FGF), on the mitosis of and collagen synthesis by lens epithelial cells (LECs) of human cataracts. METHODS: The anterior lens capsule with attached LECs was obtained by capsulotomy during cataract surgery and cultured. The cultures at 2 to 3 weeks before confluency were used for the experiments. To quantify the mitosis and collagen synthesis, the incorporation of 3H-thymidine and 3H-proline, respectively, into the LECs was measured by a scintillation counter at 48 hours and 24 hours, respectively, after addition of the cytokine at various concentrations into the incubation medium. RESULTS: IL-1 and b-FGF increased the mitosis and collagen synthesis significantly, but IL-1ra significantly decreased the mitosis while leaving the collagen synthesis intact. TGF-beta 2 decreased the mitosis significantly, but increased the collagen synthesis significantly. CONCLUSION: These cytokines may play an important role in an autocrine or paracrine pathway in the proliferation of residual LECs after cataract surgery. Elucidation of the role of these cytokines may lead to the development of new therapies for the prevention of secondary cataract.

Effect of intraocular sustained release of indomethacin on postoperative inflammation and posterior capsule opacification.

PURPOSE: To assess whether the sustained release of indomethacin significantly reduces postoperative inflammation and posterior capsule opacification (PCO). SETTING: Nishi Eye Hospital, Jinshikai Medical Foundation, Osaka, Japan. METHODS: A 7 mm diameter, 1 mm thick polylactic-polyglycolic acid disk containing 7 mg of indomethacin was implanted in five rabbit eyes after continuous curvilinear capsulorhexis and phacoemulsification. The disk and an IOL placed above it were implanted in the capsular bag. The contralateral eyes, which served as controls, received a disk without indomethacin and the same type IOL in the same manner. RESULTS: The indomethacin was fully released within 3 weeks in vitro, a release rate of about 14 micrograms/h. Postoperatively, aqueous flare intensity was significantly lower at days 2, 3, and 4 and at weeks 1, 2, and 3. Prostaglandin E2 was not detectable in the aqueous humor of the indomethacin-treated eyes on day 3 and at week 4. In the control eyes, mean concentration was 491 pg/ml +/- 54 (SD) and 990 +/- 243 pg/ml, respectively. Histopathological examination showed no significant decrease in PCO. CONCLUSION: Although sustained release of indomethacin significantly decreased inflammation, it did not reduce PCO.