Molecular Inflammation in the Contralateral Eye After Cataract Surgery in the First Eye

Abstract

Purpose: The purpose of this study was to assess the inflammatory status of the aqueous humor in the fellow eye after uneventful cataract surgery in the first eye.

Methods: At the screening stage, aqueous humor samples from 15 first-eye and 15 second-eye cataract patients were collected just before cataract surgery and assayed using human cytokine antibody array. Screened cytokines were then verified using a suspension array system with aqueous humor samples obtained from 35 first-eye and 36 second-eye cataract patients.

Results: The cytokine antibody array revealed that interleukin-1 receptor antagonist (Il-1ra) and macrophage inflammatory protein (MIP)-1a and MIP-1b were expressed at high levels in first-eye patients and were lower in second-eye patients, whereas opposite trends were found for monocyte chemoattractant protein 1 (MCP-1) and for regulated on activation, normal T expressed and secreted (RANTES) (all, P < 0.05, Student's t-test). However, only MCP-1 and IL-1ra were significantly different between the two groups after Bonferroni correction (both P < 0.00125). In the replication stage, the suspension cytokine array revealed that only MCP-1 expression was significantly greater in the aqueous humor of second-eye patients than in that of first-eye patients (P = 0.0067, Student's t-test).

Conclusions: This study revealed that expression of MCP-1, a pain-related inflammatory chemokine, was significantly increased in aqueous humor in the contralateral eye after first-eye cataract surgery. This suggests there may be a sympathetic ophthalmic type uveitis in the contralateral eye after first-eye cataract surgery and that may help to explain why second-eye phacoemulsification is often more painful. (ClinicalTrials.gov number, NCT01824927.)

Cataract surgery is one of the most common surgical procedures performed worldwide.^1,2 Advances in instrumentation and surgical techniques, such as small or micro incisions, and improved ophthalmic viscoelastic materials have reduced the risk and side effects of phacoemulsification. The wide application of topical anesthetics has also encouraged the development and clinical use of phacoemulsification.³ Topical anesthesia is now the mainstream anesthetic method in cataract surgery, as it has several advantages over regional anesthesia, including immediate visual recovery, better safety, decreased patient anxiety, and improved intraoperative patient cooperation.⁴ 

Patients having cataract surgery in the second eye under topical anesthesia often report increased pain relative to the surgery in the first eye. Ursea et al.⁵ reported a subtle increase in pain in second-eye surgery relative to that in first-eye surgery, which appears to be associated with decreased preoperative anxiety. However, other researchers have reported no significant differences between pain perceived in first-eye surgery and that in second-eye surgery,^6–8 or it could be that the patients just perceived that the second-eye surgery was more painful.⁹ 

Previous studies merely focused on subjective psychological mechanisms to explain why patients experienced pain during second-eye cataract extraction. None of those studies examined objective physiological mechanisms such as whether surgery in the first eye might cause sympathetic irritation in the second eye. This might be because cataract extraction does not appear to cause significant inflammation in the contralateral eye. However, with modern biomolecular techniques such as human cytokine antibody arrays, we were able to detect changes indicative of subclinical inflammation in the second eye. 

Pain is known to be closely related to inflammation.^10,11 Pain-related inflammatory cytokines such as interleukin (IL)-1, IL-6,¹² IL-8, spinal macrophage inflammatory protein (MIP),¹³ monocyte chemoattractant protein 1 (MCP1; also known as CCL2 or MCAF),¹⁴ and regulated on activation, normal T expressed and secreted (RANTES),¹⁵ have been extensively examined in studies of other organs. By comparing the levels of inflammatory cytokines in first-eye surgery with those in second-eye cataract surgery, we were be able to identify possible objective mechanisms of pain in second-eye cataract surgery. 

Furthermore, changes in inflammatory markers were detected in both eyes in studies of patients with sympathetic ophthalmia or bilateral uveitis after penetrating injury to one eye.^16,17 A recent study also showed that unilateral exposure to ultraviolet radiation type B was associated with inflammatory infiltration in the anterior segment of both eyes.¹⁸ Therefore, we speculated that there might be a sympathetic reaction in the contralateral eye after cataract surgery in the first eye, mimicking the process of penetrating injury and altered immune homeostasis. Accordingly, we performed the current study to determine whether such an inflammatory response was involved in this process. 

First, we used a RayBio human cytokine antibody array (Human Inflammation Array 3; product QAH-INF-3; RayBio; RayBiotech, Norcross, GA, USA) to screen the cytokines in aqueous humor and then verified the suspicious cytokines by using a Bio-Plex suspension array (Bio-Plex Suspension array system; Bio-Rad, Hercules, CA, USA). 

Methods

The Institutional Review Board of the Eye and ENT Hospital of Fudan University approved this case–control study. The study was registered (ClinicalTrials.gov identifier NCT01824927). All patients provided written informed consent before enrollment. All procedures adhered to the Declaration of Helsinki and were conducted in accordance with the approved research protocol. 

Subjects

This study was conducted at the Eye and Ear, Nose, Throat (ENT) Hospital of Fudan University between September 2013 and February 2014. We recruited 51 consecutive patients who had undergone uneventful cataract surgery one month previously and who returned for cataract surgery on the contralateral eye. We also recruited 50 patients who underwent cataract surgery for the first time as the control group. All cataract surgeries were performed by the same surgeon (YL). Eyes with glaucoma, uveitis, zonular weakness, previous trauma, fundus pathology, or diabetes-related disorders were excluded from the study. The design of this study is summarized in Supplementary Figure S1. 

Sample Collection

Eyelids and surrounding skin were swabbed with disinfectant. Samples of aqueous humor (100–200 μL) were aspirated by corneal paracentesis, by inserting a 26-gauge needle into the anterior chamber just before surgery. Samples were immediately stored at −80°C until used for proteomic analysis. 

Cytokine Antibody Arrays

We simultaneously analyzed a selection of 40 cytokines, using an antibody-based protein array (RayBio; RayBiotech). The assay was performed in accordance with the manufacturer's instructions. Briefly, the cytokine antibody array is based on the sandwich immunoassay principle. A unique set of antibodies is immobilized in specific location spots on the surface of a membrane. Array membranes were incubated with samples of aqueous humor from 15 first eyes and 15 second eyes. Cytokines present in the samples were captured by corresponding antibodies, and the bound cytokines were detected with a cocktail of biotinylated antibodies. In this study, the signals were visualized using fluorescent dye-conjugated streptavidin (cy3 equivalent) and detected using a GenePix 4000B system (Axon Instruments, Foster City, CA, USA). Densitometric analysis of each spot was then performed using a GenePix Pro version 6.0 software (Axon Instruments). 

Immune Cytokine Profiling Using a Bio-Plex Suspension Cytokine Array

Concentrations of the selected cytokine (interkeukin-1 receptor antagonist [IL-1ra]) and four chemokines (monocyte chemoattractant protein [MCP]-1, MIP-1a, MIP-1b, and RANTES) in samples of aqueous humor obtained from 35 first eyes and 36 second eyes were determined by using a human 5-plex assay (Bio-Rad) in accordance with the manufacturer's instructions.¹⁹ Samples were diluted in sample diluent and incubated for 30 minutes with capture antibody-coupled magnetic beads. After three washes in a Bio-Plex Pro washing station, the samples were incubated with the biotinylated detection antibody for 30 minutes in the dark. Each captured cytokine was detected by the addition of streptavidin–phycoerythrin, and the expression levels were measured using a Bio-Plex array reader. The cytokine concentrations were calculated using Bio-Plex Manager software (Bio-Rad). 

Statistical Analysis

All data are means ± standard deviations. The χ² test was used to examine differences in categorical variables. One-way analysis of variance (ANOVA) followed by Tukey's test was used to detect significant differences in quantitative variables. Student's t-test was used to detect differences between the first- and second-eye surgery groups. P values < 0.05 were considered statistically significant. Cytokine antibody array data were also controlled with Bonferroni correction. Because there were 40 different comparisons, P = 0.00125 (i.e., P = 0.05/40) was considered statistically significant after Bonferroni correction. All analyses were performed using SPSS software version 11.0 (SPSS, Inc., Chicago, IL, USA). 

Results

Demographic Data

Demographic characteristics of the patients are presented in Table 1. There were no statistically significant differences among the four groups in terms of age, sex, or eye(s) operated on (all P > 0.05, ANOVA and Tukey's test for age; χ² tests for sex and eye[s] operated on). 

Table 1

 

Demographic Data for All Participants

 

Cytokine Antibody Arrays

In the discovery stage, we used the antibody array technique to determine the expression profiles of 40 cytokines in the aqueous humor of patients undergoing either first- or second-eye cataract surgery. Marked changes in the aqueous humor proteome were found in second-eye patients compared with that in first-eye patients, as shown in Table 2. Of note, the expression levels of IL-1ra, MIP-1a, and MIP-1b were very high in first-eye patients and much lower in second-eye patients, whereas levels of the MCP-1 and RANTES proteins were low in first-eye patients and increased in second-eye patients (all, P < 0.05, Student's t-test) (Fig. 1). However, after Bonferroni correction, only the levels of MCP-1 and IL-1ra were significantly different between the two groups (both, P < 0.00125, Student's t-test with Bonferroni correction). 

Table 2

 

Actual Concentrations of Selected Proteins With Cytokine Antibody Array in First- and Second-Eye Patients*

 

Figure 1

 

Results of cytokine antibody arrays (discovery stage). (A) Representative results are shown for antibody arrays using 40 markers. (Left column) First eye; (right column) second eye. (B) Representative results for expression levels of MCP-1, IL-1ra, RANTES, MIP-1a, and MIP-1b proteins in first-eye and second-eye patients. (C) Expression levels of MCP-1, IL-1ra, RANTES, MIP-1a, and MIP-1b proteins in first-eye and second-eye patients (pg/mL). *P < 0.05; **P < 0.001.

Suspension Cytokine Arrays

To further validate the results of the cytokine antibody arrays, we performed suspension cytokine array tests by using a human 5-plex assay. Table 3 compares expression levels of MCP-1, IL-1ra, RANTES, MIP-1a, and MIP-1b between the first- and second-eye patients. However, the suspension cytokine arrays revealed that only the MCP-1 level was significantly different between the two groups of patients (P < 0.05, Student's t-test) (Fig. 2). 

Table 3

 

Concentrations of MCP-1, IL-1ra, RANTES, MIP-1a, and MIP-1b Proteins in First- and Second-Eye Patients With Suspension Cytokine Arrays*

 

Figure 2

 

Results of the suspension cytokine arrays (replication stage). (A) Standard curve for MCP-1, IL-1ra, RANTES, MIP-1a, and MIP-1b. (B) Expression levels of MCP-1, IL-1ra, RANTES, MIP-1a, and MIP-1b proteins in first-eye and second-eye patients (pg/mL).

Discussion

The wide application of topical anesthetic in cataract surgery has led to some controversy about whether cataract surgery is more painful in the second or first eye, even though the same procedures and anesthesia conditions are used in both procedures. However, in most of the previous studies in this setting, pain was assessed using subjective responses or visual analog scale scores.^5,6,9 In the present study, we used the cytokine antibody array technique and found that aqueous humor levels of MCP-1, a pain-related inflammatory chemokine, were significantly increased in patients undergoing cataract surgery on the second eye compared with those undergoing surgery on the first eye. These results indicate that the inflammatory status of the contralateral eye may be affected by cataract surgery in the first eye and that the increased pain reported by patients during cataract surgery on the second eye may be justified. 

Aqueous humor contains many cytokines, the levels of which change markedly in ocular disorders such as inflammation,^20–22 glaucoma,²³ and diabetic retinopathy.²⁴ However, no studies have measured the levels of cytokines in aqueous humor of the contralateral eye after cataract surgery in the first eye. Several factors have limited previous studies. For example, because of the difficulty associated with quantifying minimal inflammation in the anterior chamber by clinical observation, laser flare/cell meters had been very popular.^25,26 However, for unknown reasons, these systems have not been commercially available in recent years. In addition, the small quantities of proteins present in aqueous humor make it difficult to use standard Western blotting techniques to analyze protein expression levels in these samples. Cytokine antibody array techniques can simultaneously detect numerous biomarkers on one membrane, using samples containing very low quantities of proteins. This represents a significant advantage over other methods, and cytokine antibody arrays are therefore particularly suitable for measuring protein expression levels in the aqueous humor. 

Using this technique, we found that the expression of MCP-1 in aqueous humor was increased in patients undergoing second-eye cataract surgery compared with that in patients undergoing first-eye surgery. Monocyte chemoattractant protein 1, also known as monocyte chemotactic and activating factor (MCAF) or chemokine (C-C motif) ligand 2 (CCL2), is produced by many cell types, especially monocytes and macrophages. It was the first C-C chemokine to be discovered and is perhaps the one most extensively studied. Normally, chemokines can be classified into two main subgroups according to their inflammatory and homeostatic functions.²⁷ Monocyte chemoattractant protein 1 is an inflammatory cytokine that controls recruitment of leukocytes in inflammation and tissue injury.²⁸ Upregulation of MCP-1 has been observed in many inflammation-dependent diseases such as atherosclerosis,²⁹ arthritis,³⁰ and cancer.³¹ 

Monocyte chemoattractant protein 1 is also a pain-related cytokine. Levels of MCP-1 were reported to be closely correlated with the severity of pain in patients with fibromyalgia.³² Elevated levels of MCP-1 were also observed in intra-articular lavage samples from painful knees.³³ Monocyte chemoattractant protein 1 directly interacts with nociceptive sensory neurons in rats.^34,35 Moreover, intrathecal administration of MCP-1 was shown to cause neuropathic pain-like behavior in rats, whereas its neutralizing antibody reduced this pain.³⁶ Monocyte chemoattractant protein 1 levels in the aqueous humor of patients undergoing second-eye cataract surgery were also found positively correlated with the extent of pain in the same eye according to our previous study (Spearman ρ = 0.539, P < 0.001).³⁷ 

Monocyte chemoattractant protein 1 may also be a biomarker of sympathetic eye condition. One of the most well-known sympathetic eye conditions is sympathetic ophthalmia, a type of bilateral diffuse granulomatous panuveitis that develops after surgical or penetrating trauma to one eye, followed by the onset of uveitis in the contralateral eye. T helper 1-mediated delayed hypersensitivity is the main mechanism involved in the pathogenesis of sympathetic ophthalmia. The downstream effects of activated, delayed hypersensitivity T lymphocytes are mediated by cytokines such as MCP-1, IL-8, and macrophage migration inhibitory factor, which promote the secretion of inflammatory mediators from activated macrophages, amplifying inflammation. Other sympathetic eye conditions may also exist. Meyer et al.¹⁸ reported that unilateral exposure to ultraviolet radiation type B also causes local inflammatory reactions in both eyes. Therefore, an elevated MCP-1 level in aqueous humor of the contralateral eye following cataract surgery in the first eye may also indicate a sympathetic eye condition. 

Although expression levels of IL-1ra, RANTES, MIP-1a, and MIP-1b were not significantly different between the first- and second-eye patients in this study, these four cytokines appear to be involved in the mechanism of pain as well. IL-1ra, the specific endogenous antagonist of IL-1 receptors, regulates the action of IL-1β, a key component in the pathogenesis of inflammation³⁸ and neuropathic pain.³⁹ RANTES, a member of the chemokine supergene family, also affects eosinophils during the inflammatory process in vitro.^40,41 Recent studies have demonstrated that peripheral RANTES is implicated in the processing of pain information.⁴² Macrophage inflammatory protein–1a was classically described as a neutrophil chemoattractant and activator. It is potentially relevant to some ocular inflammatory disorders.⁴³ It also modulates induction of pain in several pathological conditions, especially models of neuropathic pain.⁴⁴ Macrophage inflammatory protein–1b is a key mediator of inflammation, and the peripheral MIP-1b–CCR5 axis mediates neurological inflammation and neuropathic pain.⁴⁵ Because MIP-1a, MIP-1b, and RANTES shared the same receptor to the b-chemokine CCR5, it is possible that these molecules interact in some way in the development of inflammation and pain.⁴⁶ 

Some studies have measured the concentrations of these cytokines, obtaining values of 23 to 3636 pg/mL for MCP-1,^47–49 0 to 356 pg/mL for IL-1ra,^47,49 0 to 93 pg/mL for RANTES,^47,49 0 to 9.1 pg/mL for MIP-1a,^21,43 and 0 to 232 pg/mL for MIP-1b.^43,47,49,50 Therefore, the concentrations of these cytokines measured in our study were within the ranges reported in previous studies. However, there are other aspects to this study that need to be interpreted with some caution. First, we did not fully evaluate the conditions that could potentially influence the composition of the aqueous humor, including systemic diseases, systemic medications, characteristics of previous surgery (duration, use of ultrasonography, surgical manipulation, surgical complications, type of IOL, postsurgical redness or pain or dry eye, and others). Second, although no ocular surface inflammatory conditions were reported either before or after the surgical procedure in our patients, caution is necessary to ensure that inflammation in the anterior chamber originates from the intraocular surgical procedure. Third, more studies should be carried out to further verify the correlation between the levels of cytokines in the anterior chamber and their subjective perception of pain. Therefore, future studies should account for these factors. 

In conclusion, using the cytokine antibody array technique, we found that the level of MCP-1, a pain-related inflammatory chemokine, was increased significantly in the aqueous humor of patients undergoing second-eye cataract surgery compared to that in patients undergoing first-eye surgery. This suggests there may be a sympathetic ophthalmic type uveitis in the contralateral eye after first-eye cataract surgery and that may help to explain why the second-eye phacoemulsification is often more painful. 

Acknowledgments

Supported by National Natural Science Foundation of China Grants 81100653, 81470613, and 81200669; National Health and Family Planning Commission of the People's Republic of China Grant 201302015; and Program of Shanghai 100 Medical Scientist Grant XBR2011056, Shanghai, China. 

Disclosure: X.-J. Zhu, None; D. Wolff, None; K.-K. Zhang, None; W.-W. He, None; X.-H. Sun, None; Y. Lu, None; P. Zhou, None