Intravitreal Injection Of Endotoxin Research Articles

Amelioration of Endotoxin-Induced Uveitis in Rabbit by Topical Administration of Tacrolimus Proglycosome Nano-Vesicles.

This work was aimed to improve the efficacy of tacrolimus in the treatment of endotoxin-induced uveitis (EIU) using propylene glycol modified lipid vesicles termed as proglycosome nano-vesicles (PNVs). PNVs were prepared by modified film hydration method. Experimental uveitis in rabbit eye was induced by an intravitreal injection of 20μL of the endotoxin solution containing 100ng of lipopolysaccharide endotoxin. Invivo efficacy of PNVs was determined by studying clinical symptoms of uveitis using slit lamp examination and by quantitatively measuring levels of tumor necrosis factor-alpha, interleukin-6, leukocytes and total proteins in aqueous humor, 24h after intravitreal injection of endotoxin. Comparison was made with healthy, untreated and tacrolimus solution treated eyes. PNVs developed were nano-sized, deformable and showed sustained release of tacrolimus over period of 12h. Invivo results indicated statistically significant difference between the effects of PNVs in the treatment of EIU compared to tacrolimus. PNV treatment not only subsides clinical symptoms of uveitis but also prevented breakdown of blood aqueous barrier. Tacrolimus loaded PNVs are potential new topical treatment for uveitis.

Determination of a No Observable Effect Level for Endotoxin Following a Single Intravitreal Administration to Cynomolgus Monkeys.

Purpose: To characterize the inflammatory response and determine the no-observable-effect level (NOEL) in cynomolgus monkey eyes after intravitreal (ITV) injection of endotoxin. Methods: The inflammatory response to endotoxin was assessed in a single-dose study in monkeys at doses of 0.01 to 0.51 endotoxin units (EU)/eye. Tolerability was assessed by clinical ophthalmic examinations, intraocular pressure measurements, fundus color photography, optical coherence tomography, and anatomic pathology. Results: ITV injection of endotoxin at ≥0.04 EU/eye resulted in a dose-related anterior segment inflammatory response. No aqueous flare or cell was noted in the 0.01 EU/eye dose group. A more delayed posterior segment response characterized by vitreous cell was observed beginning on day 5, peaking on day 15, and decreasing in some groups. Microscopic findings of mononuclear cell infiltrates in the vitreous were observed in eyes given ≥0.21 EU/eye. Conclusion: The NOEL for ITV endotoxin in cynomolgus monkeys was 0.01 EU/eye, suggesting that this species is as sensitive as rabbits to the effects of endotoxin. The vitreous cavity also appears more sensitive to endotoxin than the anterior segment/aqueous chamber. Overall, the magnitude of the inflammatory response at ≥0.04 EU/eye suggests that dose-response curve in monkeys is steeper than in rabbits. These data highlight the importance of assessing endotoxin level in ITV formulations, as levels as low as 0.04 EU/eye may confound the safety evaluations of ITV therapeutics in cynomolgus monkeys.

Evaluation of anti-inflammatory impact of dexamethasone-loaded PCL-PEG-PCL micelles on endotoxin-induced uveitis in rabbits

The aim of this study was to investigate the capability of polycaprolactone-polyethylene glycol-polycaprolactone (PCL-PEG-PCL) micelles in improving the anti-inflammatory effects of dexamethasone (DEX). A film hydration method was used for the preparation of the DEX-loaded PCL-PEG-PCL micelles. In vitro cytotoxicity of the micelles obtained was investigated on L929 cells. Cellular uptake was studied by using flow cytometry and fluorescence microscopy. Anterior uveitis was induced in a group of rabbits by intravitreal injection of endotoxin from Salmonella typhimurium. The severity of inflammation-induced was clinically graded by using Hogan’s classification method. Protein concentration in the aqueous humor was also measured. The micelles exhibited suitable compatibility on L929 cells and were taken up by the cells in a concentration- and time-dependent manner. The DEX-loaded micelles could reduce the clinical symptoms of uveitis after a lag-time. At 24 and 36 h after the LPS injection, the PCL-PEG-PCL micelles showed a better inhibitory effect on uveitis than the marketed eye drop, the differences did not reach significant levels though. This study demonstrated the potential of the PCL-PEG-PCL micelles as carriers for DEX in treating anterior uveitis. However, this concept is still to be further investigated.

Determination of a No-Observable Effect Level for Endotoxin Following a Single Intravitreal Administration to Dutch Belted Rabbits.

The purpose of this study was to characterize the inflammatory response and determine a no-observable effect level (NOEL) in rabbit eyes after endotoxin intravitreal (ITV) injection. Fifty-three naïve male Dutch Belted rabbits were treated with a single 50-μL ITV injection ranging from 0.01 to 0.75 endotoxin units/eye (EU/eye) and monitored for up to 42 days post treatment. Ophthalmic examination included slit-lamp biomicroscopy and indirect ophthalmoscopy. Laser flare photometry was performed in a subset of animals. On days 2, 8, 16, and 43, a subset of animals was necropsied and eyes processed for histopathological evaluation. Intravitreal injection of endotoxin at ≥0.05 EU/eye resulted in a dose-related anterior segment inflammation response. No aqueous flare or cell response was noted in the 0.01 EU/eye dose group. A more delayed posterior segment response characterized by vitreal cell response was observed beginning on day 5, peaking on day 9, and decreasing starting on day 16 that persisted at trace to a level of 1+ on day 43. Microscopy findings of infiltrates of minimal mixed inflammatory cells in the vitreous and subconjunctiva and proteinaceous fluid in the anterior chamber and/or vitreous were observed in eyes given ≥0.1 EU/eye. We defined the NOEL for ITV endotoxin to be 0.01 EU/eye, suggesting that the vitreal cavity is more sensitive to the effects of endotoxin than the anterior segment and aqueous chamber. These data highlight the importance of assessing endotoxin level in intravitreal formulations, as levels as low as 0.05 EU/eye may confound the safety evaluations of intravitreal therapeutics in rabbits.

Pharmacokinetics of intravenously administered tigecycline in eye compartments: an experimental study.

The purpose of this study was to evaluate the ocular distribution of intravenously administered tigecycline in a rabbit uveitis model. Tigecycline, which has a broad spectrum of activity against many gram-positive, gram-negative, and anaerobic organisms, was given intravenously to rabbits at 7 mg/kg of body weight starting 24 h after induction of uveitis by intravitreal endotoxin injection. Tigecycline concentrations were determined by high performance liquid chromatography-mass spectrometry (LC-MS/MS) assay in the aqueous humor, vitreous humor, and plasma 1, 3, 6, and 24 h after administration of a single dose. The maximum concentrations were found within 1 h after the end of the intravenously given tigecycline, and were 1,308.60 ± 301.76 ng/mL in plasma, 181.40 ± 51.32 ng/mL in vitreous humor and 145.00 ± 55.29 ng/mL in aqueous humor of the inflamed eye. After 24 h, no drug was detectable in the aqueous and vitreous of the normal eyes, whereas small amounts of drug were detectable in inflamed eyes and in plasma. Tigecycline did not reach therapeutically significant levels in the aqueous and the vitreous humor of rabbit eyes. The findings suggest a limited role for intravenously administered tigecycline in the treatment of bacterial endophthalmitis.

Clearance of Intravitreal Daptomycin in Uveitis-Induced Rabbit Model

Purpose: To investigate the elimination rate of daptomycin after intravitreal injection in uveitis-induced rabbits.Materials and methods: Intravitreal injection of the single dose of 200 μg/0.05 mL daptomycin was administered to rabbits starting 24 h after induction of uveitis by an intravitreal endotoxin injection. Aqueous humor and vitreous humor samples of eight eyes per time point were collected at selected time intervals (1, 3, 6, 24, 48, 72 and 96 h), and the in vitreous half-life was calculated. Daptomycin concentrations in vitreous and aqueous humor were assayed with high-performance liquid chromatography.Results: The vitreous concentration was noted to decline slowly with time. The mean vitreous concentration was 23.25 ± 10.99 μg/mL and 11.10 ± 3.33 μg/mL at 96 h in inflamed and normal eyes, respectively. The vitreous daptomycin concentration showed an exponential decay with a half-life of 25.67 h in normal eyes and 34.6 h in inflamed eyes. The aqueous levels of daptomycin in normal eyes were low but remained significantly higher than those of inflamed eyes.Conclusions: Given that the injected dose corresponds to several times the minimum inhibitory concentrations of organisms most involved in endophthalmitis, and that therapeutic levels are present up to 96 h after injection, intravitreal daptomycin should be considered for the treatment of endophthalmitis caused by Gram-positive bacteria.

Ocular penetration of intravenously administered colistin in rabbit uveitis model.

The purpose of this study was to evaluate the ocular distribution of intravenously administered colistin in a rabbit uveitis model. Colistin, a polypeptide antibiotic against the multidrug-resistant (MDR) Gram-negative organisms, was given intravenously to rabbits at 5 mg/kg of body weight starting 24 h after induction of uveitis by intravitreal endotoxin injection. Colistin concentrations were determined by high-performance liquid chromatography-mass spectrometry assay in the aqueous humor, vitreous humor, and plasma 0.5, 3, 6, and 24 h after administration of a single dose. The maximum colistin concentrations (mean±standard deviation) were found 0.5 h after the end of the intravenous administration and were 9.48±2.0 μg/mL in plasma and 0.62±0.07 μg/mL in the aqueous humor of the inflamed eye. After 24 h, no drug was detectable in the aqueous of the inflamed eyes. Colistin was undetectable in the aqueous of contralateral normal eyes at all time points. Drug concentrations in all the vitreous samples from both inflamed and normal eyes were undetectable, except at the 3-h inflamed eye group, and a colistin concentration of 0.02±0.01 μg/mL was found. Plasma levels of colistin fell to 0.93±0.07 and 0.24±0.08 μg/mL, after 3 and 6 h, respectively, and were not detectable 24 h after the given dose. In our model, colistin did not reach therapeutically relevant levels in the aqueous and in the vitreous humor of rabbit eyes. The findings suggest a limited role for intravenously administered colistin in the treatment of Gram-negative bacterial endophthalmitis.

Polymeric triamcinolone acetonide nanoparticles as a new alternative in the treatment of uveitis: In vitro and in vivo studies

The purpose of this work was to improve the efficacy of triamcinolone acetonide (TA) in the treatment of endotoxin-induced uveitis (EIU) using a polymeric nanoparticulate drug delivery system. Poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles were prepared using a modified emulsification/solvent diffusion method. Processing factors affecting loading and size were also studied. After physicochemical studies including in vitro release, X-ray powder diffraction, differential scanning calorimetry, and scanning electron microscopy, in vivo studies were conducted using nanoparticles sized 195nm with 3.16% drug loading. Inflammatory factors such as flare, cell, and fibrin were studied in rabbit’s eye over 96h period, using laser flare meter and slit lamp examination. Inflammatory mediators such as NO, PGE2, cell, and protein were measured quantitatively 36h after intravitreal injection of endotoxin in aqueous humor, and the therapeutic effects were compared in different groups. Results indicated statistically significant differences between the effect of nanoparticles in the treatment of EIU compared to microparticles of TA and prednisolone acetate (PA). There were no significant differences between the effects of TA injection and TA nanoparticles. In conclusion, sustain release biodegradable TA nanoparticles are potential new topical treatment options which can provide better patient compliance.

Ocular penetration of caspofungin in a rabbit uveitis model

Little is known about the ocular penetration of echinocandin antifungals. We studied the ocular distribution of systemically administered caspofungin in a rabbit uveitis model. Caspofungin (1 mg/kg per day) was given intravenously to rabbits as a single dose or as repeated daily doses on 7 days starting 24 h after induction of unilateral uveitis by intravitreal endotoxin injection. Caspofungin concentrations were determined by high-performance liquid chromatography in the cornea, aqueous humor, vitreous humor, and serum 4, 8, 16, and 24 h after administration of a single dose and 24 h after the last of seven doses. The mean caspofungin concentration in the aqueous of the inflamed eye 4 and 8 h after single-dose administration was 1.30 +/- 0.39 microg/ml and 1.12 +/- 0.34 microg/ml, respectively. Drug concentrations decreased to 0.24 +/- 0.09 microg/ml at 16 h and 0.26 +/- 0.14 microg/ml at 24 h. In the vitreous of inflamed eyes drug levels were undetectable at all time points. No drug was found in the aqueous of inflamed eyes 24 h after the last of seven repeated doses, and the vitreous only contained trace amounts. In the corneas of inflamed eyes concentrations reached 1.64 +/- 0.48 microg/g at 4 h, peaked at 2.16 +/- 1.14 microg/g at 8 h, and declined to 1.87 +/- 0.52 microg/g and 1.49 +/- 0.48 microg/g at 16 and 24 h, respectively. After repeated dosing, corneal concentrations of caspofungin were 0.8 and 1.0 microg/g and below the limit of detection in two of four animals. In non-inflamed eyes no drug was detectable in the aqueous and vitreous humor, and the corneas at any time point. In our model, caspofungin reached therapeutically relevant levels in the aqueous and cornea but not in the vitreous humor of inflamed eyes. Intraocular drug deposition was critically dependent on a disrupted blood-eye barrier. These findings suggest a limited role for caspofungin in the treatment of fungal endophthalmitis.

Corneal Concentrations following Systemic Administration of Amphotericin B and Its Lipid Preparations in a Rabbit Model

Background: Amphotericin B (AmB) and various lipid formulations of AmB are available for the treatment of fungal infections of the eye. Currently, the recommended route of administration for the treatment of fungal keratitis is by topical application. Nevertheless, because of the risk of a difficult to treat exogenous fungal endophthalmitis, a combined topical and systemic treatment is frequently given when treating deep fungal keratitis. To date, little is known about the pure corneal availability of these drugs following systemic treatment. In this study, the corneal concentration following 7 daily doses of parenteral AmB lipid complex (ABLC) or liposomal AmB (L-AmB) was compared to that of AmB deoxycholate (D-AmB) in a rabbit model. Methods: Following induction of uveitis in one rabbit eye by intravitreal injection of endotoxin, daily doses of D-AmB (1 mg/kg), ABLC (5 mg/kg) or L-AmB (5 mg/kg) were given intravenously on 7 consecutive days. Five or more rabbits per treatment were used. AmB concentrations were determined by high-performance liquid chromatography (HPLC) in corneas collected at autopsy 24 h after the 7th and final dose. Data were analyzed using the Mann-Whitney U test. Results: After 7 days of treatment, mean corneal concentrations of AmB in the inflamed eyes were significantly higher (2.38 ± 1.47 µg/g; p < 0.01) following treatment with L-AmB compared with ABLC (<0.1 µg/g) and D-AmB (0.46 ± 0.2 µg/g). No AmB could be detected in the corneas of the non-inflamed eyes. Conclusion: In our rabbit model, AmB penetration into the cornea was significantly higher after systemic administration of L-AmB compared with conventional D-AmB or ABLC.

Ocular distribution of intravenously administered lipid formulations of amphotericin B in a rabbit model.

Little is known about the ocular penetration of amphotericin B (AMB) and its lipid formulations, the current drug of choice in fungal endophthalmitis. The ocular distribution of AMB lipid complex (ABLC), liposomal AMB (L-AMB), and AMB deoxycholate (D-AMB) was studied in a rabbit model. D-AMB (1 mg/kg of body weight/day), ABLC (5 mg/kg/day), or L-AMB (5 mg/kg/day) was given intravenously to rabbits as a single dose or as repeated daily doses on 7 consecutive days after induction of unilateral uveitis by intravitreal injection of endotoxin. AMB concentrations in aqueous humor, vitreous humor, and plasma were determined by high-pressure liquid chromatography 16 h after administration of a single dose or 24 h after the last of seven doses. After single-dose administration, L-AMB achieved at least eightfold-higher AMB concentrations in the aqueous of inflamed eyes than ABLC or D-AMB (1.21 +/- 0.58 micro g/ml versus 0.14 +/- 0.04 and 0.11 +/- 0.09 micro g/ml, respectively). At that time point no drug was detectable in the vitreous. After 7 days of treatment, the concentration of AMB in the vitreous was higher after treatment with L-AMB (0.47 +/- 0.21 micro g/ml) than after treatment with ABLC (0.27 +/- 0.18 micro g/ml) and D-AMB (0.16 +/- 0.04 micro g/ml). Similarly, AMB concentration in the aqueous was higher after repeated doses of L-AMB (0.73 +/- 0.43 micro g/ml) than after repeated doses of ABLC (0.03 +/- 0.02 micro g/ml) or D-AMB (0.13 +/- 0.06 micro g/ml). No AMB was detected in noninflamed eyes. Following systemic administration, AMB distribution to the eye is inflammation dependent and occurs sequentially, first to the aqueous and then to the vitreous. Compared to D-AMB and ABLC, L-AMB reaches higher drug concentrations in both ocular compartments.

Role of oxygen and nitrogen species in experimental uveitis: anti-inflammatory activity of the synthetic antioxidant ebselen

This study was aimed at examining the role of oxygen and nitrogen reactive species in a model of experimental uveitis upon intravitreal injection of bacterial endotoxin to albino New Zealand rabbits. The inflammatory response was evaluated in terms of: (i) the integrity of the blood aqueous barrier (protein and cell content in samples of aqueous humor), (ii) histopathological changes of the eyes, (iii) clinical evaluation (with a score index based on clinical symptoms), and (iv) the concentration of malondialdehyde (MDA), in aqueous humor, as a marker of oxidative stress. Betamethasone was used as reference treatment, superoxide dismutase as quencher of superoxide anion, L-N G-nitro- l-arginine-methyl-esther (L-NAME) and chlorpromazine as nitric oxide synthase inhibitors, and ebselen, a glutathione peroxidase mimic, as peroxynitrite reductant. All the substances were injected subconjunctivally to the rabbits immediately after the intravitreal endotoxin injection. Ebselen was the only treatment able to decrease MDA concentration to control values, exerting an effect similar to that elicited by L-NAME on the rest of the parameters tested. The data presented render ebselen a notable choice for the treatment of uveitis, with implications for clinical trials.

Simultaneous in vivo imaging of leukocyte migration: Heterogeneity among iris, limbus, and choroid vessels

Purpose. Endothelial cells of different vascular systems may express site-specific adhesion molecules to attract leukocyte subsets. This study describes a method to visualize and compare leukocyte-endothelial interactions in three vascular beds within the same eye in mice. Methods. Digital in vivo fluorescence microscopy was used to record a trans-corneal iris view, a superficial limbus view, and a trans-scleral anterior choroid view of mouse tissue. Uveitis was induced by intravitreal injection of E. coli endotoxin into BALB/c mice. Leukocytes were labeled systemically with SYTO-16 or rhodamine 6G. Leukocyte rolling and sticking were quantified at baseline and 4, 6, and 24 hours after endotoxin injection. Results. In a normal animal, the limbus had 18 times the number of rolling leukocytes and 6 times the number of sticking leukocytes relative to the iris. All three vascular beds were affected by intravitreal injection of endotoxin. Although they each showed increased numbers of rolling and sticking cells, the levels and kinetics of these increases differed. Rolling peaked at 6 hours in the iris (34-fold increase from baseline) and limbus (7-fold increase) but was maximal in the choroid earlier with a 16-fold increase. Sticking was maximal at 4 hours for iris (96-fold increase) and choroid (19-fold increase) but peaked in the limbus at 6 hours (47-fold increase from baseline). Conclusions. This study demonstrates that leukocyte-endothelial dynamics are not the same in different vascular beds in the normal mouse eye. Furthermore, site-specific differences in responses to intravitreally injected endotoxin, beyond what can be readily explained by differential distribution of endotoxin, were observed. The methodology can be used to test the hypothesis that endothelial cells within the eye have site-specific patterns of adhesion molecule expression.

The contribution of nitric oxide to endotoxin-induced ocular inflammation: interaction with sensory nerve fibres.

1. The actions of nitric oxide (NO) have been investigated in an endotoxin-evoked ocular inflammatory model in the rabbit, with particular emphasis on the relationship between NO, sensory nerves (C-fibres) and the C-fibre neuropeptides, calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase activating peptide (PACAP). 2. Endotoxin, injected intravitreally, evoked inflammatory responses, i.e. conjunctival hyperaemia, miosis and protein extravasation, reflected by the aqueous flare response (AFR). In control rabbits, the maximum AFR was 66.5 +/- 9.5 (arbitrary units). Pretreatment with the NO synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NAME, 200 mg kg-1) given by intravenous injection, inhibited the endotoxin-evoked responses; the AFR was 16.5 +/- 1.9 (n = 8, P < 0.001) and the conjunctival hyperaemia was abolished. 3. Endotoxin-evoked ocular inflammation is associated with the release of CGRP and PACAP from C-fibres. In the eyes challenged with endotoxin, the concentrations of PACAP-27, -38 and CGRP in the aqueous humour were 58.2 +/- 10.9, 54.4 +/- 12.4 and 5526 +/- 519 (pmoll'), respectively. L-NAME inhibited the release of PACAP-27, -38 and CGRP; the concentrations were 14.3 +/- 2.5, 13.5 +/- 2.5 and 510 +/- 67 (pmoll-1), respectively (n = 8, P < 0.01 or 0.001). 4. Intravitreal injection of 0.3 nmol CGRP induced conjunctival hyperaemia and AFR; the maximum AFR was 140.2 +/- 11.4. L-NAME suppressed the response induced by CGRP; the AFR was 23.4 +/- 5.5 (n = 8, P < 0.001). L-NAME abolished the conjunctival hyperaemia induced by PACAP-27 and -38 (0.3 nmol) and reduced the AFR. 5. The inflammatory cells that infiltrated the uvea, cornea and aqueous humour in large numbers in response to intravitreal injection of endotoxin were found to express inducible NOS. L-NAME prevented the appearance of such cells. 6. Our findings suggest that NO plays an important role in the endotoxin-evoked ocular inflammation in the rabbit: NO activates C-fibres causing release of C-fibre neuropeptides into the aqueous humour. In addition, NO mediates scme of the ocular effects of CGRP and PACAP, since L-NAME suppressed the AFR induced by these peptides.

Additive effect of nitric oxide and prostaglandin-E2 synthesis inhibitors in endotoxin-induced uveitis in the rabbit

The involvement of nitric oxide (NO) and prostaglandin E2 (PGE2) was investigated in a model of intraocular inflammation induced by intravitreal injection of endotoxin (lipopolysaccharide, LPS, 10 ng) in rabbits. The severity of uveitis, the myeloperoxidase (MPO) activity in iris-ciliary body, and the protein concentration in aqueous humor were determined. Nitric oxide synthase (NOS) and cyclooxygenase (COX) activities were assessed respectively by nitrite and PGE2 levels in aqueous humor. Treatment with inhibitors of NOS (NG-nitro-L-arginine methyl ester, L-NAME, 50 mg/kp i.p.) or COX (diclofenac, 30 micrograms, topically), alone or in combination, were compared to a saline-treated group. Diclofenac or L-NAME alone reduced or delayed the intensity of uveitis, and partially decreased the protein concentration in aqueous humor; diclofenac, but not L-NAME, partially reduced the polymorphonuclear leukocyte infiltration in the iris ciliary body as indicated by the MPO activity. Treatment with both inhibitors in combination diminished the clinical uveitis, the disruption of the blood-aqueous barrier and the MPO activity in the iris-ciliary body. We conclude that NO and PGE2 have additive effects in endotoxin-induced uveitis in rabbits, and that the inhibition of both pathways would improve the therapeutical management of uveitis.

Evidence of cross-link formation of vitreous collagen during experimental ocular inflammation.

To determine the mechanisms of vitreous changes during ocular inflammation. We investigated vitreous changes, with special emphasis on collagen, in an experimental model of ocular inflammation induced by intravitreal injection of endotoxin (Escherichia coli) in rabbits. Inflammation caused gel contraction and loss of elasticity, accompanied by release of a water-like liquid from the gel, and increases in the amount of insoluble material and high-molecular-weight components of vitreous collagen, presumably due to extensive cross-links of the collagen molecules. Those changes were partially inhibited by intravitreal injection of superoxide dismutase. The cross-links of vitreous collagen may promote vitreous gel contraction and release of a water-like liquid from the gel. Superoxide anion may play a role in this process.

Distribution and effects of pituitary adenylate cyclase-activating peptide in the rabbit eye.

Pituitary adenylate cyclase-activating peptide (PACAP)-like immunoreactivity was demonstrated by immunocytochemistry together with calcitonin gene-related peptide (CGRP)-like immunoreactivity in small to medium-sized neurons in the trigeminal ganglion and in nerve fibers in the iris, ciliary body, cornea, choroid and sclera of the rabbit eye. The regional distribution of PACAP-27- and PACAP-38-like immunoreactivity in the eye was studied by radioimmunoassay: the highest concentrations were found in the iris sphincter and ciliary body. The distribution pattern resembled that of CGRP-like immunoreactivity, which is a well-known constituent of sensory C-fibre neurons. Intravitreal injection of PACAP-27 or PACAP-38 induced conjunctival hyperemia, swelling of the anterior segment of the eye, miosis and breakdown of the blood-aqueous barrier, manifested as a marked aqueous flare response. Tetrodotoxin pretreatment inhibited the conjunctival hyperemia, the swelling of the anterior segment of the eye, and the miosis but not the aqueous flare response. The concentration of PACAP-like immunoreactivity in the aqueous humor was increased greatly following infrared irradiation of the iris, topical application of formaldehyde to the cornea, or intravitreal injection of endotoxin or bovine serum albumin. Also the concentration of CGRP-like immunoreactivity in the aqueous humor was increased greatly. Both in vivo and in vitro studies showed that capsaicin caused a parallel release of PACAP-like immunoreactivity and CGRP-like immunoreactivity from the uvea. Injection of PACAP-27 and PACAP-38 resulted in the release of CGRP-like immunoreactivity (and PACAP-like immunoreactivity) into the aqueous humor and PACAP-27 and PACAP-38 were also found to evoke tachykinin-mediated contractions of the isolated iris sphincter muscle, indicating that PACAP induces positive feedback on C-fibres. Thus, PACAP is a sensory neuropeptide in the eye. Since the PACAP-induced ocular responses mimicked the symptoms of inflammation, and since the PACAP-like immunoreactivity concentration in the aqueous humor was greatly increased following noxious stimulation, we suggest that it takes part in the inflammatory responses of the rabbit eye.

Cellular response to intravitreal injection of endotoxin and xanthine oxidase in rabbits.

In the present study, the ocular inflammatory response to intravitreally injected endotoxin and xanthine oxidase was studied and the cellular response of the anterior and posterior segments was contrasted. There was a clear dose response relationship to both compounds in aqueous humor protein concentration and aqueous and vitreous humor white cell number. Xanthine oxidase and low doses of endotoxin (0.25 and 1.0 ng) produce a mainly mononuclear response in the anterior segment. Higher doses of endotoxin (10 and 100 ng) produced a predominantly neutrophilic response. Cellular infiltration into the posterior segment differed qualitatively and quantitatively from the anterior segment in response to the same stimuli. Myeloperoxidase (MPO) activity (a marker for neutrophils) of the iris-ciliary body was increased only in those eyes with a large neutrophilic response and thus is not recommended for use as a definitive index of the ocular inflammatory response, but may be a useful adjunct for such studies.

Does the lens serve as a ‘sink’ for iron during ocular inflammation?

Twenty-four hours after induction of ocular inflammation by intravitreal injection of endotoxin (10 ng), the intraocular fluid (IOF, aqueous and vitreous humors) concentration of iron (Fe) increased. This was presumably due to entry of the plasma Fe-binding protein, transferrin, into the IOFs through disrupted blood ocular barriers. After 1 day of inflammation the Fe concentration in lenses from the inflamed eye was 60% greater than that measured in contralateral control lenses. By day 15, lens Fe concentration had returned to levels of the contralateral control lenses. There was a distinct relationship between the dose of endotoxin used and the amount of Fe accumulated by the lens. The Fe concentration in lenses from eyes injected with 100 ng endotoxin was 0·376±0·027 μg g −1 wet weight compared to 0·214±0·014 μg g −1 in lenses from eyes injected with 0·25 ng endotoxin. In a previous study, copper (Cu) concentration in the IOFs was elevated to the same extent as Fe in response to intravitreal injection of endotoxin. However, in the present study, lenticular Cu concentration was unaltered at the highest (100 ng) dose of endotoxin. Since the increase in lens uptake was selective for Fe, there may be a specific Fe uptake mechanism in this ocular tissue. The physiological reasons for and possible pathological consequences of such a process are discussed.

Copper and aspirin treatment increase the antioxidant activity of plasma

Copper (Cu) complexes of a number of compounds have increased anti-inflammatory efficacy over the native compound. In the present study, Cu and acetylsalicylic acid (ASA) were administered to rabbits over a one week period. At the end of this time, ocular inflammation was induced by intravitreal injection of endotoxin. ASA caused a slight reduction in the inflammatory response at 24 hours, but this response was not further decreased when Cu was added to the treatment regimen. However, there was a gradual and significant increase in antioxidant activity in the plasma of animals in the combined treatment group which could not be accounted for by a Cu-ASA complex, an increase in the concentration of known plasma protein antioxidants (ceruloplasmin and transferrin) or an increase in superoxide dismutase activity. The compound(s) responsible for this increased activity has a molecular weight greater than 10,000 Da. A large increase in plasma antioxidant activity may have a protective effect in other models of inflammation and provide a mechanism for the proven increase in anti-inflammatory efficacy of Cu-chelates of many compounds.