Ocular Immunology & Inflammation, Early Online, 1–8, 2014! Informa Healthcare USA, Inc.

ISSN: 0927-3948 print / 1744-5078 online

DOI: 10.3109/09273948.2014.949779

ORIGINAL ARTICLE

Levels of Selected Aqueous Humor Mediators(IL-10, IL-17, CCL2, VEGF, FasL) in Diabetic Cataract

Sanja Mitrovic, MD1, Tomislav Kelava, MD, PhD

2, Alan Sucur, MD2, and

Danka Grcevic, MD, PhD2

1Department of Ophthalmology, General Hospital, ‘‘Dr. J. Bencevic,’’ Slavonski Brod, Croatia; andOphthalmology Clinic, Slavonski Brod, Croatia and 2Department of Physiology and Immunology and Laboratory

for Molecular Immunology, University of Zagreb School of Medicine, Zagreb, Croatia

ABSTRACT

Purpose: To compare levels of selected mediators in serums and aqueous humor (AH) of type 2 diabetes mellituscataract patients with senile cataract patients, and to determine their association with postoperative cornealedema (CE).

Methods: Patients (32 senile and 29 diabetic cataract) undergoing standardized phacoemulsification combinedwith intraocular lens implantation were recruited. CE was assessed using an ordinal scale (grade 0 to 3). IL-10,CCL2, IL-17, FasL, and VEGF were measured by ELISA.

Results: Diabetic patients had higher AH levels of VEGF (p = .042) and IL-10 (p = .021), lower AH levels of FasL(p = .048), and higher serum levels of CCL2 (p = .002). AH levels of CCL2 were higher in diabetic patients withmore severe CE at the first postoperative day (p = .012).

Conclusions: We found disturbed AH microenvironment in diabetic cataract, with significant changes for VEGF,IL-10, and FasL. Higher CCL2 was associated with the development of early postoperative CE in diabeticpatients.

Keywords: Aqueous humor, cataract, chemokines, cytokines, type 2 diabetes mellitus

The prevalence of type 2 diabetes mellitus (T2DM) israpidly increasing, with severe socioeconomicimpacts, since it ultimately causes damage of variousorgans, leading to diabetic complications.1 Cataract,diabetic retinopathy (DR), and diabetic macularedema (DME) are considered to be the most frequentdiabetes-related eye complications and major causesof visual impairment. Cataract is one of the earliestcomplications of T2DM and diabetic patients developcataracts at an earlier age, 2–5 times more often thanthe general population.2–4 It has been estimated thatup to 20% of all cataract surgery is performed ondiabetic patients.5

Although the pathogenesis of diabetic cataract isstill not fully understood, it is generally accepted that

the accumulation of sorbitol, a polyol produced fromglucose by the aldose reductase enzyme, is aninitiating mechanism, creating intracellular hyperos-molarity that causes an influx of fluid and lensswelling. The so-formed osmotic stress leads to acollapse and liquefaction of lens fibers and inducesapoptosis of lens epithelial cells, which results in theformation of lens opacities. Furthermore, the oxida-tive stress caused by glycation of lens proteins mightaccelerate cataract formation.6,7

A growing number of reports suggest that anactivation of the immune system might contribute tocataract pathogenesis in diabetic patients and prolongtheir postoperative recovery as well.8,9 Disturbedprotein content of aqueous humor (AH) is caused by

Correspondence: Tomislav Kelava, Department of Physiology and Immunology, University of Zagreb School of Medicine, Salata 3, 10000Zagreb, Croatia. E-mail: tkelava@mef.hr

Received 3 April 2014; revised 24 July 2014; accepted 25 July 2014; published online 14 October 2014

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the blood–humor barrier leakage, accumulation ofinflammatory cells, and local intraocular cytokineproduction. AH samples from cataract diabeticpatients have higher concentrations of various proin-flammatory cytokines, such as interleukin (IL)-1, IL-6,and IL-12.10–12 Furthermore, elevated levels of che-mokines, including IL-8 and CCL2 (monocyte chemo-tactic protein-1), were also reported.12–14 IL-6 andvascular endothelial growth factor (VEGF) in AHhave been associated with the severity of DR andDME. VEGF plays an essential role in ischemic retinaland choroidal neovascularization.15 In addition to thechanges in AH, high vitreous levels of VEGF, IL-1b,IL-6, tumor necrosis factor-a, and intercellular adhe-sion molecule-1 (ICAM-1) were related to increasedvascular permeability in DME and neovascularizationin proliferative DR.10,16,17

Protein profiling of immune mediators in AH wasmostly performed in patients who, besides cataract,also had proliferative DR or DME, focusing on theoutcome of these entities. The data about theimmunoregulatory mediators in T2DM patients whohave not developed DR or DME are still relativelyscarce. Furthermore, for mediators which previousstudies indicated should be included in cataractpathogenesis (VEGF, IL-10, CCL2) the data on theirpossible association with postoperative outcome, interms of CE development, are lacking. In addition,IL-17, a proinflammatory cytokine produced by Th17lymphocytes, has been found to be increased in someeye diseases (such as uveitis and dry eye syndrome).18

In the mouse model of choroidal neovascularization,proapoptotic mediator Fas ligand (FasL) effectivelyprotected eye structures from inflammation andneovascularization.19 Nevertheless, the levels ofIL-17 and FasL have not been investigated in diabeticcataract.

Therefore, the major aim of our study was tocompare the levels of selected mediators—proinflam-matory IL-17 and CCL2, immunosuppressive IL-10,angiogenic VEGF, and proapoptotic FasL—in serumand AH between T2DM cataract patients, withoutclinically evident DR or DME, and nondiabeticcataract patients. Due to the complex interplay ofcytokines, chemokines, growth, and apoptotic factorsduring immune response, we hypothesized thatconcurrent changes in the level of these mediatorscontribute to cataract development in diabeticpatients. Since a disturbed microenvironment in thediabetic eye may contribute to an increased frequencyof complications related to intraocular lens (IOL)implantation, we tested the associations of localand systemic concentrations of selected mediatorswith intraoperative and postoperative parameters.Particularly, we focused on the development ofcorneal edema (CE) as one of the main causes oflow visual acuity in the immediate postoperativeperiod after IOL surgery.20,21

SUBJECTS AND METHODS

Patient Enrollment

After obtaining approval from the institutional ethicscommittee and informed consent from participants,29 diabetic (T2DM) and 32 nondiabetic patients(Ctrl) undergoing phacoemulsification combinedwith IOL implantation were recruited into the study(registered on ClinicalTrials.gov under the identifierNCT01832311). The study was conducted in accor-dance with the Declaration of Helsinki principles.Inclusion criteria for the diabetic group were durationof T2DM for 10–15 years, therapy with oral hypogly-cemic agents for glycemic control, and no other ocular(retinal) or systemic complications of T2DM exceptcataract. Blood biochemical test for glycosylatedhemoglobin (HbA1c) level was performed. For thecontrol nondiabetic cataract group, we selected age-and sex-matched patients with fasting blood glucoselevel within the normal range and diagnosedsenile cataract, the most common age-related eyecomplication.5,22

Before surgery, all patients underwent completeophthalmologic examination, including determinationof the cataract grade by Wisconsin grading method,23–

25 visual acuity with and without correction (Snellencharts), measuring of intraocular pressure (IOP) usinga Goldmann applanation tonometer, slit-lamp biomi-croscopic examination of the anterior eye segment,fundus examination in mydriasis using Goldmannlens, keratometry, and B-scan ocular ultrasoundbiometry (Nidek US-3000 Ultrasonic A/B scanner,Nidek). Presence and severity of nuclear cataract wasdefined on a five-level scale by comparison with a setof four standard slit-lamp photographs as describedby Mitchell et al.25 Different involvement of cortical orposterior subcapsular opacity was present in somepatients, equally distributed between groups. Patientswho had cataract that could result from some otherocular conditions, systemic disease (except T2DMfor the diabetic group) or trauma were excludedfrom the study, as well as patients with immunedisease, local or systemic inflammatory condi-tions, which could affect cytokine concentrationsin serum or AH. Furthermore, patients (both nondia-betic and diabetic) were randomized into a groupreceiving topical nonsteroidal anti-inflammatory drug(NSAID) ketorolac 0.5%, dosed 4 times a day, starting3–7 days before surgery and ending 4–5 weeks aftersurgery, respectively, and into a group not receivingNSAID.

Surgical Procedure

Cataract surgery was performed using a standardizedphacoemulsification procedure combined with IOL

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implantation. A single ophthalmologist performed allthe surgical interventions in this study (S. Mitrovic).During surgery, the following parameters wererecorded for each patient: duration and intensity(EPT, equivalent phaco time) of the applied ultra-sound (U/S, seconds), duration of irrigation/aspira-tion (I/A, seconds), type of implanted lens, andviscoelastic. There were no intraoperative complica-tions, such as rupture of the posterior capsule, irisprolapse, or loss of vitreous mass.

Postoperatively, patients were examined at severaltimepoints (days 1 and 8, week 3, and month 3). Eachfollow-up examination included a determination ofthe uncorrected and corrected distance visual acuityby Snellen chart, IOP measurement, slit-lamp biomi-croscopy examination of the anterior segment, andfundus examination. CE was assessed using anordinal scale as grade 0 to 3 (0 = no edema, 1 = mini-mal edema of the central cornea, 2 = moderate edemawith Descemet’s folds, 3 = large edema of the entirecornea), modified according to Crandall et al.26

Sample Collection

AH sampling was performed during the cataractsurgery. AH samples were collected by paracentesisimmediately prior to phacoemulsification procedure,subsequently performed using the same paracentesisport. A cannula was used to manually aspirate 0.15–0.25 mL of AH into an insulin syringe.13 Samples weretransferred into microcentrifuge tubes, centrifuged at300 g for 5 min, and supernatants were harvested foranalysis. Immediately before the surgery, peripheralblood samples were drawn by cubital venipuncture,centrifuged at 400 g for 15 min, and serums (2 mL)were harvested. Collected samples were aliquotedand frozen at �80 �C until the final analysis.

ELISA

The serum and AH concentrations of IL-10(Quantikine High Sensitivity Immunoassay, R&Dsystems, Minneapolis, MN, USA), CCL2, IL-17, FasL,and VEGF (Quantikine Immunoassay, R&D systems)were determined according to the manufacturerinstructions. Briefly, samples were added to mono-clonal antibody-precoated plates and incubated for2–3 h at room temperature, washed 5 times, andincubated for the next 2 h with horseradish peroxidaseconjugated (for CCL2, IL-17, FasL, and VEGF) oralkaline phosphatase conjugated (for IL-10) specificantibodies. After 5 further washes, the reaction wasvisualized with substrate solution (tetramethylbenzi-dine for CCL2, IL-17, FasL, and VEGF) or substrate

and amplifier solutions (NADPH and amplifierenzymes for IL-10), and halted with hydrochloric orsulfuric acid, respectively. Optical density was deter-mined within 15 min on the microplate reader (Bio-Rad, Hercules, CA, USA) set to excitation wavelengthof 450 nm (for CCL2, IL-17, FasL, and VEGF) or490 nm (for IL-10).

Statistical Analysis

Clinical parameters and cytokine concentrations wereexpressed as means ± standard deviation or medianwith interquartile range (IQR), according to datadistribution. Group difference was compared usingnonparametric Mann-Whitney or Kruskal-Wallis test.Data were correlated using Spearman’s coefficient ofrank correlation rho (�) with its 95% confidenceintervals (CI). The receiver operating characteristic(ROC) curve analysis was expressed as area undercurve (AUC) with its 95% CI and used to determinethe efficacy of analyzed cytokines to discriminatebetween diabetic and nondiabetic groups. Diagnosticefficacy for cytokine concentration values wasassessed through sensitivity and specificity at thecutoff point. The minimal false discovery rate level(Storey’s q) was calculated for multiple test correc-tion.27 Statistical analysis was performed using theMedCalc software package (version 12.5, MedCalc,Mariakerke, Belgium) and the R project for statisticalcomputing (available at www.r-project.org/). For allexperiments, results with p5.05 and q5.05 wereconsidered statistically significant.

RESULTS

Patient Characteristics

A total of 61 cataract patients (32 with senile cataractand 29 with diabetic cataract) were recruited into thestudy (Table 1). One diabetic patient developed DMEafter surgery and was excluded from further analysis.Nuclear cataract was present in all patients, mostlywith opacity grade 3 (12/32 Ctrl and 11/29 T2DM) or4 (9/32 Ctrl and 11/29 T2DM) by the Wisconsincataract grading method and no statistical differencebetween groups. Median HbA1c level was 9.6%(range 8.9–11.7%) for the T2DM group and 5.4%(range 5.0–5.9%) for the control group. In both groups,patients were further divided into NSAID-treated andnontreated subgroups. The surgical procedure ofphacoemulsification and IOL implantation wereuneventful in all patients, with no significant differ-ence in EPT, U/S, and A/I time, and the degree ofpostoperative CE between groups (Table 1).

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Levels of Immune Mediators in Serumand Aqueous Humor

To test anterior chamber microenvironment of theselected mediators in T2DM patients, we determinedtheir levels in AH of diabetic and nondiabetic group(Figure 1A). Diabetic patients had higher AH levels ofVEGF (p = .042) and IL-10 (p = .021), and lower levelsof FasL (p = .048) compared with nondiabetic controls.

In parallel, systemic levels of the same mediatorswere determined in serum of diabetic and nondiabeticcataract patients (Figure 1B). In contrast to AH levels,there were no significant differences between groups,except for CCL2, which was significantly higher inserum of diabetic patients compared with nondiabeticcontrols (p = .002). IL-17 levels were similar in diabeticand nondiabetic groups, both in AH and serum, withvery low overall concentrations (close to assaydetection sensitivity). The AH levels of the selectedmediators were not in positive correlation with thecorresponding serum levels, except for CCL2 in thenondiabetic group of patients (�= 0.44 (95% CI 0.05–0.72); p = .034).

Since several proinflammatory cytokines mayinduce a cascade production of downstream media-tors that act cooperatively in cataract pathogen-esis,13,16 we tested the association between the AHlevels of the selected mediators (Figure 2). Significantnegative correlations between AH levels of CCL2 andIL-10 (p = .016), and VEGF and IL-10 (p = .005) wereobserved in nondiabetic patients, whereas in thegroup of diabetic patients we could not confirm anysignificant correlation.

Finally, we evaluated the ability of the intraopera-tive levels of analyzed mediators in serum and AH todiscriminate between patients with senile and diabeticcataract by ROC curve analysis. The two groups ofpatients could be distinguished based on the AH levelof VEGF (AUC = 0.68, 95% CI 0.52–0.81, p = .029), FasL(AUC = 0.66, 95% CI 0.52–0.79, p = .033), and IL-10(AUC = 0.68, 95% CI 0.54–0.80, p = .013) as well asserum level of CCL2 (AUC = 0.75, 95% CI 0.62–0.86,p5.001) (Figure 3).

TABLE 1. Demographic and clinical characteristics of patientswith senile and diabetic cataract.

Variablea

Senile cataract(control)

n = 32

Diabeticcataractb

n = 29

Age, years 69.0 ± 4.5 70.5 ± 4.9Female/Male 18/14 15/14Cataract grade 3.4 ± 1.1 3.6 ± 0.8EPT, s 1.8 ± 1.5 1.8 ± 1.6U/S, s 123.5 ± 56.1 125.2 ± 46.7I/A, s 22.6 ± 9.5 20.2 ± 8.0Corneal edema

Day 1 1.1 ± 0.6 1.1 ± 0.5Day 8 0.2 ± 0.4 0.2 ± 0.4

NSAID, yes/no 15/17 17/12

aValues are presented as means ± standard deviation. EPT,equivalent phaco time; U/S, ultrasound; I/A, irrigation/aspiration; NSAID, nonsteroidal anti-inflammatory drug.Corneal edema is presented for days 1 and 8, graded usingthe ordinal scale: 0 = no edema, 1 = minimal edema of thecentral cornea, 2 = moderate edema with Descemet’s folds,3 = large edema of the entire cornea.bOne diabetic patient developed diabetic macular edema afterthe surgery and was excluded from further analysis.

FIGURE 1. Levels of immunoregulatory/angiogenic mediatorsin aqueous humor and serum of nondiabetic (control) anddiabetic (T2DM) cataract patients. Concentrations of mediatorswere determined in aqueous humor (AH) (A) and serum (B) byenzyme-linked immunosorbent assay. Boxes indicate themedian with interquartile range; lines indicate the minimumand maximum values and individual points indicate outliers.Group-to-group comparisons were performed using nonpara-metric Mann-Whitney test; p5.05 and q5.05 are consideredstatistically significant. Ctrl, nondiabetic cataract patients;T2DM, cataract patients with type 2 diabetes mellitus; VEGF,vascular endothelial growth factor; FasL, Fas ligand; IL-10,interleukin-10; IL-17, interleukin-17.

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Association of Clinical Parameters andImmune Mediators with the Degree ofCorneal Edema

Development of CE, as one of the most importantindicators of postoperative recovery in uneventfulcataract surgery,20,21 was analyzed in the context of

cataract severity and intraoperative parameters. Aclinically detectable CE was present in 28 out of 32nondiabetic and 25 out of 29 diabetic patients atpostoperative day 1, and in only 6 patients from eachgroup at postoperative day 8, with no significantdifference between groups (Table 1). CE was com-pletely resolved in all patients at month 3postoperatively.

More severe CE at postoperative day 1 wasassociated with longer EPT and U/S time, as well aswith higher cataract grade in both groups of patients(Figure 4A). However, at day 8, the degree of CEremained significantly associated with U/S time(p = .012) only in the diabetic group of patients (notshown).

FIGURE 2. Association between aqueous humor levels ofimmunoregulatory/angiogenic mediators in nondiabetic (con-trol) (A) and diabetic (B) cataract patients. Concentrations ofmediators were determined in aqueous humor (ah) by enzyme-linked immunosorbent assay. Values were correlated using rankcorrelation Spearmans coefficient � (95% confidence intervalfor �); p5.05 and q5.05 are considered statistically significant.Ctrl, nondiabetic cataract patients; T2DM, cataract patients withtype 2 diabetes mellitus; VEGF, vascular endothelial growthfactor; FasL, Fas ligand; IL-10, interleukin-10.

FIGURE 3. Discriminatory ability of aqueous humor (A) andserum (B) levels of immunoregulatory/angiogenic mediatorsbetween nondiabetic (control) and diabetic cataract patients.Receiver operating characteristic (ROC) curves for aqueoushumor (ah) and serum (s) concentrations of CCL2, VEGF, FasL,and IL-10. Diagnostic efficacy for those values was assessedusing the sensitivity and specificity at a cutoff point. ROC curveanalyses; p5.05 and q5.05 are considered statistically signifi-cant. VEGF, vascular endothelial growth factor; FasL, Fasligand; IL-10, interleukin-10.

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To further confirm the importance of the intrao-perative AH profile of selected mediators, we ana-lyzed their concentrations in relation to thedevelopment of CE. Among tested factors, only the

increased AH level of CCL2 was associated with moresevere CE in diabetic cataract (Figure 4A, p = .012).Therefore, we analyzed if preoperative NSAID topicalapplication would affect CCL2 level and found lowerCCL2 concentration in the NSAID-treated subgroupof diabetic patients compared to the nontreateddiabetic patients, but the difference was not statisti-cally significant (Figure 4B, p = .101).

DISCUSSION

In our study we confirmed that the AH levels ofVEGF, IL-10, and FasL in cataract diabetic patientswithout clinically evident DR or DME are signifi-cantly different compared to those of nondiabeticcataract controls, indicating changes in anteriorchamber microenvironment associated with T2DM.The levels of analyzed mediators in AH weregenerally not associated with the correspondingvalues in plasma, suggesting that they are producedintraocularly and that the blood–AH barrier is stillintact.

Compared to patients with senile cataract, VEGFlevel was higher in AH of diabetic patients (althoughthe difference was not highly significant, p = .042),whereas there was no significant difference in thelevel of CCL2. Several previous studies have observedthat both CCL2 and VEGF levels are elevated indiabetic patients with proliferative DR.28–30 Cheunget al. studied cytokine levels in nondiabetic cataractpatients compared with two groups of diabeticcataract patients (with or without developed DR)and reported an elevated VEGF level in both groupsof diabetic patients, but the CCL2 level was elevatedonly in the group with developed DR.13 Our results,together with results of Cheung et al.,13 support theconclusion that VEGF becomes upregulated in dia-betic cataract at an earlier timepoint (before thedevelopment of DR), whereas the upregulation ofCCL2 occurs later, with the development of clinicallyevident DR. Moreover, we found that diabetic patientshad higher CCL2 serum levels compared withnondiabetic patients, confirming that CCL2 plays animportant role in the systemic manifestation ofT2DM.31

Development of cataract is associated with theapoptotic death of lens epithelial cells.7,32,33 It wasreported that diabetic cataract patients with prolif-erative DR have more apoptotic cells and higherexpression of Fas mRNA in lens epithelium thannondiabetic controls.32 Moreover, Kim et al. recentlydemonstrated that expression of Bax, another proa-poptotic molecule, in lens epithelial cells increaseswith the duration of diabetes.33 On the other hand,FasL may also induce apoptosis of infiltrating Fas-expressing immune cells, thereby protecting the

FIGURE 4. Association of preoperative characteristics andintraoperative clinical parameters with the degree of cornealedema in nondiabetic (control) and diabetic (T2DM) cataractpatients. (A) Association of intraoperative parameters (equiva-lent phaco time (EPT), ultrasound (U/S)), grade of nuclearcataract (NG), and aqueous humor levels of CCL2 (ahCCL2)with the degree of corneal edema (CE). CE was determined atfirst postoperative day using an ordinal scale (grade 0 to 3).Boxes indicate the median with interquartile range; linesindicate the minimum and maximum values and individualpoints indicate outliers. Group-to-group comparisons wereperformed using nonparametric Kruskal-Wallis test; p5.05and q5.05 are considered statistically significant. (B) Effect ofnonsteroidal anti-inflammatory drug (NSAID) on ahCCL2.Group-to-group comparisons were performed using nonpara-metric Mann-Whitney test. Ctrl, nondiabetic cataract patients;T2DM, cataract patients with type 2 diabetes mellitus.

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ocular integrity and maintaining immune privilege ofthe eye.34,35 We found lower levels of the soluble formof FasL in AH of diabetic patients, suggesting adecreased level of apoptosis. However, this observa-tion should be taken cautiously since the differencewas not highly significant (p = .048), and we deter-mined the soluble form of FasL that does not alwaysreflect the membrane (functional) form of FasL.36

Besides FasL, immunosuppressive cytokines mayalso prevent inflammatory ocular damage.14 Theelevated level of IL-10 in diabetic eyes, which wefound, might be a compensatory response to theenhanced expression of proinflammatory mediators.Although Nolan et al. reported that VEGF stimulatesIL-10 production in cultured macrophages,37 weobserved negative correlation of VEGF and IL-10levels in AH, but only in the nondiabetic group.Moreover, Dong et al. reported an increase in CCL2level and a decrease in IL-10 level in T2DM cataractpatients with the progression of DR, indicatingthat deregulation of both mediators is associatedwith advanced manifestations of diabetic ocularcomplications.14

It is known that postoperative complications, suchas inflammation and macular edema, occur moreoften in diabetic cataract patients. However, we foundno difference in the degree of CE between diabeticand nondiabetic patients. Similarly, Altintas et al.concluded that, in terms of corneal thickness, uncom-plicated phacoemulsification is safe in diabetic andnondiabetic cataract patients.20 Also, CE was notaffected by the AH sampling procedure (approxi-mately 85% incidence of early postoperative CE forsenile cataract with and without AH sampling,personal data). However, we observed that higherAH levels of CCL2 were associated with greaterdegree of CE in diabetic patients, which indicates thatCCL2 may prolong postoperative recovery in cataractpatients burdened with chronic proinflammatoryactivity.38 NSAID pretreatment, mostly applied as aprophylaxis for macular edema,39,40 was able to lowerthe CCL2 level in the diabetic group of patients,although the difference was not statistically signifi-cant (p = .101), indicating a need for further investiga-tion. In addition, we were able to confirm thatdiabetic and nondiabetic patients may be distin-guished based on the AH levels of VEGF, IL-10, andFasL, which could serve as possible therapeutictargets in the preoperative treatment in order toalleviate postoperative complications in diabeticcataract patients.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authorsalone are responsible for the content and writing ofthe paper.

This work was supported by a grant from theCroatian Ministry of Science, Education and Sports(108-1080229-0142).

We thank Ivan Kresimir Lukic for statistical editing.

REFERENCES

1. Wild S, Roglic G, Green A, et al. Global prevalence ofdiabetes: estimates for the year 2000 and projections for2030. Diabetes Care. 2004;27:1047–1053.

2. Benson WE. Cataract surgery and diabetic retinopathy.Curr Opin Ophthalmol. 1992;3:396–400.

3. Klein BE, Klein R, Moss SE. Prevalence of cataracts in apopulation-based study of persons with diabetes mellitus.Ophthalmology. 1985;92:1191–1196.

4. Nielsen NV, Vinding T. The prevalence of cataract ininsulin-dependent and non-insulin-dependent-diabetesmellitus. Acta Ophthalmol (Copenh). 1984;62:595–602.

5. Hamilton AMP. Epidemiology of diabetic retinopathy.In: Hamilton AMP, Ulbig MW, Polkinghorne P, eds.Management of Diabetic Retinopathy. London: BMJ; 1996:1–15.

6. Obrosova IG, Minchenko AG, Vasupuram R, et al. Aldosereductase inhibitor fidarestat prevents retinal oxidativestress and vascular endothelial growth factor overexpres-sion in streptozotocin-diabetic rats. Diabetes. 2003;52:864–871.

7. Pollreisz A, Schmidt-Erfurth U. Diabetic cataract—patho-genesis, epidemiology and treatment. J Ophthalmol. 2010;2010:1–8.

8. Funatsu H, Yamashita H, Noma H, et al. Prediction ofmacular edema exacerbation after phacoemulsificationin patients with nonproliferative diabetic retinopathy.J Cataract Refract Surg. 2002;28:1355–1363.

9. Chu L, Wang B, Xu B, et al. Aqueous cytokines aspredictors of macular edema in non-diabetic patientsfollowing uncomplicated phacoemulsification cataract sur-gery. Mol Vis. 2013;19:2418–2425.

10. Funatsu H, Yamashita H, Noma H, et al. Increased levelsof vascular endothelial growth factor and interleukin-6in the aqueous humor of diabetics with macular edema.Am J Ophthalmol. 2002;133:70–77.

11. Gverovic Antunica A, Karaman K, Znaor L, et al. IL-12concentrations in the aqueous humor and serum ofdiabetic retinopathy patients. Graefes Arch Clin ExpOphthalmol. 2012;250:815–821.

12. Jonas JB, Jonas RA, Neumaier M, et al. Cytokine concen-tration in aqueous humor of eyes with diabetic macularedema. Retina. 2012;32:2150–2157.

13. Cheung CM, Vania M, Ang M, et al. Comparison ofaqueous humor cytokine and chemokine levels in diabeticpatients with and without retinopathy. Mol Vis. 2012;18:830–837.

14. Dong N, Xu B, Wang B, et al. Study of 27 aqueous humorcytokines in patients with type 2 diabetes with or withoutretinopathy. Mol Vis. 2013;19:1734–1746.

15. Kwak N, Okamoto N, Wood JM, Campochiaro PA (2000)VEGF is major stimulator in model of choroidal neovascu-larization. Invest Ophthalmol Vis Sci. 41:3158–3164.

16. Funatsu H, Yamashita H, Sakata K, et al. Vitreous levels ofvascular endothelial growth factor and intercellular adhe-sion molecule 1 are related to diabetic macular edema.Ophthalmology. 2005;112:806–816.

17. Demircan N, Safran BG, Soylu M, et al. Determination ofvitreous interleukin-1 (IL–1) and tumour necrosis factor

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(TNF) levels in proliferative diabetic retinopathy. Eye(Lond). 2006;20:1366–1369.

18. Kang MH, Kim MK, Lee HJ, et al. Interleukin-17 in variousocular surface inflammatory diseases. J Korean Med Sci.2011;26:938–944.

19. Roychoudhury J, Herndon JM, Yin J, et al. Targetingimmune privilege to prevent pathogenic neovasculariza-tion. Invest Ophthalmol Vis Sci. 2010;51:3560–3566.

20. Altintas AG, Yilmaz E, Anayol MA, et al. Comparison ofcorneal edema caused by cataract surgery with differentphaco times in diabetic and non-diabetic patients. AnnOphthalmol (Skokie). 2006;38:61–65.

21. Amon M, Menapace R, Scheidel W. Results of cornealpachymetry after small-incision hydrogel lens implant-ation and scleral-step incision poly(methyl methacrylate)lens implantation following phacoemulsification. J CataractRefract Surg. 1991;17:466–470.

22. Stefek M, Karasu C. Eye lens in aging and diabetes: effectof quercetin. Rejuvenation Res. 2011;14:525–534.

23. Panchapakesan J, Mitchell P, Tumuluri K, et al. Five yearincidence of cataract surgery: the Blue Mountains EyeStudy. Br J Ophthalmol. 2003;87:168–172.

24. Wong WL, Li X, Li J, et al. Cataract conversion assessmentusing lens opacity classification system III and Wisconsincataract grading system. Invest Ophthalmol Vis Sci. 2013;54:280–287.

25. Mitchell P, Cumming RG, Attebo K, et al. Prevalence ofcataract in Australia: the Blue Mountains eye study.Ophthalmology. 1997;104:581–588.

26. Crandall AS, Zabriskie NA, Patel BC, et al. A comparisonof patient comfort during cataract surgery with topicalanesthesia versus topical anesthesia and intracamerallidocaine. Ophthalmology. 1999;106:60–66.

27. Storey JD. A direct approach to false discovery rates. J RStat Soc. 2002;64:479–498.

28. Oh IK, Kim SW, Oh J, et al. Inflammatory and angiogenicfactors in the aqueous humor and the relationship todiabetic retinopathy. Curr Eye Res. 2010;35:1116–1127.

29. Schoenberger SD, Kim SJ, Sheng J, et al. Increasedprostaglandin e2 (pge2) levels in proliferative diabeticretinopathy, and correlation with VEGF and inflammatorycytokines. Invest Ophthalmol Vis Sci. 2012;53:5906–5911.

30. Tashimo A, Mitamura Y, Nagai S, et al. Aqueous levels ofmacrophage migration inhibitory factor and monocytechemotactic protein-1 in patients with diabetic retinopathy.Diabet Med. 2004;21:1292–1297.

31. Mine S, Okada Y, Tanikawa T, et al. Increased expressionlevels of monocyte CCR2 and monocyte chemoattractantprotein-1 in patients with diabetes mellitus. BiochemBiophys Res Commun. 2006;344:780–785.

32. Okamura N, Ito Y, Shibata MA, et al. Fas-mediatedapoptosis in human lens epithelial cells of cataractsassociated with diabetic retinopathy. Med ElectronMicrosc. 2002;35:234–241.

33. Kim B, Kim SY, Chung SK. Changes in apoptosis factors inlens epithelial cells of cataract patients with diabetesmellitus. J Cataract Refract Surg. 2012;38:1376–1381.

34. Stein-Streilein J. Immune regulation and the eye. TrendsImmunol. 2008;29:548–554.

35. Griffith TS, Yu X, Herndon JM, et al. CD95-inducedapoptosis of lymphocytes in an immune privileged siteinduces immunological tolerance. Immunity. 1996;5:7–16.

36. Kovacic N, Grcevic D, Katavic V, et al. Targeting Fas inosteoresorptive disorders. Expert Opin Ther Targets. 2010;14:1121–1134.

37. Nolan A, Weiden MD, Thurston G, et al. Vascular endo-thelial growth factor blockade reduces plasma cytokines ina murine model of polymicrobial sepsis. Inflammation.2004;28:271–278.

38. Daniele G, Guardado Mendoza R, Winnier D, et al. Theinflammatory status score including IL–6, TNF-a, osteo-pontin, fractalkine, MCP-1 and adiponectin underlieswhole-body insulin resistance and hyperglycemia in type2 diabetes mellitus. Acta Diabetol. 2014;51:123–131.

39. Almeida DR, Johnson D, Hollands H, et al. Effect ofprophylactic nonsteroidal antiinflammatory drugs oncystoid macular edema assessed using optical coherencetomography quantification of total macular volume aftercataract surgery. J Cataract Refract Surg. 2008;34:64–69.

40. Almeida DR, Khan Z, Xing L, et al. Prophylactic nepafenacand ketorolac versus placebo in preventing postoperativemacular edema after uneventful phacoemulsification.J Cataract Refract Surg. 2012;38:1537–1543.

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