Trabeculectomy is commonly performed in patients with uncontrolled glaucoma and with the use of antimetabolites and surgical technique modification is successful in the majority of patients [
To understand the mechanism of filtering surgery failure the research has mainly focused on conjunctiva. Changes in the number of conjunctival fibroblasts and inflammatory cells were associated with increased risk of trabeculectomy failure. Long-term use of antiglaucoma drops with preservative benzalkonium chloride (BAK) induces subclinical inflammation of conjunctiva with over expression of human leukocyte antigen- (HLA-) DR on epithelial cells. Several cytokines were found to stimulate proliferation of Tenon's capsule fibroblasts, cells with a key role in scarring process, such as TGF-
In the prospective study we examined the effect of five cytokines in the aqueous humour on the outcome of trabeculectomy. We also investigated the superior bulbar conjunctiva overlying the area of filtration area for the expression of HLA-DR, the markers of subclinical inflammation, on conjunctival epithelial cells and CD80 positive cells (antigen-presenting cells) during follow-up to determine whether changes in the expression of these molecules are associated with the outcome of surgery.
Thirty patients (30 eyes) with uncontrolled intraocular pressure on maximal topical antiglaucoma medication who needed glaucoma surgery were included in this prospective study. The study protocol was approved by National Ethics Committee and all patients after receiving complete information signed informed consent form for inclusion in the study.
At the beginning of surgery after local anaesthesia aqueous humour was obtained through the side incision made by
Patients were scheduled for control visits 1 week, 1 month, 2, 3, 6, and 12 months after surgery, and more often if deemed necessary.
The collection of conjunctival cells was performed by the imprinting of the ocular surface onto Millipore filter paper (versus 0.22
Surgical success was defined as intraocular pressure (IOP) less than 21 mm Hg without antiglaucoma medication and was evaluated at month 3, 6, and month 12 follow-up visits.
A BD Human Inflammation CBA (Cytometric Bead Array) Kit was used to quantitate IL-8, IL-1
Monoclonal antibodies conjugated with fluorochromes, CD80 (FITC), Anti-Cytokeratin (FITC) and Anti-HLA-DR (PE), were purchased from BD Pharmingen (San Jose, CA, USA). A two-parameter analysis was performed to determine the expression of HLA-DR on cytokeratin positive cells (epithelial cells) and on CD80 positive cells (antigen-presenting cells). Flow cytometric analysis was performed using BD FACSCanto cytometer. At least 3000 events were collected per sample. Cells were analyzed with BD FACSDiva software. The FACS data are reported as mean fluorescence intensities (MFI) and as percentages of positive cells.
For statistical analysis SPSS 15.0 software for Windows was used. Because the data were not normally distributed, median, minimum, and maximum values were calculated for the levels of cytokines and for the expression of HLA-DR by epithelial and antigen-presenting cells. Mann-Whitney
Aqueous humour specimens from 30 patients undergoing trabeculectomy (13 females, 17 males) with the mean age 63.6 years (standard deviation 10.3 years) were analyzed for cytokines. The levels of cytokines in all patients as well as the values regarding the type of glaucoma are summarized in Table
Median levels of cytokines (ranges) in the aqueous humour of 30 eyes (30 patients) and with respect to the type of glaucoma.
Cytokines (pg/mL) | All eyes ( | POAG ( | EXG ( | |
---|---|---|---|---|
IL-8 | 36 (19–177) | 41 (19–177) | 33 (28–53) | .76 |
IL-1 | 22 (15–48) | 21 (15–48) | 22 (18–27) | .74 |
IL-6 | 27 (9–809) | 38 (9–809) | 15 (14–96) | .16 |
IL-10 | 10 (3–19) | 10 (3–19) | 10 (9–12) | .80 |
TNF- | 9 (4–21) | 9 (4–21) | 8 (6–9) | .07 |
IL-12p70 | 14 (6–39) | 14 (10–39) | 12 (6–15) | .05 |
Mann-Whitney
POAG: primary open-angle glaucoma.
EXG: exfoliative glaucoma.
At 3 months of follow-up 23 eyes (patients) had successful surgery (defined as IOP less than 21 mm Hg without antiglaucoma medication), and at 6 and 12 months there were 21 eyes with successful surgery. The median values and ranges for the percentages of HLA-DR positive cytokeratin cells (epithelial cells) and CD80 positive cells (antigen-presenting cells), with their median fluorescence intensity are shown in Table
Percentage of HLA-DR positive epithelial and CD80 positive cells (antigen-presenting cells) and mean fluorescence intensity (MFI) of ocular surface inflammatory molecules obtained by impression cytology at month 1 and 3 of follow-up in eyes with successful and failed surgery.
% of HLA-DR positive cells and their MFI median values (minimum-maximum) | 3 months after surgery | 6 months and 12 months after surgery | ||||
Success ( | Failure ( | Success ( | Failure ( | |||
% of HLA-DR positive epithelial cells at month 1 | 52 (11–97) | 83 (65–83) | .058 | 58 (11–97) | 52 (32–83) | .960 |
MFI of HLA-DR on epithelial cells at month 1 | 14834 (2742–55237) | 16444 (3019–34050) | .906 | 21369 (2742–55237) | 8094 (3019–16444) | |
% of HLA-DR positive CD80 cells at 1 month | 64.5 (26–97) | 82 (70–96) | .128 | 65 (26–97) | 68 (44–85) | .926 |
MFI of HLA-DR on CD80 positive cells at 1 month | 2686 (592–23661) | 2082 (1861–22194) | .844 | 2728 (592–23661) | 2114 (1459–11540) | .514 |
% of HLA-DR positive epithelial cells at month 3 | 56 (7–93) | 56 (30–84) | .762 | 55 (7–88) | 69 (30–93) | .402 |
MFI of HLA-DR on epithelial cells at month 3 | 5178 (533–20735) | 3450 (835–8747) | .493 | 6150 (533-20735) | 2885 (1835–18634) | .082 |
% of HLA-DR positive CD80 cells at 3 month | 67 (48–99) | 67 (43–82) | .401 | 66 (48-83) | 73 (43–99) | .920 |
MFI of HLA-DR on CD80 positive cells at 3 month | 4456 (1028–17943) | 6106 (1445–17808) | .595 | 6050 (1028–17943) | 2755 (1340–17808) | .188 |
Mann-Whitney
Box plot of expression of HLA-DR on conjunctival epithelial cells (MFI-mean fluorescence intensity) collected by impression cytology 1 month after surgery in eyes with surgical success and failure at 6 months (
The expression of HLA-DR on conjunctival epithelial cells (MFI-mean fluorescence intensity) collected by impression cytology 3 months after surgery in eyes with surgical success and failure at 6 months of follow-up (
Aqueous levels of cytokines in the eyes with surgical success and failure are summarized in Table
Concentration of cytokines in aqueous humour in patients with successful and failed surgery at 3, 6, and 12 months of follow-up.
Cytokines, median (minimum-maximum) (pg/mL) | 3 months after surgery | 6 months and 12 months after surgery | ||||
Success ( | Failure ( | Success ( | Failure ( | |||
IL-8 | 31 (19–177) | 43 (29–70) | .478 | 36 (11–177) | 34 (28–137) | .910 |
IL-1 | 22 (15–48) | 19 (17–41) | .357 | 22 (15–48) | 21(17–41) | .504 |
IL-6 | 18 (9–809) | 106 (26–375) | 20 (9–809) | 75 (15–629) | .072 | |
IL-10 | 10 (4–19) | 11 (3–15) | .983 | 11 (4–19) | 9 (3–15) | .341 |
TNF- | 9 (4–21) | 10 (8–15) | 9 (4–21) | 10 (8–15) | .101 | |
IL-12p70 | 14 (6–39) | 13 (11–24) | .983 | 14 (6–39) | 13 (12–24) | .696 |
Mann-Whitney
Aqueous levels of IL-6 in eyes with surgical success and failure 3 months after surgery (
Aqueous levels of TNF-
The expression of HLA-DR on epithelial cells was significantly lower 3 months compared to 1 month after surgery (paired sample
Box plot of expression of HLA-DR on conjunctival epithelial cells 1 month and 3 months after surgery in eyes with successful and failed trabeculectomy (
Several studies have investigated conjunctival biopsy specimens from patients with glaucoma receiving long-term antiglaucoma medication and after previous ocular surgery before undergoing filtration surgery. They have shown increased number of conjunctival fibroblasts and inflammatory cells when compared to eyes without previous ocular surgery or only briefly treated-primary surgery eyes [
Conjunctival epithelium has been studied in impression cytology specimens by flow cytometry. In eyes treated over long term without clinical inflammation, overexpression of inflammatory marker HLA-DR and IL-6, IL-8, and IL-10 was documented [
In our study, we used impression cytology specimens from the superior bulbar conjunctiva and flow cytometry to measure ocular surface inflammation 1 month and 3 months after trabeculectomy in an attempt to correlate ocular surface changes with the outcome of surgery. There was a trend of increased number of HLA-DR positive conjunctival epithelial cells collected one month after surgery in failures when compared to successes at 3-month follow-up (Table
Because the imprints of ocular surface were not collected before surgery, it remains unknown whether there was any difference in preoperative HLA-DR expression between the eyes with successful and failed surgery. In both groups there was a reduction of HLA-DR expression on epithelial cells over time, indicating diminished postoperative inflammation with longer follow-up
The aqueous humour is known to contain several cytokines capable of affecting fibroblast activity [
In this study, we used flow cytometry to measure cytokines in the aqueous humour of patients undergoing trabeculectomy to evaluate whether preoperative changes in the level of cytokines could predict the outcome of surgery. The proinflammatory cytokines IL-1
Tumour necrosis factor-
Interleukin-6 is a macrophage-derived cytokine but is also thought to be produced by ocular parenchymal cells [
Both TNF-
Interleukin-1
The limitation of our study is a small number of patients included. Also, aqueous humour was collected at one time point only, at the beginning of trabeculectomy, and we do not know about the changes in cytokine levels during follow-up, which could support our results. Other cytokines not investigated in our study may influence the outcome of surgery as well as interactions between different cytokines, which cannot be studied in vivo. However, the study was prospective with a 12-month follow-up, a period where most of the surgical failures occur and also studied ocular surface at the site of surgery.
The increased percentage and expression of HLA-DR on conjunctival epithelial cells and antigen-presenting cells were not associated with surgical failure. In contrast to other authors the expression of inflammatory marker HLA-DR on epithelial cells was elevated in eyes with successful trabeculectomy when compared to eyes with surgical failure [
In our study, inflammation of superior bulbar conjunctiva detected as overexpression of inflammatory molecules one and three months after surgery was not associated with subconjunctival fibrosis. Lower levels of TNF-