The groups of potent inflammatory mediators that have gained interest in recent years are nitric oxide (NO) and the cytokines, especially interleukin-1-beta (IL-1β), interleukin-2R (IL-2R), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α).1–5
Nitric oxide and cytokines, including IL-1β, IL-2R, IL-6, and TNF-α, play a crucial role in inflammatory and immunologic response. Any immunologic or inflammatory stimuli induce the production of NO by the expression of the inducible isoform of the nitric oxide synthase (NOS).1 Interleukin-1 is synthesized by various ocular and lens epithelial cells and has been detected in the aqueous humor after intraocular lens (IOL) implantation in rabbits.2 The presence of IL-2 and TNF has been found in patients with uveitis, suggesting a possible contribution to the inflammation.3,5 Interleukin-6 is a proinflammatory cytokine produced by a variety of cells including monocytes, macrophages, keratinocytes, endothelial cells, and fibroblasts.4 Various cytokines have been detected in the eyes of patients with intraocular inflammation, and experimental animal studies have shown that injection of recombinant cytokines into eyes can cause uveitis.6–8
We performed an experiment in rabbits to evaluate the efficacy of NG-nitro L-arginine (L-NAME) and topical steroidal and nonsteroidal drugs on aqueous levels of NO and cytokines in inflammation after cataract surgery. To our knowledge, this is the first analysis of the effectiveness of these substances after experimental cataract surgery.
Materials and Methods
Fifteen rabbits weighing 1.5 to 2.0 kg were used in this experiment. All animals were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The rabbits were anesthetized with ketamine hydrochloride (5 mg/kg) and xylazine hydrochloride (2 mg/kg). Pupil dilation was achieved by repeated instillation in each eye of cyclopentolate hydrochloride 1% and phenylephrine hydrochloride 10% 2 to 3 times at approximately 5 minute intervals. Topical proparacaine hydrochloride 0.5% (Alcaine®) was used for local anesthesia.
A 2.0 to 3.0 mm corneal incision was made with a Superblade. Sodium hyaluronate (Healon®) was introduced into the anterior chamber. After an anterior capsulotomy was made with a 30 gauge cystotome, intercapsular phacoemulsification was performed. Healon was removed by irrigation, and the corneal incision was closed by 1 or 2 X-sutures of 9-0 nylon.
After surgery, 5 mg of gentamicin and 1 mg of betamethasone sodium phosphate were injected subconjunctivally. An antibiotic ointment and atropine sulfate 1% drops were instilled. Then, the rabbits were divided into 3 treatment groups: Group 1, treated with topical prednisolone acetate 1% drops 5 times a day for 1 week; Group 2, flurbiprofen 0.03% drops 5 times a day for 1 week; Group 3, a 0.1 cc subconjunctival injection of 150 mg/kg L-NAME 1 and 3 days postoperatively. Three rabbits, serving as controls, received a subconjunctival injection of an equal volume of balanced salt solution (BSS®) at the same times as the L-NAME injections.
Anterior Chamber Puncture
Aqueous humor was collected preoperatively before the corneal incision and 1, 3, 7, and 30 days postoperatively. The animals were anesthetized. After a paracentesis was made, a sample of the aqueous humor was obtained from the anterior chamber with a 30 gauge needle.
Precaution was taken at each anterior chamber puncture to ensure that the aqueous humor was not contaminated by bleeding and to avoid contact with anterior chamber structures. The test sample of aqueous humor was not diluted or stained, and it was stored at –40°C until use. Nitric oxide levels were measured according to the method described by Orpana et al.9 Immunoreactive levels of cytokines, IL-1β, IL-2R, IL-6, and TNF-α were determined by enzyme-linked immunoadsorbent assay using commercially available kits (Diagnostic Products Corp.). The Mann–Whitney U test was used for statistical analysis.
There were no statistically significant differences in preoperative levels of NO and cytokines among the groups (P > .05). The mean preoperative levels of IL-1β, IL-2R, IL-6, TNF-α, and NO were 5.68 pg/mL ± 2.1 (SD) (range 3.7 to 7.1 pg/mL), <50 U/mL, 12.13 ± 3.7 pg/mL (range 9.9 to 16.1 pg/mL), 11.29 ± 4.8 pg/mL (range 9.8 to 16.6 pg/mL), 28.59 ± 11.1 μmol/L (range 17 to 35 μmol/L), respectively.
One day postoperatively, IL-1β and IL-6 levels were lower in Groups 1 and 3 than in Group 2 and the control group; however, the levels were high in all groups compared with those preoperatively. The differences were only statistically significant between Groups 3and 2 and between Group 3 and the control group(P < .05). The TNF-α and NO levels were significantly lower in Groups 1 and 3 than in Group 2 and the control group (P < .05) (Table 1).
Three days postoperatively, IL-1β and IL-6 levels in Group 2 and the control were the highest, with a peak value of 35.86 ± 13.2 and 55.17 ± 12.7 pg/mL and 41.18 ± 13.5 and 59.12 ± 16.2 pg/mL, respectively. By 30 days, the levels had gradually decreased to 13.16 ± 3.8 and 17.14 ± 9.1 pg/mL and 18.61 ± 12.1 and 31.60 ± 12.8 pg/mL, respectively (Tables 2 to 4). The difference was significantly higher in the control group than the other groups at 3 days only (P < .05). There was no statistical significance later (P > .05). Although the levels of IL-1β and IL-6 were lower in Groups 1 and 3 than in Group 2 and the control group, the differences were not statistically significant (P > .05). The TNF-α and NO levels in Groups 1 and 3 were the lowest: 9.12 ± 2.3 pg/mL and 112.12 ± 66.7 μmol/L in Group 1 and 8.86 ± 2.8 pg/mL and 58.62 ± 18.3 μmol/L in Group 3. The levels increased slightly at 30 days: Group 1, 14.16 ± 3.8 pg/mL and 127.16 ± 48.7 μmol/L; Group 3, 21.66 ± 12.1 pg/mL and 78.42 ± 61.8 3 μmol/L. The differences were statistically lower in Groups 1 and 3 than in Group 2 and the controls 3 and 7 days postoperatively; there were no statistically significant differences at 30 days (P > .05). Although slight differences were observed between Groups 2 and 4 at all times, the differences were not statistically significant (P > .05). The levels of IL-2R in all groups were below detection limits of the assay(<50 U/mL) at all times.
The cellular and biochemical events triggered by uveitis involve a complex array of cells and a heterogenous network of mediators of intraocular inflammation. These mediators of immune and inflammatory responses may play a central role in uveitis as a result of cell priming because IL-1, TNF, and other cytokines are also being generated.10 The concomitant accumulation of these mediators in various parts of the eye promotes the intraocular inflammatory response and cell damage. The identification of the cellular events that cause the accumulation of networks of mediators of inflammation and their effects have important therapeutic implications in inflammation.11
Increasing attention has been paid to the role of NO synthase in various physiological and pathologic situations such as diabetes, hypertension, low-tension glaucoma, retinal ischemia, uveitis, and keratitis.12–15 Nitric oxide may play a key role in exacerbating the inflammation. Many immunologic or inflammatory stimuli induce the production of NO. Mandai et al.16 reported that there is a significant increase in NO synthase activity in ocular tissues during the inflammatory process in the anterior chamber.
Nitric oxide synthase is induced in many types of cells, including macrophages, neutrophils, and vascular smooth muscles, and is thought to be associated with some pathologic conditions involving these cell types.17 Mulligan and coauthors18 found that the inhibition of inducible NO synthase successfully decreased both tissue damage and inflammation without affecting neutrophil infiltration in a vasculitis model. Nitric oxide is generated from L-arginine by the enzyme NOS, which is inhibited effectively in vitro or in vivo by analogs of L-arginine such as L-NAME and N-iminoethyl L-ornithine (L-NIO).19
Two types of NOS have been identified, one constitutive and Ca2+-dependent (c-NOS) and the other inducible and Ca2+ -independent (i-NOS). L-NAME is more selective for c-NOS than for i-NOS, and it can cause irreversible inactivation of c-NOS.20 In vivo, L-NAME can be considered a potent inhibitor of c-NOS and L-NIO, of i-NOS.
There is a close relationship between NO synthase and cytokines. Inducible NOS can produce large amounts of NO once induced by mediators such as IL-1β, IL-2, IL-6, and TNF-α. Inducible NOS is present in vascular smooth muscle cells and infiltrating inflammatory cells.
Interleukin-1, IL-2, IL-6, and TNF are all cytokines, the general term for a group of peptide mediators that function as up and down regulators of immunologic, inflammatory, and reparative host responses to an injury.21 These cytokines have been shown to interact synergistically, overlapping biologic activities in nonocular tissues.4,22 Some cytokines such as IL-1 increase prostaglandin E2 (PGE2) synthesis, which disrupts the blood–aqueous barrier23; PGE2 plays a major role in postoperative inflammation after IOL implantation.24
The primary, known physiologic effect of IL-2 is to act as a T lymphocyte growth factor. Some earlier observations implicate IL-2 as a potential contributor to postoperative inflammation. The serum IL-2 levels of patients with ocular Behçet's disease were found to be significantly different from the levels in a control group.25
The molecular structure and biological properties of these cytokines are homologous in humans and rabbits.26 The presence of IL-1β and TNF might play a role in the pathogenesis of the ocular barrier when the blood–ocular barrier has been breached and ocular antigens have been exposed to the systemic immune system.27
Interleukin-6 can be induced by IL-1 and TNF.28 Serum levels of IL-6 are increased in patients with acute bacterial infections.29 Interleukin-6 was detected in the aqueous humor after IOL implantation in humans.30 Malecaze et al.30 observed that levels of IL-6 in aqueous humor of patients with moderate uveitis after cataract surgery dramatically increased over preoperative values. Interleukin-6 caused an acute inflammation subsequent to the intravitreal injection and is known to be induced by IL-1 in the inflammatory response.31 Since both IL-1 and TNF can induce IL-6 production, there is speculation that many in vivo effects of these molecules are mediated by IL-6. Furthermore, observations have shown low IL-6 levels in the aqueous humor, suggesting local ocular production.30 Despite extensive evidence implicating these cytokines in the intraocular inflammatory response, little is known about their levels and influences after cataract surgery.
In this study, we assessed the therapeutic efficacy of L-NAME and steroidal drugs on aqueous levels of NO and cytokines. Blockade of NO synthesis with the competitive inhibitor L-NAME reduced TNF-α and NO levels, but no inhibitory effect was observed on IL-1β and IL-6 levels in rabbits. Corticosteroids reduce the induction of NOS in many organs. This finding suggests that, in part, NO may be responsible for tissue damage of the anti-inflammatory and immunosuppressive actions of corticosteroids. Our results also show that NO and TNF-α levels after cataract surgery were suppressed by L-NAME and corticosteroids. The suppression was stronger in the L-NAME-treated group than the steroid-treated group. Both drugs inhibited the levels of IL-1β and IL-6. Finally, this observation may cause the development of new methods designed to block this inflammatory response more specifically and effectively.
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