Effect of COX inhibitors on VEGF-induced retinal vascular leakage and experimental corneal and choroidal neovascularization

The primary objective of this study was to evaluate the effect of cyclooxygenase (COX) inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs), in two in vivo models of VEGF-dependent corneal and choroidal angiogenesis and two in vivo models of VEGF-mediated vascular leakage. Non-selective COX inhibitors (the NSAIDs indomethacin and flunixin, p.o. or i.p.), the COX-1 selective inhibitor SC-560 (s.c. or i.p.), and the COX-2 selective inhibitor NS-398 (s.c. or i.p.) were evaluated in four experimental models. Choroidal neovascularization was induced in Brown Norway rats by argon laser photocoagulation and measured after ten days. Corneal neovascularization was induced by alkaline cautery in Sprague-Dawley rats and measured after four days. VEGF protein levels in the cornea were quantified by ELISA. VEGF-induced intradermal extravasation of Evans blue dye (EBD)-albumin was assayed in Hartley guinea pigs. Intravitreal VEGF-induced blood-retinal barrier breakdown was assayed by scanning ocular fluorophotometry in Dutch Belt rabbits. Indomethacin (1 or 3 mg kg(-1) day(-1), p.o.), SC-560 (20 mg kg(-1) day(-1), s.c.), and NS-398 (20 mg kg(-1) day(-1), s.c.) failed to inhibit laser-induced CNV. CNV was inhibited, however, by the corticosteroid dexamethasone (0.5 mg kg(-1) day(-1); p.o. or s.c.; 99% or 90% inhibition; p<0.01 or p<0.001, respectively). In contrast, cautery-induced corneal angiogenesis was inhibited partially by the NSAID indomethacin and the COX-2 selective inhibitor NS-398. Indomethacin, 3.5 or 7 mg kg(-1) day(-1), inhibited corneal neovascularization by 56% (p<0.001) or 68% (p<0.001) respectively. Similar partial inhibition of angiogenesis in the cornea model was observed with NS-398 (10 or 20 mg kg(-1) day(-1), s.c. or i.p.; 54% inhibition, p<0.001), but not with the COX-1 selective SC-560 (10 or 20 mg kg(-1) day(-1), s.c.). In the cornea, VEGF protein is dramatically upregulated 24 and 48 hr after cautery, and both indomethacin and NS-398-but not SC-560-significantly inhibited this VEGF upregulation. In experimental models of VEGF-induced vascular leakage, COX inhibitors had no effect on dermal or retinal vascular responses to VEGF. The NSAIDs indomethacin (7.5 or 20 mg kg(-1), p.o. or i.p.) and flunixin (12.5 mg kg(-1), i.p.) failed to inhibit VEGF-induced dermal extravasation of EBD-albumin in guinea pigs. In contrast, L-NAME (25 or 50 mg kg(-1), p.o.)-an anti-vasodilatory inhibitor of nitric oxide synthase-dose-dependently inhibited up to 64% (p<0.001) of this dermal vascular leakage. VEGF-mediated retinal vascular leakage was not blocked by COX inhibition. Intravitreal VEGF-induced BRB breakdown--which was completely blocked by VEGF neutralizing s-Flt-1/Fc protein (intravitreal co-administration; p<0.001)--was not inhibited by indomethacin (20 mg kg(-1) day(-1), s.c.). Although COX inhibitors were ineffective at blocking experimental CNV, both non-selective and COX-2 selective inhibitors partially blocked severe inflammatory corneal angiogenesis and its concurrent upregulation of VEGF protein. These results suggest that eicosanoids produced by inducible COX-2 are among multiple mediators that modulate VEGF expression as a stimulus in inflammation-associated angiogenesis. The lack of effect with COX inhibitors on either VEGF-mediated dermal extravasation or VEGF-mediated blood-retinal barrier breakdown indicates that COX activity is not required for vascular leakage responses to VEGF.     

Inhibition of COX in ocular tissues: an in vitro model to identify selective COX-2 inhibitors.

The aim of this work was to study the regulation of LPS-stimulated PGE 2 synthesis by traditional NSAIDs (piroxicam and diclofenac) and a selective COX-2 inhibitor (NS-398), in cultured bovine corneal endothelial cells and retinal pigmentary epithelial cells. The IC50 values of piroxicam and diclofenac were compared with IC50 values of NS-398, diclofenac, in both types of cells, showed higher potency than piroxicam. Diclofenac seemed to be a COX-2 inhibitor because its IC50 values were similar to the IC50 values of NS-398. We suggest that this in vitro cell assay system could be useful for identifying compounds that selectively inhibit COX-2 in ocular tissues.     

Effects of salicylate and steroid on neutrophil migration and corneal blood vessel growth

Cauterization of the cornea results in emigration of neutrophils from the limbal blood vessels into the corneal tissue. Blood vessel proliferation follows, the stimulus for which is unknown. In this study, 0.1 M sodium salicylate drops administered topically to cauterized rat corneas over a 48-h period had an inhibitory effect on the migration of neutrophils from the limbal vessels 6 h after injury, but this was not maintained at 48 h. After 6 days of treatment, the salicylate had no effect on vessel growth into the cauterized rat cornea. Application of prednisolone disodium phosphate ointment to cauterized corneas also inhibited neutrophil migration at 6 h, but increased the extravascular neutrophils at 48 h. After 6 days of treatment, corneal blood vessel growth was significantly reduced. It was concluded that there is no consistent relation between the number of extravascular neutrophils at the corneal limbus and the extent of corneal blood vessel growth.     

Involvement of cyclooxygenase-2 in rat models of conjunctivitis

PURPOSE: Using two animal models to determine which isoform of cyclooxygenase (COX), constitutive COX-1 or inducible COX-2, is involved in the progression of anterior ocular inflammation. METHODS: Lambda-carrageenan (500 mg/eye) or bacterial lipopolysaccharide (LPS; 3 mg/eye) was injected into rat conjunctiva to induce conjunctivitis. Vascular permeability in inflamed conjunctiva was measured by uptake of systemic Evans blue. Changes in mRNA for COX-1 and COX-2 in conjunctiva were detected by RT-PCR. Changes in COX-2 protein were detected by immunoblotting after immunoprecipitation. To assess involvement of COX-2 in carrageenan and LPS-induced conjunctivitis, NS-398 (a selective COX-2 inhibitor) or indomethacin (non-selective COX inhibitor) was topically administrated at 15 and 30 minutes before inflammatory stimulator-injection. RESULTS: In the carrageenan-injected model, the dye content of conjunctiva (12.4 +/- 2.8 mg/eye) was significantly increased 4 hours after injection compared to saline-injected control rats (3.7 +/-1.1 mg/eye). mRNA for COX-2 was significantly increased by 2 hours and gradually increased until 24 hours; COX-1 mRNA did not show major changes until 24 hours after injection. COX-2 protein was markedly elevated 4 hours after injection of carrageenan. COX-2 protein levels were well correlated with increased mRNA levels. In the LPS-injected model, the dye content of conjunctiva (5.8 +/- 1.2 mg/eye) was significantly increased 4 hours after injection compared to saline-injected control rats (3.1 +/- 0.6 mg/eye). Expression of COX-2 mRNA was increased 1 hour after injection, peaked at 2 hours, and decreased at 4 hours. mRNA for COX-1 did not change by 24 hours. COX- 2 protein increased 2 hours after injection of LPS. COX-2 protein levels were well correlated with increased mRNA. Topical administration of 1% NS-398 exhibited strong inhibition of dye-leakage into conjunctiva 4 hours after injection of carrageenan or LPS, since 59% or 83% of dye-uptake were inhibited, respectively. 1% of indomethacin eye drops showed only a minimal effect. CONCLUSIONS: These results suggest that the mechanism for anterior ocular inflammation may be due to up-regulation of COX-2.     

Piroxicam与NS-398对角膜新生血管抑制作用及对兔角膜上皮毒性作用的比较

目的:比较Piroxicam(环氧化酶-1选择抑制剂)和NS-398(环氧化酶-2选择性抑制剂)对角膜新生血管的抑制作用并比较两者对角膜上皮细胞的毒性作用。方法:将含20μg脂多糖的缓释小丸植入兔角膜基质以诱导角膜新生血管。使用不同浓度的Piroxi-cam和NS-398滴眼剂滴眼,植入10d后分析角膜新生血管的面积。采用MTT法分析Piroxicam和NS-398对角膜上皮细胞的毒性作用。结果:0.01μmol/LNS-398和0.01μmol/LPiroxi-cam组兔角膜新生血管生长浓密,其它组因抑制剂的不同及浓度的不同角膜新生血管面积有不同程度的减低。在0.5,5及50μmol/L时,NS-398较Piroxicam对角膜上皮细胞的毒性低。结论:环氧化酶-1特异性抑制剂和环氧化酶-2特异性抑制剂对角膜新生血管形成均有抑制作用。相同浓度时,NS-398的抑制效果强于Piroxicam。在高浓度时NS-398较Piroxicam对角膜上皮细胞的毒性低。     

Corneal stimulation of MMP-1, -9 and uPA by platelet-activating factor is mediated by cyclooxygenase-2 metabolites

PURPOSE: This study was undertaken to evaluate the significance of cyclooxygenase-2 (COX-2) activity on urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs)-1 and -9 induction in cornea following platelet-activating factor (PAF) treatment. METHODS: Corneal organ cultures were pre-treated with increasing concentrations of COX-2-specific inhibitors NS398 or nimesulide prior to PAF stimulation. To determine the effect of exogenous prostaglandins (PGs) on uPA, MMP-1 and MMP-9 levels, corneas were pre-treated with COX-2 inhibitors followed by the addition of 2.5 microM PGD2, PGE2 or PGF2alpha. The levels of uPA and MMP-9 were assayed by casein and gelatin zymography, respectively. MMP-1 levels were determined by Western Blot analysis. RESULTS: The increase in uPA, MMP-9 and MMP-1 levels detected in corneal organ cultures treated with 100 nM cPAF was blocked by 5 microM NS398 and 10 microM nimesulide, concentrations at which these inhibitors selectively inhibit COX-2 activity. Furthermore, pre-incubation with COX-2 inhibitors, followed by supplementation with PGD2, PGE2 or PGF 2alpha, increases uPA, MMP-9 and MMP-1 levels in corneas similar to and in some cases greater than that produced by cPAF treatment alone. CONCLUSIONS: During corneal injury and inflamation, PAF is an important factor in the activation of proteolytic cascades, which could lead to corneal epithelial defects and ultimately ulceration. One important goal in treating these defects is to modulate the activity of enzymes that destroy the extracellular matrix. Our results suggest that COX-2 induction following PAF stimulation and subsequent eicosanoid release may play a crucial role in the induction of uPA, MMP-1 and MMP-9 enzymes. Specific COX-2 inhibition could therefore block the actions of PAF when inflammation is sustained.     

Effect of irradiation on vascularization of corneas grafted onto chorioallantoic membranes

Several studies have shown that total body irradiation decreases the angiogenic response to corneal cauterization. This inhibition could be due to alterations in angiogenic stimuli within injured corneas and/or to a decreased ability of irradiated animals to respond to such stimuli. To determine whether total body irradiation specifically affects angiogenic stimuli within injured corneal tissue, cauterized corneas from mice exposed to 900 rads of total body irradiation and from non-irradiated controls were grafted onto the chorioallantoic membranes (CAM) of chick embryos and their abilities to stimulate the ingrowth of healthy embryonic blood vessels were compared. Cauterized corneas incorporated into CAM mesenchymal tissue were invaded by blood vessels in 34.6% of the irradiated group and in 75% of the non-irradiated controls. This difference in the two groups was statistically significant (P less than 0.03). Total body irradiation significantly decreased the frequency of vascular invasion of cauterized corneal tissues by healthy CAM blood vessels. This finding suggests that total body irradiation can specifically affect the stimulus for angiogenesis within cauterized corneas.     

Expression of integrins and MMPs during alkaline-burn-induced corneal angiogenesis

PURPOSE: To determine in a corneal alkaline burn model of angiogenesis whether the expression of integrins and MMPs is consistent with a VEGF-induced angiogenic response. METHODS: Neovascularization in female Sprague-Dawley rats was induced by alkaline cauterization of the central cornea. RT-PCR for integrins alpha(1), alpha(2), beta(3), and beta(5); the endothelial marker CD31; and metalloproteinases MMP-2 and MT1-MMP was performed on naive corneas and on cauterized corneas 72 and 288 hours after cautery. Analyses of protein and MMP expression were conducted on naive corneas and on cauterized corneas 24, 72, 120, and 168 hours after cautery by immunofluorescence microscopy and gelatin zymography. RESULTS: RT-PCR indicated a correlation between the induced angiogenic response and the expression of alpha(1) and beta(3) integrin subunits and MT1-MMP. Immunohistochemical analysis indicated that alpha(1), alpha(2), alpha(5), and beta(5) integrins and MMP-2 and MT1-MMP were expressed on the newly developing vasculature. The beta(3) integrin was preferentially expressed on platelets. CONCLUSIONS: Integrin expression during neovascularization of rat corneas in response to alkaline injury correlates with an angiogenic response that uses the VEGF/alpha(v)beta(5) pathway. MMP-2 and MT1-MMP, but not MMP-9, are expressed in a pattern consistent with their involvement in the angiogenic response.     

Topical indomethacin effect on neovascularisation of the cornea and on prostaglandin E2 levels.

The effect of indomethacin 1% drops against a placebo (NaCl 0.9% solution) was tested in rabbits' corneas injured by a standardized chemical burn. The corneas treated with indomethacin 1% had less neovascularisation and a much smaller increase in the PGE2 levels as compared with the control fellow eyes treated with a placebo. It appears that indomethacin 1% drops have a clear effect on the inflammatory response of the cornea and deserve a clinical trial in selected patients suffering from corneal inflammatory changes.     

Effect of inhibition of inflammatory mediators on trauma-induced stromal edema

PURPOSE: To determine the specific biochemical pathways involved in the initial-phase inflammatory response that causes stromal edema after epithelial debridement of the rabbit cornea. METHODS: Adult New Zealand White rabbit corneas were treated with 2 mM synthetic inhibitor of metalloproteinase (SIMP)-1, 1 mM DFU (a specific cyclooxygenase [COX]-2 inhibitor) in 50/50 dimethyl sulfoxide (DMSO)/Ringer's solution, 300 KIU aprotinin (a serine protease inhibitor), 0.05% or 0.10% IL-1 receptor type II solution, 1 mM gliotoxin (a Ras farnesyltransferase inhibitor), or vehicle alone (the control). These were applied topically in vivo in five doses over a 3-hour period except IL-1 receptor type II, which was applied in vitro. After rabbits were killed, the corneas were mounted in perfusion chambers with the endothelium bathed in a modified Ringer's solution and the epithelium bathed with silicone oil. Corneal thickness was measured with an automatic specular microscope. The corneal thickness typically stabilized 1 hour after mounting. After stabilization, the corneal epithelium was removed with a rotating bristle brush and stromal thickness monitored for 1 hour. Paired control corneas were treated similarly. RESULTS. Stromal swelling after epithelial debridement was significantly less in most treated corneas, compared with untreated controls: 18.4 +/- 5.3 microm vs. 28.6 +/- 7.7 microm (n = 6, P = 0.004); SIMP-1, 18.7 +/- 10.2 microm vs. 34.3 +/- 10.2 microm (n = 7, P = 0.02); DFU, 19.3 +/- 10.2 microm vs. 23.5 +/- 8.4 microm (n = 6, P = 0.01); and IL-1 receptor type II (0.05%), 26.2 +/- 5.6 microm vs. 30.4 +/- 5.6 microm (n = 5, P = 0.03) and (0.10%), 26.6 +/- 5.6 microm vs. 32.1 +/- 7.4 microm (n = 8, P = 0.03). Gliotoxin was not effective (21.5 +/- 8.0 microm vs. 21.9 +/- 6.2 microm; n = 5, P = 0.94). CONCLUSIONS: The reduction of stromal edema after topical administration of the inhibitors demonstrates the involvement of the COX-2 enzyme, the matrix metalloproteinase family, plasminogens, and the IL-1 system in the trauma-induced inflammatory response of the rabbit cornea. The inflammatory process in the cornea associated with trauma can proceed along multiple redundant parallel pathways.     

Effects of intravenous administration of FR122047 (a selective cyclooxygenase 1 inhibitor) and FR188582 (a selective cyclooxygenase 2 inhibitor) on prostaglandin-E2-induced aqueous flare elevation in pigmented rabbits

PURPOSE: Two isoforms of cyclooxygenase (COX-1 and COX-2) exist. To determine in vivo effects of the intravenous administration of FR122047 (a selective COX-1 inhibitor), FR188582 (a selective COX-2 inhibitor), diclofenac sodium or dexamethasone phosphate disodium on prostaglandin-E2 (PGE2)-induced aqueous flare elevation and mRNA levels for COX-1 and COX-2 in pigmented rabbits. METHODS: To produce aqueous flare elevation in rabbits, PGE2, 25 microg/ml, was applied to the cornea with the use of a glass cylinder. FR122047, FR188582, diclofenac sodium or dexamethasone phosphate disodium was intravenously injected before PGE2 application. Aqueous flare was measured with a laser flare-cell meter. The mRNA levels for COX-1 and COX-2 in the iris-ciliary body were determined by real-time polymerase chain reaction. RESULTS: FR122047, FR188582 and diclofenac sodium (15 micromol/kg each) injected intravenously 30 min before PGE2 application inhibited 29 +/- 5, 40 +/- 12 and 50 +/- 9% of aqueous flare elevation, respectively. Simultaneous injection of FR122047 (15 micromol/kg) and FR188582 (15 micromol/kg) 30 min before PGE2 application inhibited 61 +/- 8% of flare elevation. Dexamethasone phosphate disodium (15 micromol/kg) injected intravenously 300 min before PGE2 application inhibited 68 +/- 8% of aqueous flare elevation. Less than 3-fold changes in mRNA levels for COX-1 and COX-2 in the iris-ciliary body were noted after PGE2, FR122047, FR188582, diclofenac sodium or dexamethasone phosphate disodium treatment. CONCLUSION: It is possible that enzyme activities of both COX-1 and COX-2 may be involved in the mechanism of PGE2-induced aqueous flare elevation in pigmented rabbits.     

The effect of hypoxia on endogenous corneal epithelial eicosanoids

PURPOSE: Injury to the corneal epithelium increases arachidonic acid (AA) metabolism through the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 pathways. The authors used the rabbit corneal organ culture model to demonstrate the effect of hypoxia on the endogenous formation of 12-hydroxy-5,8,11,14-eicosatetraenoic acid (12-HETE), 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), and prostaglandin (PG) E2 by the intact cornea in the absence of exogenously added cofactors or substrate. METHODS: Rabbit corneas were isolated and cultured for 24 hours in normoxia or hypoxia. After culture, PGE2 in media was quantitated by enzyme immunoassay. 12-HETE and 12-HETrE were extracted from culture media and corneal epithelium and quantitated by negative chemical ionization-gas chromatography-mass spectrometry. COX-1 and -2 protein expression in corneal epithelium was determined by Western blot. Acute (2 hours) COX activity in normoxia and hypoxia was determined as the conversion rate of [14C]AA to [14C]PGE2, quantitated through reverse-phase-high-performance liquid chromatography and radiodetection. RESULTS: In the media of cultured rabbit corneas, both 12-HETE and 12-HETrE were detected, with 12-HETrE levels being four times higher. Hypoxia did not significantly increase extracellular 12-HETE or 12-HETrE; however, it caused more than 90% inhibition of PGE2 synthesis. Intracellular 12-HETE and 12-HETrE were undetectable in normal corneas but increased to 7.7+/-1.3 and 2.2+/-0.4 ng/mg protein, respectively, after 24 hours in culture. Culture in hypoxia further increased intracellular 12-HETE threefold but had no additional effect on 12-HETrE. CONCLUSIONS: Hypoxia creates an environment in which epithelial COX activity is severely suppressed, whereas cytochrome P450-AA and/or 12-LOX metabolizing activity is maintained or enhanced. Additionally, the findings suggest that 12-HETE produced by the corneal epithelium acts intracellularly to promote corneal edema, whereas 12-HETrE acts in a paracrine manner to initiate an inflammatory cascade that can elicit neutrophil chemotaxis and neovascularization of the cornea.     

Drug modification of angiogenesis in a rat cornea model

PURPOSE: To evaluate the influence of some widely used antiglaucoma agents on angiogenesis in a novel rat cornea model. METHODS: Angiogenesis was induced in 32 rats by slow-release polymer pellets containing basic fibroblast growth factor (bFGF) placed in a corneal micropocket. Angiogenesis was later measured and compared in groups of rats given one of four antiglaucoma drug therapies and one control group. The drugs were commercially available preparations of prostaglandins, beta-blockers, alpha-2 agonists, and carbonic anhydrase inhibitors given for 7 days in a manner similar to that used in humans. Growth was measured by calculating the maximum linear vessel growth divided by pellet-limbus distance. RESULTS: Biomicroscopic observation disclosed that all tested animals showed an induction of neovascular reactions in their corneal stroma. The growth index results for the control, latanoprost, dorzolamide, brimonidine, and timolol malate groups were 1.65 +/- 0.16, 1.98 +/- 0.18, 1.85 +/- 0.19, 2.03 +/- 0.38, and 1.65 +/- 0.14, respectively, confirming the hypothesis that topically delivered antiglaucoma drugs modify the normal angiogenic response. Of them, the prostaglandins showed the most prominent angiogenic stimulatory effect (P = 0.03). CONCLUSIONS: This modified micropocket assay of corneal angiogenesis in rats demonstrated the stimulatory effect of several widely used topically delivered antiglaucoma medications on the angiogenic process. The results indicate that the selection of drugs for treating different ophthalmic diseases should take into account their influence on angiogenic processes.     

Suppressive effects of indomethacin on thermally induced neovascularization of rabbit corneas.

In 16 rabbits with bilateral corneal burns, indomethacin was administered topically to one eye on each day after the lesion was made to determine the effect of a prostaglandin inhibitor on the corneal neovascular response to experimental thermal burns. Comparison of the two eyes showed a reduction of both hyperemia and neovascularization in indomethacin-treated eyes during the first five days after injury. Histologic observations during this period showed a reduction in polymorphonuclear cell infiltration in the treated corneas. Inhibition of prostaglandin synthesis by indomethacin apparently led to a reduction in the inflammatory response and the subsequent corneal neovascularization.     

Peripheral adrenergic stimulation and indomethacin in experimental ocular shedding of HSV

The application of 6-hydroxydopamine to the cornea by iontophoresis, followed by topical epinephrine, effectively induces herpes simplex virus (HSV) shedding from the external eye of latently infected rabbits. In this study the beta adrenergic blocker, Timolol, reduced virus shedding when applied immediately before the epinephrine, but continued administration resulted in increased viral shedding. While indomethacin, a prostaglandin synthesis inhibitor decreased HSV replication in cell culture, it failed to decrease virus shedding when applied topically to the eye in adrenergically stimulated animals. Timolol may act then by its effect on the peripheral cells of the eye rather than by stimulation of virus production in ganglionic neurons. These same animals were subsequently tested for latent infection of the trigeminal and superior cervical ganglia and corneas 14 months after primary infection. Only 2 of 14 animals had virus in the trigeminal ganglia, a finding which suggests that latent virus may be depleted by repeated reactivations. Virus was recovered from corneas of five rabbits by co-cultivation so it is possible that corneal latency occurs in this rabbit model as it does in humans.     

NS398对白介素1α诱导兔角膜基质细胞环氧化酶2表达的抑制

目的探讨氮-2,环己氧-4,硝基苯-甲基磺胺（NS398）对白介素1α（IL-1α）诱导的兔角膜基质细胞环氧化酶2（COX-2）表达的影响。方法体外培养兔角膜基质细胞,实验组分别以含0、25、50、100、200μmol／LNS398的培养液孵育2h后加入IL-1α诱导COX-2表达,24h后实时荧光定量聚合酶链反应（Real-Time PCR）检测兔角膜基质细胞中COX-2基因表达的差异,四甲基偶氮唑盐（MTT）比色法检测不同浓度NS398对细胞生长的影响,并与对照组进行比较。结果Real—Time PCR结果显示IL—1α诱导后24h各组COX-2 mRNA表达量差异有统计学意义（F=988．45,P〈0．01）;对照组与各实验组以及各实验组之间COX-2 mRNA表达量进行多重比较可见,0μmol／LNS398浓度组与对照组之间差异无统计学意义（q=1．3322,P〉0．05）,其他NS398浓度组与对照组相比差异均有统计学意义（q=34．7896,48．3298,65．8010,70．8131,P〈0．01）,随培养液中NS398浓度的增加,IL-1α刺激后兔角膜基质细胞中COX-2 mRNA表达量不断下降（q=36．1218,49．6620,67．1332,72．1453,13．5402,31．0114,36．0235,17．4712,22．4833,5．0121,P〈0．01）;MTT法检测结果显示,100μmol／L、200μmol／L的NS398对兔角膜基质细胞生长均有明显的抑制作用（q=12．7693,20．9087,P〈0．01）。结论NS398能够有效抑制IL-1α诱导的兔角膜基质细胞COX-2的表达,但超过一定浓度会对细胞产生明显毒性作用。     

Activation of the epidermal growth factor receptor in optic nerve astrocytes leads to early and transient induction of cyclooxygenase-2

PURPOSE: The epidermal growth factor receptor (EGFR) appears in astrocytes after neural injury. The authors' laboratory has reported the presence of EGFR in glaucomatous optic nerves. The activation of EGFR is often associated with induction of cyclooxygenase (COX)-2. In this study, the induction of COX-2 pathway in rat optic nerve astrocytes was investigated. METHODS: Induction of COX-2 was determined by immunoblot and immunocytochemistry in optic nerve astrocytes stimulated with EGF. EGF-induced prostaglandin (PG)E(2) release into the culture medium was assayed by ELISA. The effects of the EGFR tyrosine kinase inhibitor, AG1478, were studied on COX-2 expression and PGE(2) synthesis. In rat optic nerve transection and a rat optic nerve explant culture model, the relationship between the expression of COX-2 and activation of EGFR was examined. RESULTS: Activation of EGFR caused the rapid and transient induction of COX-2 in optic nerve astrocytes. The level of COX-2 was rapidly upregulated in optic nerves after axotomy and in an optic nerve explant culture model. When induced, COX-2 localized to the nuclear membrane of the astrocytes. When COX-2 was induced in response to activation of EGFR, the activated astrocytes produced and released the proinflammatory mediator, PGE(2), in a time-dependent manner. EGF-stimulated induction of COX-2 protein and synthesis of PGE(2) were abolished by the EGFR tyrosine kinase inhibitor AG1478. The stimulatory action of EGF on release of PGE(2) was inhibited by the COX-2-selective inhibitor NS398. CONCLUSIONS: The data demonstrate that the activation of EGFR in optic nerve astrocytes leads to the induction of the immediate early gene COX-2 and subsequent signaling through the synthesis of PGE(2). This early signal of neural tissue damage may be important in setting up secondary events in the damaged tissue.     

氟康唑眼液滴眼的眼内药代动力学实验研究

目的　研究 0 5 %氟康唑滴眼液在兔角膜的渗透性及其在角膜和房水中的药代动力学行为。方法　将新西兰大白兔双眼滴入 0 5 %氟康唑眼液后 ,分别于不同时间处死家兔 ,将获取的房水和角膜组织用反相高效液相色谱法 (highperformanceliquidchromatography ,HPLC)进行定量测定。色谱条件 :色谱柱为SpherisorbC18,粒径为 5 μm ,色谱柱长度和直径为 2 0 0 0mm× 4 6mm ,柱温为 4 5℃ ,流动相为 0 0 5mol/L磷酸二氢钾 甲醇 (6 5∶35 ) ,流速为 1ml/min ,检测波长为 2 6 5nm。采用非线性最小二乘法进行计算机拟合求得药代动学参数。结果　采用反相HPLC法可以将氟康唑同其他杂质很好分离 ,最低定量浓度为 0 1mg/L ,氟康唑在组织中的回收率平均为 90 6 %。 0 5 %氟康唑滴眼后的各时间点角膜组织及房水中均可检测出氟康唑的含量 ,其中角膜组织中于滴药后 2min时含量最高为 (15 2 0± 1 95 ) μg/g ,房水中 15min时的含量最高为 (2 39± 0 92 )mg/L。角膜上皮渗透系数为1 0 6× 10 -5。角膜组织中药物浓度半衰期为 6 3 96min ,房水中为 4 2 14min。结论　氟康唑可用于真菌性角膜炎的局部抗真菌治疗 ,是否用于治疗真菌性眼内炎尚待研究     

Roles of thrombospondin-1 and -2 in regulating corneal and iris angiogenesis

PURPOSE: Thrombospondin (TSP)-1 and -2 are important antiangiogenic factors thought to be involved in maintaining corneal avascularity (angiogenic privilege). This study was undertaken to investigate whether deficiencies of these factors altered developmental and inflammation-induced angiogenesis in the cornea and developmental angiogenesis of the iris of mice. METHODS: Expression of TSP-1 and -2 mRNA and protein was assayed in cornea and iris stroma by RT-PCR and Western blot. Corneas and irides of TSP-1(-/-), TSP-2(-/-), and TSP-1,2(-/-) mice aged 2, 3, and 6 months, and wild-type control mice, were analyzed for spontaneous angiogenesis biomicroscopically, histologically, and with CD31 immunohistochemistry. The mouse model of suture-induced, inflammatory corneal neovascularization was used to evaluate the lack of TSP-1,2 and both TSPs on induced-corneal angiogenesis. Seven days after intrastromal placement of three 11-0 sutures, vascularized areas were analyzed morphometrically on CD31-stained corneal flatmounts. RESULTS: Corneas and irises from normal mouse eyes constitutively expressed TSP-1 and -2 mRNAs and proteins. Corneas of TSP-1(-/-), -2(-/-), and -1,2(-/-) mice displayed no evidence of spontaneous developmental-postnatal angiogenesis, although irises of these mice contained significantly increased iris vessel density compared with wild-type animals (P < 0.01). One week after suturing, corneas of all TSP(-/-) mice had significantly greater corneal angiogenesis than those of control mice (P < 0.05). TSP-1(-/-) had a significantly greater effect on induced corneal neovascularization than did TSP-2(-/-), with the opposite being the case in developmental iris angiogenesis (P < 0.01). CONCLUSIONS: Corneal avascularity during development is redundantly regulated, shown by the fact that lack of the antiangiogenic factors TSP-1 and/or -2 resulted in no spontaneous corneal angiogenesis. By contrast, TSP-1, more than TSP-2, helps to suppress inflammation-induced corneal angiogenesis postnatally, implying that angiogenic privilege in the cornea is actively maintained.