Abstract
Purpose:
Lipoxins exert potent anti-inflammatory and pro-resolving actions by reducing polymorphonuclear neutrophil (PMN) infiltration. This study describes the effect of lipoxin A4 and a stable analog on the resolution of ocular inflammation induced by intravitreal injection of lipopolysaccharides (LPS) in rats.
Methods:
Six- to eight-week-old male Sprague Dawley (SD) rats were injected intravitreally with 2.5 µL physiologically balanced solution (LPS) containing 5 ng LPS, or 5 ng LPS + 50 ng LXA4 or 5 ng LPS + 50 ng 15-epi-LXA4 analog. Rats were anesthetized with intraperitoneal injection of a ketamine and xylazine cocktail. At 24 h, the animals were again anesthetized and the eyes examined for clinical signs of inflammation. The animals were then euthanized by CO2 inhalation and aqueous humor was collected in heparinized saline. Aqueous humor PMNs were counted using an Improved Neubauer Hemocytometer, and the protein concentration was determined by standard procedure. After enucleation, the eyes were dissected to remove the lens and the ocular tissues were frozen in liquid nitrogen and stored at −80°C. Myeloperoxidase assay was done by a standard procedure.
Results:
Compared to untreated LPS-injected controls, rats treated with either LXA4 or its stable analog had lower clinical inflammation score, significantly reduced aqueous humor PMN cell counts, aqueous humor protein levels, and the MPO values. The difference between the mean values of aqueous humor protein and MPO in the LXA4 and the analog injected eyes was not statistically significant, but PMN cell counts were significantly different.
Conclusions:
The ocular inflammatory response to intravitreally injected LPS in rats is significantly reduced by simultaneous injection of LXA4 or its analog. This finding supports an earlier independent observation of the ocular anti-inflammatory effect of LXA4. Further investigation of lipoxins in the eye might offer a novel therapeutic approach to treating ocular inflammation in man.
Introduction
D
Inflammation is a multistep process of which the final step is its resolution. However, not all types of inflammation, particularly chronic inflammation, resolve without damaging tissues and their functional integrity. It is now known that endogenous molecules such as lipoxin A4 (LXA4), resolvin Es, and resolvin Ds mediate the resolution of inflammation, primarily by reducing extravasation of polymorphonuclear neutrophils (PMNs).8, 9 At the later phase of initiation of inflammation, lipoxins (LXs) are formed from arachidonic acid by 15-lipoxygenase during PMN and vascular endothelium interactions.10–12 Lipoxin (LX) A4 and aspirin-triggered lipoxins (ATLs) promote resolution of inflammation by regulating excessive leukocyte trafficking.13–15 These agents are unstable as they are rapidly inactivated by prostaglandin dehydrogenase.16, 17 Recently, a stable analog was developed to prevent the metabolic inactivation by modifying the C15–C20 region of LXA4 and ATLs18 and is effective as an anti-inflammatory agent.19 Anti-inflammatory effects of LXA4 and 15-epi-LXA4 stable analogs in a variety of tissues have been extensively reviewed by Serhan and Chiang.9 Studies on the impact of treatment with these compounds on ocular inflammation are limited.
We therefore examined the anti-inflammatory effect of LXA4 on ocular inflammation in rats induced by intravitreal injection of lipopolysaccharide (LPS). We also compared anti-inflammatory effects of LXA4 and its stable analog 15-epi (5S,6R,15R)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid).
Methods
The lipoxin compounds were obtained from Calbiochem (San Diego, CA). Six- to eight-week-old male Sprague Dawley (SD) rats, weighing 200–300 g, procured from Harlan (Indianapolis, IN) were used in this study. All procedures on animals were conducted in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research using a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Louisville.
Experiments were performed to compare the effects of LXA4 and of 15-epi LXA4 analog. Each experiment included an independent LPS control. Groups consisting of 6 rats were anesthetized by intraperitoneal injection of 1 mL/kg body weight of ketamine (37.5 mg/mL) and xylazine (5 mg/mL) mixture. One drop of local anesthetic proparacaine hydrochloride (Bausch & Lomb, Aliso Viejo, CA) was given topically to the eye immediately before intravitreal injections. All intravitreal injections of 5 ng LPS with or without 50 ng LXA4 or 50 ng of its analog performed with a volume of 2.5 µL phosphate-buffered saline (PBS). After 24 h, the animals were anesthetized, and the eyes were examined for clinical signs of vasodilation and exudates using a slit lamp. The level of inflammation was scored using a scale from 0 to 4 (0, Normal; 1, mild; 2, moderate; 3, severe; and 4, very severe). After euthanizing the animals by CO2 inhalation, the anterior chamber of the eye was punctured with a 30 gauge needle and aqueous humor was collected in heparinized saline (1% heparin in physiological saline) using a 10 µL disposable micropipettes (Fisher Scientific, Pittsburgh, PA) for protein estimation and PMN cell counts. After enucleation, the eyes were dissected to remove the lens and the ocular tissues were frozen in liquid nitrogen and stored at −80°C for myeloperoxidase (MPO) assay.
Polymorphonuclear neutrophil counts
PMNs in aqueous humor were counted using an Improved Neubauer Hemocytometer (Hausser Scientific, Horsham, PA).
Estimation of protein
The protein concentration in the aqueous humor was determined by following standard procedure.20 In 100 µL of the diluted (1:7) sample, 2.0 mL of working solution (50 parts of 2% Na2CO3 in 0.1 N NaOH + 1 part of 1% NaK tartrate + 1 part of 0.5% CuSO4) was added and left at room temperature for 10 min. To each tube 0.2 mL of 1 N Folin-Ciocalteu’s phenol (Sigma-Aldrich, St. Louis, MO) solution was added, vortexed, and left for 30 min at room temperature, and the tubes were read at 750 nM on the spectrophotometer (Beckman, model DU 640). Bovine serum albumin (1 mg/mL) was used as the standard.
Myeloperoxidase (MPO) assay
The inflammation of the uvea is characterized by infiltration of PMN cells into the ocular tissue. Assaying the MPO, an enzyme in the azurophilic granules of PMN21, 22 activity from the ocular tissues allows an accurate quantification of the inflammatory cells.23 The assay for detection of MPO per milligram of ocular tissues was done by a standard procedure.24 Ocular tissues were weighed and homogenized in 800 µL of N-ethylmaleimide (NEM) buffer using a “Tissue Tearor®” (Model 985370, Biospec Products Inc., Bartlesville, OK). After centrifuging at 12,000g for 30 min at 4°C, the supernatant was decanted and the procedure was repeated once more. The sediment was resuspended in 500 µL of 0.5% hexadecyltrimethylammonium bromide in potassium phosphate buffer (50 mM, pH 6.0), and sonicated 3 times for 10 s each in Branson Sonifier® (Branson Ultrasonics Corp., Danbury, CT). The sample was freeze–thawed three times and centrifuged at 12,000g for 30 min at 4°C, and the supernatant was taken.
Finally, 200 µL supernatants were added to 2.8 mL of working buffer containing 0.167 mg/mL O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide in potassium phosphate buffer and were read immediately in a Spectrophotometer (Beckman, model DU 640).
Statistical analysis
Results were expressed as mean ± SD. Group means were compared by a Student’s t-test. Significance was determined with P values of 0.05. The power of the study was analyzed using SAS 9.1 (SAS Institute Inc., Cary, NC).
Results
Injection of LPS into the vitreous body induced inflammation in the anterior uvea characterized by vasodilation, protein exudation, and PMN infiltration of iris–ciliary body and anterior chamber (Table 1). The clinical vasodilation score in the LPS control animals was 2.7 ± 0.75 compared with 1.9 ± 0.9 in LXA4 and 1.3 ± 0.45 in the 15-epi-LXA4 analog-treated animals. Aqueous humor PMN cell count in the control animals 24 h after LPS injection was 11,400 ± 396 compared with 4,585 ± 2,769 and 2,645 ± 989 in LXA4 and 15-epi-LXA4 analog-treated animals, respectively. Myeloperoxidase, specific to PMN granules, in the iris–ciliary body of LPS-injected eyes was 3.3 ± 0.81 and that in LXA4 and the 15-epi-LXA4 analog-treated eyes were 2.2 and 2.1, respectively. These results are summarized in the Table 1. LXA4 and the 15-epi-LXA4 analog-treated animals had significantly (P < 0.001 to P < 0.05) reduced aqueous humor cell counts and protein and MPO values compared with the LPS control. Between the 2 drug treatments, the difference in aqueous humor protein and MPO values was not significant (P > 0.05). However, the aqueous humor PMN counts in the 15-epi-LXA4 analog-treated group was significantly lower (P < 0.05) compared with the LXA4-treated group. The power achieved for each one of the tests is summarized in the Table 2.
E
Values are mean ± SD. No. of observations are within parenthesis.
Abbreviations: LPS, lipopolysaccharide; MPO, myeloperoxidase; PMN, polymorphonuclear neutrophil.
P
Abbreviations: LPS, lipopolysaccharide; MPO, myeloperoxidase; PMN, polymorphonuclear neutrophil.
Discussion
Progressions to chronic inflammation, scarring, and fibrosis or complete resolution are the natural outcome to inflammation.24 During inflammation, prostaglandins and leukotrienes and a variety of pro- and anti-inflammatory mediators are formed in tissues and cells. Lipoxin A and B are synthesized during cell–cell interactions in an environment of neutrophils, platelets, and vascular endothelium.25 Furthermore, in recent years, resolvin Es and resolvin Ds derived, respectively, from eicosapentaenoic acid and docosahexaenoic acid have been reported to form endogenously during inflammation.8, 9 A number of studies have established that lipoxins and resolvin Ds act as anti-inflammatory agents and mediate the resolution of inflammation in human diseases and in animal models of inflammation.25–27 Studies on the role of lipoxins and resolvins in the resolution of ocular inflammation are limited. It has been reported that lipoxins accelerated corneal re-epithelialization28 and suppressed corneal angiogenesis in mice.29 Another interesting study by Connor and colleagues.30 demonstrated that dietary intake of omega-3-polyunsaturated fatty acids reduced retinal angiogenesis in mouse model implicating resolvin Es in the resolution of vascular inflammation in the retina.
In the present study, LXA4 and the 15-epi-LXA4 analog administered intravitreally in rats significantly reduced protein exudation into the anterior chamber, aqueous humor PMN counts, and MPO activity in the iris–ciliary body. The difference between the mean values of these parameters of the groups treated with the above compounds was not significant. This observation suggests that lipoxin A4, unstable in tissues and cells containing prostaglandin dehydrogenase, was relatively stable in the vitreous body. Otherwise, its inhibitory effects on the inflammatory responses would have been significantly less than that of the stable analog.
This suggests that relative instability of LXA4,16, 17 because of the metabolic inactivation by PGDH, does not seem to be a major factor in LPS-induced uveitis model used in this study. However, the apparent significant difference (P < 0.05) in PMN cell counts but not in protein and MPO values between the LXA4 and LXA4 analog-treated groups suggests the limitations of using aqueous humor PMN cell counts only as a criterion for assessing the degree of inflammation. It is reported that only a fraction of the PMN infiltrates into the aqueous humor in endotoxin-induced uveitis,23 because of the firm adherence of the cells to the inner surface of the iris venules.31 This means the number of PMN cells counted in aqueous humor represents only a fraction of the cells infiltrated into the ocular tissues. On the other hand assaying the MPO, an enzyme in the azurophil granules of PMN,21 allows an accurate quantification of the number of infiltrated PMN cells23 in the ocular tissues. Therefore based on the results of aqueous humor protein and MPO values, it is concluded that there is no difference in the inhibitory effects of LXA4 and its analog against LPS-induced uveitis. The power of the study as analyzed using SAS 9.1 achieved a significantly high value of 92% and over for all the tests except vasodilation score between LPS vs. LPS+LXA4 groups.
The results of our study demonstrated that lipoxin and its analog inhibited ocular inflammatory responses. A recent study32 reported very similar anti-inflammatory effect of topical administration of LXA4 and their analogs administered before or after LPS injection. These authors also reported an effect of lipoxins in the same order as that of prednisolone. Both of these studies are important addition showing the anti-inflammatory effects of lipoxin A4 and 15-epi-lipoxin A4 analog in a model of anterior uveitis induced by LPS. Lipoxins and their analogs might prove to be a therapeutic alternative to the currently used anti-inflammatory drugs against ocular inflammatory diseases.
Footnotes
Acknowledgments
Supported by an unrestricted grant from Research to Prevent Blindness, Inc, New York, NY. We thank Betty Nunn and Kathy Schaeffer for the technical assistance, and Ms. Savitri N. Appana, Department of Bioinformatics and Biostatistics, University of Louisville, for her help with the Power analysis.
Author Disclosure Statement
The authors have no proprietary interest in the products or no competing financial interests.