Subjects
This study was conducted in accordance with the tenets of declaration of Helsinki, and it was approved by the Institutional Review Board of the Shanghai General Hospital. Patients were recruited from Dec 1, 2017 to Mar 31, 2018, prospectively and consecutively. Written and oral informed consent was obtained from each patient and their families, respectively.
All participants underwent a thorough ophthalmic examination including slit-lamp biomicroscopy, IOP measurement by Goldmann applanation tonometry, ultrasound biomicroscopy (UBM) as well as gonioscopy and fundus examinations. Enrolled APAC patients had experienced an APAC attack within one month before hospital admission. Even once the pupillary block was released, they still had uncontrolled IOP under standardized anti-glaucomatous medications. Patients with secondary angle closures, other ocular diseases or histories of previous ocular surgeries were excluded. Cataract patients, who were enrolled as the control group, had no history of IOP higher than 21 mmHg or glaucomatous optic neuropathy.
Surgical Technique and Postoperative Care
For all of the previous APAC patients, trabeculectomy combined with cataract surgery was performed by one experienced surgeon [17]. First, a fornix-based conjunctival flap was made, a limbus-based 4×4-mm scleral flap was dissected, and then a sponge soaked with 5-fluorouracil (25 mg/mL) was placed underneath the conjunctival flap for 3 minutes. Next, 250 mL balanced salt solution was used to wash the surgical area. Then, cataract extraction was performed by phacoemulsification and intraocular lens implantation according to the surgeon’s preferred technique. Finally, a trabeculectomy and a basal iridectomy were performed. After the procedure, the scleral flap was sutured using two 10-0 nylon sutures at the corners, and the centers of the two sides were closed using two adjustable sutures to allow minimal leakage during anterior chamber reconstruction. Finally, the conjunctiva was closed using a 10-0 nylon suture. A topical antibiotic and topical nonsteroidal anti-inflammatory medication were given four times daily for 1 week. Topical corticosteroids were used every 2 to 3 hours for the first week and then tapered over the next 1 to 2 months. Postoperative interventions were performed for bleb formation. At first, ocular massages were performed. Then adjustable sutures were loosened with forceps. If the bleb became encapsulated and associated with a rise in IOP, needing was carried out without antimetabolites. In the case of postoperative IOP measurements > 21 mmHg, despite receiving an ocular massage and having any adjustable sutures loosened (if needed), IOP-lowering medication was added. In cases of inadequate IOP control, additional surgical procedures were performed as required.
Aqueous Humor Collection and Analysis
Generally, 80-120μl aqueous humor was needed for an examination and analysis. The 30-gauge needle and tuberculin syringe were used to withdraw the aqueous humor via limbal paracentesis. Aqueous humor samples from APAC and cataract patients were collected at the start of the trabeculectomy and cataract surgery, respectively. The aqueous humor was collected with caution to avoid touching any intraocular tissue. The collection was frozen in liquid nitrogen immediately and, then, transferred within 24 hours into a -80℃ environment until the analysis was completed.
EPO and PDGF family members (PDGF-AA, PDGF-BB, PDGF-CC and PDGF-DD) were detected by the Luminex Screening Human Magnetic Assay (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions of the Bio-Plex 200 System (Bio-Rad Laboratories, Inc., Hercules, CA). The standard curve was automatically produced using the Bio-Plex Manager 5.1.0.0 software. The fluorescence intensities of both the sample and background were detected after the aqueous humor samples had been diluted in a 1:2 ratio. The final levels of the EPO and PDGF family members were analyzed after subtracting the background levels.
Outcome Measures
The IOPs of all the participants were analyzed a half hour before each surgical procedure, and all procedures were performed during a similar time frame in the afternoon. After operation, all APAC patients were followed up at 1 week and 1, 3, 6, 12 and 18 months. Goldmann tonometry and slit lamp biomicroscopy were performed at each post-operative follow-up. The surgical failure was defined as IOP >21 mmHg or administration of anti-glaucoma medications after 3 months or an additional glaucoma surgery [18,19]. The ocular massage, loosening of adjustable sutures and bleb needling were performed at the slit lamp and were not considered as an additional glaucoma surgery. If the IOP of one visit was lower than 21 mmHg without the use of topical anti-glaucoma medication, the visit was defined as a “success”.
Statistical Analysis
Commercially available SPSS software (version 26.0.0.0, SPSS Inc., Chicago, IL, USA) was used to analyze the results. The Kolmogorov-Smirnov test was used to test the normality of the continuous variable. The median (M) value and interquartile range (IQR) were used to describe the distribution of EPO, PDGF-AA, PDGF-BB, PDGF-CC and PDGF-DD. A Mann-Whitney U test was used to perform the comparisons of target proteins and the clinical characteristics between the two groups. A Spearman correlation test was performed to determine the correlation between target proteins and associated factors.
After operation, a Mann-Whitney U test was used to perform the comparisons of EPO and PDGF families between success and failure groups. If any target protein was detected to have a significant difference between the two groups, it was included in the multiple linear regressions to characterize the effects after adjustment. If any protein showed significance after multiple linear regressions, the patients would be divided into two groups according to the protein level. The Kaplan Meyer survival curves were used to display the success rate of the end points over time between the low and high protein groups. Also, in the failure group, a Spearman test was used to examine the correlation between significant-protein(s) and the IOP and the time of failure before the use of any drug or surgical treatment. A p-value less than 0.05 was regarded as being statistically significant.