Study population
This study conducted in the Hospital “Nuestra Señora de la Luz” specialized in ophthalmology and High Medical School of National Polytechnic Institute (Mexico). This study was performed in accordance with the Helsinki Declaration. 120 patients (54 men and 66 women) took part in the study, selected from individuals who went to the “Nuestra Señora de la Luz” Hospital for pterygium resection surgery from 2015 to 2019 and had been given a clinical diagnosis of primary or recurrent pterygium. The tissue samples evaluated represent five conditions: the control (healthy conjunctiva) and four patient groups, including untreated primary pterygium, recurrent pterygium, and primary pterygium treated topically or systemically with NAC. The following were criteria for non-inclusion: 1) history of disease or trauma to the ocular surface involving the sampling area; 2) ocular surgery during the three months prior to the pending pterygium surgery; 3) oral or topical immunosuppressive treatment during four weeks before the pending pterygium surgery; 4) systemic diseases such as diabetes, rheumatoid arthritis, another autoimmune disease or neoplasia; 5) chronic addictions to substances (e.g., tobacco or alcohol). Finally, during the four years of the study, 28 patients with primary pterygium (12 women and 16 men) were excluded.
The number of patients by group and their age was presented in Table 1.
Table 1. Age (mean ± SD) of the study population by group and gender.
Groups
|
C
|
P
|
R
|
Ps
|
Pt
|
all patients age
|
61.3 ± 6.0
|
56.9 ± 10.5
|
46.1 ± 6.3**
|
53.0 ± 10.3
|
55.4 ± 9.3*
|
N
|
19
|
36
|
19
|
17
|
29
|
men age
|
61.9 ± 5.9
|
53.8 ± 8.7*
|
45.6 ± 7.1**&
|
50.1 ± 8.0**
|
56.5 ± 13.4&
|
N
|
7
|
18
|
10
|
8
|
11
|
women age
|
60.8 ± 5.4
|
62.1 ± 13.0
|
46.8 ± 4.9**
|
55.5 ± 8.9
|
52.5 ± 10.3*&
|
N
|
12
|
18
|
9
|
9
|
18
|
C, control group; P, untreated primary pterygium group; R, recurrent pterygium group; Ps, systemic-treated primary pterygium group; and Pt, topic-treated primary pterygium group. Treatments were with NAC. * P < 0.05 versus the control; & P < 0.05 comparing the primary pterygium group to other pathological groups.
The average age of individuals in the recurrent pterygium group was lower than in the control group. Upon comparing the primary pterygium group to the other pathological groups, some moderate differences were detected (principally in men). Taking into account all patients with primary pterygium that used the service of the hospital (including those excluded from the study), the percentage of recurrence was 17% for all patients, 19% for men and 16% for women. Among the pterygium groups with a limited quantity of patients, recurrence occurred in 2 with systemic treatment (1 man and 1 woman, 13% and 11%, respectively, of the treatment group considered by gender), and in 3 with topical treatment (1 man and 2 women, 9% and 11%, respectively, of the treatment group by gender). However, it is incorrect to make a statistical comparison of these data with those from all patients because the treatment groups had the low quantity of patients.
Tissue samples obtaining
The pterygium samples were obtained during pterygium resection surgery. The surgery was performed by dissecting the head of the pterygium of the cornea, followed by resection of the body in a block. During the process, the conjunctiva was taken together with the subconjunctival fibrovascular tissue. The bare sclera was covered with a conjunctival autograft taken from the upper or lower conjunctiva and fixed with nylon 10.0 sutures in separate stitches. The control samples were obtained from the resection of conjunctiva resulting from extracapsular cataract extraction. During resection by this technic, conjunctiva contained minimal quantity (10-20 mg) of healthy tissue that was separated for control samples. The tissue extracted was stored in 2 ml flat bottom Eppendorf tubes and immediately frozen in liquid nitrogen, then stored at -70° C to await processing.
Treatments
For systemic treatment of primary pterygium, NAC was administered at 600 mg/day for one month prior to surgery. For respiratory diseases, the same dose is commonly used in systemic treatments aimed at increasing the level of GSH in tissues [16]. Topical treatment of primary pterygium, also lasting one month before surgery, involved the application of one drop of a 10% solution of NAC four times per day. The treatment groups do not represent a clinical trial. Rather, they are a scientific instrument for exploring the origin of high levels of GSH in recurrent pterygium. The reason for choosing NAC as the present treatment is of its prior use as a therapy for different pathologies including ocular disorders.
Samples processing
Samples were processed periodically, when approximately 20 had accumulated, resulting in an average storage time of 2-3 weeks after collection. Samples of homogenates were obtained by placing the tissue in a 30 mmol cold phosphate buffer solution (pH 7.2) and adding 0.1% of Triton 100 (1 mg of tissue per 10 μl buffer). Tissues were homogenized and centrifuged at 10 000 rpm for 15 min at 4 °C, and the supernatants were stored at -70 °C to be processed within two weeks. Regarding the tissue homogenates, assay kits (Cayman Chemical, MI, USA) were employed for measurement of total proteins (TP, No.704002), NO (nitrate/nitrite colorimetric assay kit, No.780001), total reduced and oxidized GSH (glutathione assay kit, No. 703002) and CAT (CAT assay kit, No. 707002). The level of 3NT (3-nitrotyrosine Elise kit, Abcam, No. ab116691, UK) was established in homogenates by the enzyme-linked immunosorbent assay (ELISA). The values of NO, GSH, GSSG and 3NT are expressed as nmol/mg of TP/ml, corresponding to a µmol concentration. The degree of GSH oxidation was calculated by the following equation: GSSG% = GSSG/2GSH x 100. Photometer Eliza Stat Fax 4200 was used for colorimetric measurements.
Western blot assay
From the different groups with pterygium and control group, 100 µg of protein were subjected to 10% SDS-PAGE under non-reducing conditions and transferred to polyvinylidene fluoride membranes (Immobilon PVDF, 0.45 μm; Millipore, USA). The membranes were then blocked with 5% albumin serum bovine in TBS and 0.1% Tween 20 (TBS-T, pH 7.4) for two hours. Subsequently, they were washed three times with TBS-T and incubated with the primary antibody for eNOS (Cat. SC-5302) at 4 °C for 18 h under continuous agitation. After adequately washing with TBS-T, the membranes were incubated with the secondary antibody (Cat. SC-516102) for 2h under constant agitation. Detection was then carried out by the enhanced chemiluminescence method (Western Blotting Luminol Reagent, Cat. 2048, Santa Cruz, CA, USA). Membranes were photographed and the image digitalized to perform densitometric analysis on Imagen Studio Lite software (LI-COR Biosciences). The relative presence of each protein was normalized with β-actin as the housekeeping protein. Before implementation of Western blot, the study groups were separated into subgroups of six samples each. Three groups had an average of 3x6 samples each: the control, recurrent pterygium and primary pterygium with systemic treatment. The untreated primary pterygium group had 6x6 determinations and the topic-treated primary pterygium group 4x6 determinations.
Statistical analysis
Data was expressed as the mean values ± standard deviations for each group and examined on GraphPad Prism software version 6 (GraphPad Software Inc., La Jolla, CA, USA). The results were analyzed by ANOVA, followed by Tukey post hoc test, considering significance at p<0.05. The bivariate Pearson correlation was also used to compare the parameters between groups.