PMN accumulation in aqueous humor and iris-ciliary body during intraocular inflammation

The accumulation of polymorphonuclear leukocytes (PMNs) was determined in the aqueous humor and iris-ciliary body following an intravitreal injection of endotoxin in the albino rabbit. PMN accumulation in the iris-ciliary body was quantified by measuring myeloperoxidase (MPO) activity in homogenates of this tissue. Leukocyte appearance in the aqueous humor was determined by counting the number of PMNs in diluted aspirates of aqueous humor and also by measuring MPO activity in the same aspirates. Twenty-four hours following an intravitreal injection of endotoxin, there was marked vasodilatation in the iris, breakdown of the blood-aqueous barrier, and infiltration of PMNs into the aqueous humor. There was, however, no correlation between MPO activity in the iris-ciliary body and the number of PMNs or the MPO activity in the aqueous humor. Furthermore, the number of PMNs in the aqueous humor did not increase with increasing amounts of intravitreally injected endotoxin, whereas MPO activity in the iris-ciliary body increased in a dose-dependent manner. The results of this study suggest that quantification of leukocytes in the aqueous humor does not represent a meaningful index of intraocular inflammation.     

Intraocular production of a cytokine (CINC) responsible for neutrophil infiltration in endotoxin induced uveitis.

AIMS/BACKGROUND: The subcutaneous injection of bacterial endotoxin in Lewis rats produces an acute intraocular inflammation evolving over a 24 hour period. This endotoxin induced uveitis (EIU) is characterised by a biphasic protein exudation and a cellular infiltrate composed of macrophages and polymorphonuclear neutrophils (PMNs). This model was used to study the mechanism of cellular infiltration in ocular inflammation. METHODS: EIU was induced by a subcutaneous injection of lipopolysaccharide (LPS) (S typhimurium) at 350 micrograms/kg. The levels of cytokine induced neutrophil chemoattractant (CINC) were measured every 2 hours in the serum and in the aqueous humour by ELISA. The intraocular inflammation was quantified by protein measurement and leucocyte counting. RESULTS: The kinetics of CINC production in the systemic circulation showed a rapid rise, peaking 2 hours after LPS injection, followed by a progressive decline over the next 8 hours. In the eye, the CINC levels increased above the serum levels 10 hours after EIU induction corresponding to the time of cellular infiltration. When leucocyte entry in the eye was inhibited by 56% and 64% with an antiadhesion molecule antibody, there was only a slight reduction in the aqueous humour CINC levels of 9% and 16%, respectively, indicating that CINC was produced by ocular tissue cells. The specific effect of CINC in the eye was confirmed when a direct intraocular injection of 250 ng of purified CINC was followed by significant PMN infiltration, in the absence of protein exudation. CONCLUSION: The data indicate that the production of the CINC chemotactic factor by ocular tissue participates in the inflammatory reaction in EIU.     

The insensitivity of the chicken eye to the inflammatory effects of x-rays in contrast to its sensitivity to other inflammatory agents.

The effects of x-rays and three chemical agents, known to cause intraocular inflammation in mammalian eyes, were studied on the chicken eye because this species was reported to be insensitive to the cataractogenic effects of x-rays. Intravitreal injection of Shigella endotoxin and topical and/or intravitreal administration of PGE2, PGF2alpha, or arachidonic acid caused a breakdown of the blood-aqueous barrier, as indicated by flare and increased protein concentration in the aqueous humor. Following endotoxin injection, there was also a large accumulation of cells in the anterior chamber. The ocular inflammatory effects of endotoxin and arachidonic acid were inhibited by indomethacin. Thus the chicken eye reacts to these inflammatory agents in a manner similar to that previously described for the rabbit. In contrast, the inflammatory response which was reported to occur in the rabbit eye 3 to 4 hr after exposure to 500 or 1000 rads of x-rays was not observed in the chicken eye even after expsoure to 10,000 rads. Minimal flare and a small cellular infiltration were observed in some eyes only after extensive swelling of the surrounding tissues had developed. It is concluded that the insensitivity of the chicken eye to x-rays is due to some unique difference in the chain of events which mediates, or prevents, the effects of ionizing radiation rather than to a general insensivity to inflammatory agents.     

Characteristics of polymorphonuclear leukocyte infiltration into the alkali burned eye and the influence of sodium citrate

Polymorphonuclear leukocytes (PMNs) are considered to play a central role in the corneal ulceration process subsequent to an alkali burn. We have described the time course of PMN infiltration into the ocular tissues following an alkali burn. In addition, we examined the effect of sodium citrate upon the accumulation of PMNs in the alkali burned cornea. The accumulation of PMNs into the cornea and iris-ciliary body was quantified by measuring myeloperoxidase (MPO), an enzyme marker for these inflammatory cells. Leukocytes in aqueous humor aspirates and corneal washes were counted directly under a microscope. In the alkali burned cornea, we found an initial transient, yet substantial, infiltration of PMNs, peaking at about 12-24 hr and limited to the peripheral cornea; this subsided by about 72 hr. By 14 days, the MPO activity, and hence the number of leukocytes, had risen again, and by 21 days the level had increased by several fold. Qualitatively similar biphasic patterns of leukocyte infiltration were observed in the iris-ciliary body and aqueous humor. Leukocyte numbers in corneal washes only increased between 4-24 hr following the alkali burn. The exceptionally high degree of leukocyte infiltration into the cornea at 21 days corresponded with the presence of ulceration. Topical administration of sodium citrate (10%) inhibited the early and late phase of PMN accumulation in the alkali burned cornea, i.e. at 24 hr (-63%) and 21 days (-92%). The inhibition of PMN infiltration by sodium citrate correlates with the reduced corneal ulceration observed following treatment with this compound.     

Time course of rabbit ocular inflammatory response and mediator release after intravitreal endotoxin

An inflammatory response was elicited in the rabbit eye by intravitreal injection of endotoxin. The appearance in aqueous humor of selected metabolites of arachidonic acid metabolism at various times was correlated with the influx of protein and myeloperoxidase activity in the iris-ciliary body. After intravitreal injection of endotoxin, aqueous humor protein levels increased substantially within 2 hr. This aqueous humor protein increase occurred before a significant appearance of prostaglandin E2 (PGE2) in the aqueous humor. Myeloperoxidase activity in the iris-ciliary body, a measure of polymorphonuclear leukocyte (PMN) infiltration, showed little elevation until 6 hr after endotoxin injection and then increased rapidly through 24 hr. The appearance of the leukotriene B4 (LTB4) followed a similar time course: levels in the aqueous humor were partially elevated until 6 hr after endotoxin injection, when levels begin to rise rapidly. These findings are interpreted to demonstrate the dependence of PMN infiltration on the release and accumulation of LTB4; the initial breakdown of the blood-aqueous barrier and influx of protein appears to be independent of significant release of PGE2.     

Ascorbic acid inhibits the activity of polymorphonuclear leukocytes in inflamed ocular tissues

Myeloperoxidase (MPO) is present at high levels in polymorphonuclear leukocytes (PMNs) and has been used as a marker to quantify the accumulation of PMNs in inflamed tissues. MPO activity in inflamed ocular tissues was inhibited by aspirates of aqueous humor. This inhibition could be duplicated by the addition of ascorbic acid at concentrations equivalent to those present in the aliquots of aqueous humor. Similarly, aqueous humor and ascorbic acid inhibited MPO from isolated rabbit leukocytes. Therefore, ascorbic acid appears to inhibit the functional activity of the peroxidase in PMNs, thus preventing potential tissue damage by this enzyme when released during leukocyte degranulation in inflammation. Ascorbic acid might fulfill a role as an endogenous anti-inflammatory agent in the eye.     

Quantification of stromal destruction in the inflamed cornea

An intrastromal injection of endotoxin lipopolysaccharide (LPS) in one eye of New Zealand albino rabbits induced a prominent keratitis characterized clinically and microscopically by edema and infiltration. Polymorphonuclear leukocytes (PMNs) constituted the primary invading leukocytic element. Collagen synthesis was measured by pulsing the corneas with 3H-proline before inducing inflammation. The invasion of the cornea by leukocytes did not alter the conversion of proline to hydroxyproline significantly in the stroma during the 14-day observation period, signifying that there were only negligible changes in the rate of collagen synthesis. However, the percentage of total stromal protein represented by collagen (ie, collagen/total protein) was only 50% of that in comparable corneas receiving an injection of phosphate-buffered saline. Some animals were rendered leukopenic by intravenous nitrogen mustard before intrastromal LPS injection caused a less severe corneal inflammatory response, characterized microscopically by fewer infiltrating leukocytes. Similarly, in nonleukopenic rabbits, topical therapy with 1% prednisolone acetate markedly reduced the corneal inflammatory response which also was characterized by fewer invading leukocytes. In neither instance was there extreme collagen loss, suggesting that the loss of stromal collagen is related to PMN infiltration.     

Time course for prostaglandin synthesis by rabbit lens during endotoxin-induced ocular inflammation

Three hours to 14 days following the intravitreal injection of 10 ng of E. coli endotoxin into the vitreal chamber of one eye of the New Zealand white rabbit, ocular inflammation was evaluated by clinical and biochemical criteria and prostaglandins were measured in the intraocular fluids and in the incubation medium of the intact lens. Increased synthesis of PGE2 was detected for lenses from inflamed eyes beginning at 18 h post-endotoxin injection. Lenticular PGE2 synthesis remained above control levels for the duration of the time course. Lenses also exhibited increased PGF2 alpha synthesis, which began at 18 h and returned to control levels by day 7. At the times of peak production, aqueous humor PGE2 concentration correlated with lenticular PGE2 synthesis and with aqueous humor leukocyte number. No correlations were found for lenticular PGE2 vs. cell number, or vitreous humor PGE2 vs. aqueous humor PGE2. These results suggest that during ocular inflammation, aqueous humor PGE2 may be derived, at least in part, from the lens and leukocytes.     

Endotoxin-induced uveitis in rats: morphological and biochemical study

Inflammation induced by systemic injection of endotoxin can be a good model for endogenous uveitis since ocular inflammation is induced without manipulating the eye. We carried out morphological and biochemical studies in Lewis rats to evaluate the breakdown of the blood-ocular barrier following injection of endotoxin (1 mg/rat) in footpads. Vasodilation was observed as early as 3 hours and became maximum at 18-24 hours after the injection. Unlike in the eye, no inflammatory changes were observed in other organs. Protein and cell contents in the aqueous humor increased significantly as early as 3 hours after the injection and reached a peak level at 24 hours. The protein content returned to the normal level in the following several days, while cells in the aqueous humor remained at a high level even 1 week after the injection. The time-course of the pupillary size was very similar to that of the protein concentration. Furthermore, we examined leukotrienes (LTs) levels in aqueous humor by high-pressure liquid chromatography. LTD4 was detected in the aqueous humor at 6 hours and reached its peak level at 24 hours. The present data indicates that the systemic injection of endotoxin causes the disruption of the blood-ocular barrier soon after the injection and inflammation becomes maximum in 18-24 hours. This model can be used for studying endogenous uveitis and the disruption of the blood-ocular barrier without direct trauma to the eye.     

Ocular blood flow and vascular permeability in endotoxin-induced inflammation

Blood flow was measured in several tissues of the rabbit eye following intravitreal injection of a dose of endotoxin that induces an inflammatory response. In separate experiments, the vascular permeability of the inflamed eye was estimated by iris fluorescein angiography and by measuring protein influx into the aqueous humor. The effect of topical corticosteroid treatment upon blood flow and vascular permeability was also measured. Following intravitreal endotoxin injection, minor changes in blood flow occurred in retina and optic nerve head. Marked changes were observed in blood flow in iris, ciliary processes, and choroid. Steroid treatment had no effect upon the increased blood flow 24 h after the endotoxin injection, although from a clinical standpoint the steroid-treated eyes appeared less inflamed. Fluorescein angiography demonstrated a massive increase in iris vessel permeability 6 and 24 h following endotoxin injection. Topical steroid treatment reduced fluorescein entry into the anterior chamber at both time periods. On the other hand, the increase in protein influx into the aqueous humor in the endotoxin-inflamed eye was not inhibited by steroid pretreatment. It is suggested that corticosteroids have a selective effect upon the permeability of different components of the blood-aqueous barrier, namely the ciliary processes and the iris vasculature.     

Efficacy of low concentrations of ketorolac tromethamine in animal models of ocular inflammation.

PURPOSE: To determine if topical ophthalmic application of ketorolac tromethamine concentrations below 0.5% can block the biochemical and physiological processes associated with chemically induced ocular inflammation in rabbits. METHODS: Ocular inflammation was induced in rabbits by intravenous (i.v.) injection of endotoxin (2.5 microg/kg) isolated from Salmonella typhimurium, or by a topical application of arachidonic acid (1.0%). The effect of ketorolac (at concentrations ranging from 0.001%-0.5%) on ocular inflammation was determined by measuring changes in the blood-aqueous barrier, using fluorophotometry (dextran-isothiocyanate-fluorescein; FITC-dextran 2%) and by measuring changes in aqueous humor protein concentrations. Changes in aqueous humor prostaglandin E(2) (PGE(2)) concentrations were also measured. RESULTS: Ketorolac 0.01%-0.5% produced substantial decreases in endotoxin-induced fluorescein leakage into the aqueous humor. The decrease produced by ketorolac 0.1% was comparable to that produced by ketorolac 0.5%. Ketorolac 0.1%-0.5% produced substantial decreases in endotoxin-induced increases in prostaglandin concentrations in the aqueous humor, and in arachidonic acid-induced protein leakage into the aqueous humor. CONCLUSIONS: Topical application of ketorolac concentrations as low as 0.01%-0.1% significantly reduce chemically induced ocular inflammation in rabbits.     

A protective role for ascorbic acid during inflammatory episodes in the eye

During an experimentally induced inflammatory response in the rabbit eye, the decrease in the ascorbic acid concentration within the aqueous humor corresponded, in large part, to the infiltration of leukocytes into this ocular fluid. Additional in vitro studies demonstrated that activated leukocytes reacted with ascorbic acid, diminishing the concentration of this vitamin in the surrounding medium. We suggest that the very high concentrations of ascorbic acid found in the aqueous humor of a number of species affords extracellular protection for the ocular tissues against oxygen radicals and metabolites released by infiltrating leukocytes during ocular inflammation.     

Dynamics of ascorbate in the aqueous humor and tissues surrounding ocular chambers

The level of ascorbate in the aqueous humor and surrounding intraocular structures in enucleated arterially perfused rabbit eye was investigated. The enucleated eye preparation was shown to be capable of secreting ascorbate from the perfusate into the aqueous humor. Ascorbate in the iris, ciliary body and cornea was released into the aqueous humor when the eye was perfused with ascorbate-free solution. Failure to obtain aqueous flow rates from the decay of ascorbate in the anterior chamber was due to the contribution of ascorbate from these ocular tissues during the perfusion. Histochemically, ascorbate was localized in the pigmented epithelial layer in the valleys between the ciliary processes and the pars plana of the ciliary body and in the iris. In the cornea, distinct localization of ascorbate was observed in the endothelium and basal cell layer of the epithelium.     

Downregulation of endotoxin-induced uveitis by intravitreal injection of vasoactive intestinal Peptide encapsulated in liposomes

PURPOSE: To reestablish the immunosuppressive microenvironment of the eye, disrupted by ocular inflammation during endotoxin-induced uveitis (EIU), by means of intravitreal injection of vasoactive intestinal peptide (VIP) in saline or encapsulated in liposomes, to increase its bioavailability and efficiency. METHODS: EIU was induced in Lewis rats by subcutaneous injection of lipopolysaccharide (LPS). Simultaneously, animals were intravitreally injected with saline, saline/VIP, VIP-loaded liposomes (VIP-Lip), or unloaded liposomes. EIU severity and cellular infiltration were assessed by clinical examination and specific immunostaining. VIP concentration was determined in ocular fluids by ELISA. Ocular expression of inflammatory cytokine and chemokine mRNAs was detected by semiquantitative RT-PCR. Biodistribution of rhodamine-conjugated liposomes (Rh-Lip) was analyzed by immunohistochemistry in eyes and regional cervical lymph nodes (LNs). RESULTS: Twenty-four hours after intravitreal injection of VIP-Lip, VIP concentration in ocular fluids was 15 times higher than after saline/VIP injection. At that time, EIU clinical severity, ocular infiltrating polymorphonuclear leukocytes (PMNs), and, to a lesser extent, ED1(+) macrophages, as well as inflammatory cytokine and chemokine mRNA expression, were significantly reduced in VIP-Lip-injected rats compared with rats injected with saline/VIP, unloaded liposomes, or saline. Rh-Lip was distributed in vitreous, ciliary body, conjunctiva, retina, and sclera. It was internalized by macrophages and PMNs, and VIP colocalized with liposomes at least up to 14 days after injection. In cervical LNs, resident macrophages internalized VIP-Rh-Lip, and some adjacent lymphocytes showed VIP expression. CONCLUSIONS: VIP was efficient at reducing EIU only when formulated in liposomes, which enhanced its immunosuppressive effect and controlled its delivery to all tissues affected by or involved in ocular inflammation.     

AL-2512, a novel corticosteroid: preclinical assessment of anti-inflammatory and ocular hypertensive effects.

The anti-inflammatory efficacy and ocular hypertensive effect of AL-2512 were characterized in rodent and feline models of ocular inflammation. Neutrophil influx into ocular tissue following topical ocular administration of test drugs was evaluated in models of endotoxin-induced uveitis. In rats, the anti-inflammatory efficacy of AL-2512 was compared with that of 0.1% dexamethasone. Test drug or vehicle was administered topically before subplantar injection of endotoxin. Neutrophil influx was assessed at 24 hours. Feline eyes, injected intravitreally with endotoxin, were treated topically with 0.1% AL-2512, 1.0% prednisolone acetate or vehicle at various timepoints before and after endotoxin injection. At 12 hours, protein concentration and leukocyte count in aqueous humor were determined. In the feline intraocular pressure (IOP) model, after baseline IOP values were established, AL-2512, dexamethasone, or vehicle was administered topically to both eyes of cats. IOP was measured daily before and during treatment. Topical ocular administration of AL-2512 inhibited endotoxin-induced leukocyte influx in rodent and feline models of uveitis. In rats, AL-2512 significantly inhibited neutrophil influx by 89%, compared with 93% by dexamethasone. In feline eyes, AL-2512 significantly (p < 0.05) inhibited leukocyte infiltration of aqueous humor by 59%, compared to 37% inhibition by prednisolone acetate. Intraocular pressure in cats treated for 32 days with AL-2512 or dexamethasone increased 6% and 18%, respectively. The ocular anti-inflammatory effect of AL-2512 was equivalent to dexamethasone and superior to prednisolone acetate in rat and feline models of ocular inflammation, respectively. This steroid provides anti-inflammatory efficacy equivalent to dexamethasone with a reduced risk of inducing ocular hypertension.     

Inflammation-induced changes in the iron concentration and total iron-binding capacity of the intraocular fluids of rabbits

Changes in iron (Fe) concentration and total-iron-binding capacity (TIBC) of the intraocular fluids were measured during endotoxin-induced ocular inflammation in rabbits over a 3-week time course. In the aqueous humor, both Fe and TIBC increased to peak levels 24 h after intravitreal injection of endotoxin (10 ng) and gradually decreased to baseline levels by 3 weeks. In the uninflamed eye, the TIBC of the aqueous was only 23% saturated. During inflammation the TIBC became more highly saturated over time, reaching 50% at 3 weeks. In the vitreous humor the picture was more complicated due to the presence of slight hemorrhage. Noncellular Fe and TIBC increased to peak levels by 7 days, while TIBC approached 100% saturation. Both returned to baseline by 21 days. The influx of the partially saturated plasma protein transferrin through disrupted blood-ocular barriers most likely accounts for the increased TIBC in the inflamed eye and could provide some protection against the potentially harmful effects of Fe arising from tissue necrosis and hemolysis subsequent to hemorrhage. Under conditions of the model of inflammation studied here, the TIBC was not exceeded at any time during the 3 weeks. However, with more severe and long-lasting inflammation or when there is greater hemorrhage, the TIBC could be exceeded. This could lead to greater, and perhaps irreversible, damage to ocular tissues.     

Endotoxin-induced uveitis in the rat

Experimental studies of ocular inflammation induced by endotoxin have been performed almost exclusively in rabbits. Although the rat has been considered refractory to both the systemic and local effects of endotoxin, the present study has shown that intravitreal injection of endotoxin evoked a characteristic acute inflammatory response which was reproducible and dose-dependent. In addition, a consensual ocular response in the control eye occurred which was less severe but also dose-dependent. Preliminary investigations support the view that consensual responses of this nature may be mediated by a nervous reflex arc mechanism. Sequential histological analysis revealed a marked polymorphonuclear cell infiltration of most ocular tissues in the early stages, while mononuclear cells predominated after 3 days. Widespread intravascular microthrombi were also observed, particularly in the ciliary vessels. Recovery, however, was usually complete by 7 days.This study was supported by National Institute of Health Grants EY-02648-01 and EY-01747 from the National Eye Institute     

Captopril suppresses inflammation in endotoxin-induced uveitis in rats

Captopril is an inhibitor of angiotensin-converting enzyme (ACE) that is largely used in the treatment of cardiovascular diseases. Several previous studies have demonstrated that captopril exhibits a wide variety of biological activities, including an anti-inflammatory action, on which we focused our attention. The aim of the present study was to investigate the efficacy of captopril on endotoxin induced uveitis (EIU) in rats. We investigated its effect upon cellular infiltration and protein leakage, as well as on the concentration of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), prostaglandin E2 (PGE2), monocyte chemoattractant protein-1 (MCP-1) in the anterior chamber. In addition, we checked its effect on activation of nuclear factor kappa B (NF-kappaB) in iris and ciliary body (ICB) cells in vivo. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). One hour after the LPS inoculation, either 1mg/kg, 10mg/kg or 100mg/kg captopril were injected intravenously. 24h later, the aqueous humor was collected from both eyes, and the number of infiltrating cells and protein concentration in the aqueous humor were determined. Levels of TNF-alpha, PGE2, NO and MCP-1 were determined by enzyme-linked immunosorbent assay. On some eyes, after enucleation, immunohistochemical staining with a monoclonal antibody against activated NF-kappaB was performed. Captopril treatment significantly decreased the inflammatory cells infiltration, the level of protein, concentrations of TNF-alpha, PGE2, NO and MCP-1 in the aqueous humor. The number of activated NF-kappaB-positive cells was lower in ICB of the rats treated with captopril 3h after the LPS injection. The present results indicate that captopril suppresses the inflammation in EIU by inhibiting the NF-kappaB-dependent pathway and the subsequent production of pro-inflammatory mediators.     

The effect of sodium citrate on the stimulation of polymorphonuclear leukocytes

Topical administration of sodium citrate reduces the incidence of corneal ulceration and perforation following an alkali burn to the eye. The specific mechanism by which sodium citrate prevents the ulceration is not understood, although citrate does inhibit the infiltration of polymorphonuclear leukocytes (PMNs) into the cornea following an alkali burn. In the present study, the effects of sodium citrate and another calcium chelator, ethylene glycol bis (beta-aminoethylether)-N,N'tetraacetic acid (EGTA), upon PMN oxygen consumption and lysosomal enzyme release were determined. Oxygen consumption was measured polarographically using a Clark-type oxygen electrode, and lysosomal enzyme release was determined by intra- and extra-cellular measurements of myeloperoxidase activity. Opsonized zymosan and N-formylmethionylleucylphenylalanine (FMLP) were used to stimulate neutrophil oxygen consumption and lysosomal release. Both sodium citrate and EGTA inhibited PMN oxygen consumption and lysosomal enzyme release in response to opsonized zymosan. In contrast, neither sodium citrate nor EGTA reduced PMN oxygen consumption or lysosomal enzyme release in response to FMLP. Therefore, the ability of sodium citrate (and EGTA) to inhibit PMN stimulation is dependent upon the choice of stimulus. Until the inflammatory mediators involved in the ulcerative process following an alkali burn to the eye are delineated, the impact of sodium citrate upon PMN stimulation in vivo cannot be resolved.