A total of 29 healthy individuals were recruited from students at Tongji Medical College, Huazhong University of Science and Technology. All participants signed written informed consent before entering the study, and the study was conducted in accordance with the tenets of the Declaration of Helsinki. All subjects underwent an ophthalmic examination, and data from their right eyes were included in the study.
The criteria for inclusion of subjects were: (1) at least 18 years old; (2) IOP of 10-21 mmHg; (3) with a normal anterior chamber depth and open angle. Further, subjects should not have ingested caffeine for at least 24 h before the studies started, and should have no history of receiving any medicines affecting the circulatory system within 1 month prior to evaluation.
Exclusion criteria were as follows: (1) systemic diseases (e.g., hypertension, diabetes, and severe cardiopulmonary insufficiency), or a family history of these conditions; (2) current ocular diseases or previous ocular surgery; (3) refractive error (RE) ≤－6.0 D and RE ≥3.0 D, or best corrected visual acuity <0.5 (to ensure that the subjects had good central fixation); (4) best corrected visual acuity (BCVA) <0.5; (5) abnormal pupil reflexes; and (6) poor compliance in performing VM correctly.
Every subject struck a correct sitting pose on the measuring instrument before blowing, and held positions during the whole VM, after which the measurements were taken. All examinations were performed following standard operating procedures. In this study, no contact was required to avoid the influence of corneal contact on the parameters and participants’ health.
Standardized Valsalva maneuver
Every subject was trained to perform a standardized VM. Subjects were asked to exhale into a mouthpiece connected to a mercury manometer, and to maintain an expiratory pressure of 40 mmHg for approximately 15s to complete the image acquisition process. After training, every individual was able to manage the maneuver well. The resting state before breath holding, the continuous blowing state, and the immediate recovery of normal breathing state were recorded as baseline, phase 2, and phase 4, respectively of the VM. Each phase took 15 s. Participants were given a short break of at least 5 min between every 2 VMs. Each individual performed the maneuver a total of 5 times.
Measurement of blood pressure, HR, and electrocardiograms
The systolic blood pressure (SBP) and diastolic blood pressure (DBP) at baseline, phase 2, and phase 4 of the VM for each individual were measured using an automatic sphygmomanometer (OmronHEM-7201; Omron, Dalian, Liaoning, China). The mean arterial pressure (MAP) was calculated by the equation: MAP = DBP + (SBP － DBP)/3. Electrocardiograms were monitoring in real-time during the entire process, including in the resting state, continuous blowing state and immediately recovered normal breathing(15 s per period). HR was determined by measuring the R-R intervals. The heart rate variability (HRV) parameters of individuals were calculated in each state (baseline, phase 2, and phase 4 of the VM) by using software (Kubios HRV premium v 2.2; University of Eastern Finland).
Anterior optical coherence tomography imaging
In a sitting position, all participants received an anterior optical coherence tomography (AS-OCT) examination (Visante OCT; Carl Zeiss Meditec, Dublin, USA.). Rectangular AS-OCT scans of the frontal, nasal, and temporal sides were collected in 3 phases. For frontal scans, the scan angle was horizontal (with nasal and temporal angles at 0°-180°) across the center of the pupil in 1 single image, while the subject stared at the internal fixation point. All AS-OCT tests were performed under standardized darkroom photopic condition (approximately 3.5 lux).
Measurements of SC and pupil diameter
Anterior chamber depth (ACD), the angle opening distance at 500 μm from the scleral spur (AOD500), the angle recess area at 750 μm from the scleral spur (ARA750), trabecular iris angle at 500 μm from the scleral spur (TIA500), and trabecular-iris space area at 500 μm from the scleral spur (TISA500) were measured by the built-in 2-dimensional analysis function of the Visante OCT. ACD was defined as the length of the central perpendicular line between the posterior surface of the cornea and the anterior surface of the lens. The anterior chamber angle was defined as the arms of the posterior cornea and opposite peripheral iris, with its apex in the angle recess (Fig. 1). The SC was defined as observable when a thin, black, lucent space was detected in the images (Fig. 2). The area of the SC (SCAR; μm2) in the same location of the nasal and temporal sides was measured using imaging software (Image J v1.45S; National Institutes of Health, Bethesda, MD, USA). The mean SCAR was calculated as the averaged SCAR of the nasal and temporal regions. The distance from 1 side of the pupillary tip of the iris to the opposite side on images acquired by AS-OCT was measured as the pupil diameter (PD). Measurements of SCAR and PD were performed by 2 observers, and the data were recorded and stored for later statistical analysis.
Measurement of IOP
The IOP at baseline, phase 2, and phase 4 of the VM were measured using a noncontact tonometer (NIDEK RT-2100; Nidek, Co., Ltd, Gamagori, Japan). The averaged IOP was calculated from measurements and recorded as the result. The mean ocular perfusion pressure (MOPP) was calculated as: MOPP = 2/3MAP－IOP .
All statistical analyses were performed using statistical software ( SPSS v 22.0; Inc., Chicago, IL, USA), and data were plotted with graphing software (GraphPad Prism v7.0; GraphPad Software, USA). The intraclass correlation coefficients test was used to analyze the re-test reliability of the measurements of SCAR and PD, which were performed by 2 observers. All applicable data are presented as the mean ± standard deviation. Repeated measures analysis of variance was used to detect differences between every 2 different phases. Univariate linear regression analysis was adopted to examine the relationship between SCAR (mean) and HF, LF/HF and IOP. All tests were 2-tailed, and statistical significance was defined as a P value < 0.05.