Animals and vitro eyeball
We choose purebred New Zealand White rabbits of 2.5 kg weight as eye donors, provided by the Experimental Animal Center of Capital Medical University. Implement excessive anesthesia death on rabbits by rapid injection of urethane into the ear vein. Then bluntly dissect the bulbar conjunctiva, cut the rectus, and finally cut together the central retinal artery, vein and the optic nerve bundle after ligation to isolate the eyeball. Save the vitro eyeball temporarily in saline solution at room temperature. All the animal experiments reported in our study were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of China.
Particle image velocimetry technology
Tracer particles
Polystyrene particles with 10 um diameter are selected as tracer particles in our study. They can be uniformly dispersed with a similar density to water and show a good following feature in intraocular fluid[14–16].
Experimental apparatus
In our study, the pivotal device are particle image velocimetry system, including Laser, CCD camera, and Synchronization and the micro-injection pump (Nd:YAG Pulse laser with single pulse energy up to 200 mJ; CCD camera, 1600 × 1200 pixels,with Sampling Frequency of 30 fps; HARVARD PHD 2000 Syringe pump); Softwares are Dynamic Studio developed by Denmark DANTEC Company and Tecplot visualization; main materials include the fluorescent particle solution of 10um particle diameter, the no-leaking vitro rabbit eyeball, 20G × 29 mm intravenous catheter, 20 ml syringe, slender and transparent tube, glass tank, and saline.
Velocity measurement principle of PIV
Flow situations are captured and recorded by PIV with proper parameters of CCD camera, laser intensity (0~200 mJ) and pulse frequency (0~10000 us). Finally the corresponding velocity distributions are calculated from successive particle images.
As shown in Figure 1, the average velocity components of the tracer particle during a period along the x and y directions are as follows[17, 18]:
Where, dt is a time period, dx and dy are the displacements of the particle along the x and y directions respectively.
In the formula above, when dt is sufficiently small, V_x and V_y can accurately reflect the instantaneous velocities of the fluid particle. PIV technology realizes the measurement of the two-dimensional flow field by measuring the instantaneous average velocity of tracer particles.
Flow field measurement with PIV method of intraocular aqueous flow in vitro eyeball
Drive mode of aqueous humor production in vitro eyeball
A proper inflow point in PC should be determined to better simulate the production of aqueous humor. Two typical inflow positions in PC were considered: along the pupillary margin; at the root of PC. Figure 2 is the scheme of these two conditions, in the left diagram, inflow point is along the pupillary margin; in the right diagram, inflow point is at the root of PC[13, 19].
As shown in Figure 2 (a), intravenous catheter was needled at the limbus peripheral 1 mm obliquely upward in the direction of 15°, and was slided underbelow the iris in the direction of the pupil center until its tip reached the pupil edge. Then pull out the metal needle, and fix well the trocar, ensuring that the inflow point is along the pupillary margin. In the circumferential direction of 180° to the inflow point, puncture at the angle of AC as the outflow pathway for intraocular liquid, exerting a 10 cm high saline water column pressure to represent the episcleral venous pressure resistance on physiological humor outflow. This entire device was then fixed into saline to eliminate the refractive error in the measurement. Fluorescent particle solution was pumped uniformly to the pupillary margin to drive the intraocular flow by Micro-injection pump and the flow process was captured and recorded by PIV.
In Figure 2 (b), intravenous catheter was needled at the limbus peripheral 1 mm, penetrating the sclera, and was slided underbelow the iris. Pull out the metal needle for 2 mm, and continually slide the trocar until its tip reaches the root of iris. Extract the whole metal needle and fix well the trocar to ensure that the injection point is at the root of PC. Puncture at one point of angle of AC as the outflow pathway for intraocular fluid, exerting a 10 cm high saline water column pressure to represent the episcleral venous pressure resistance on physiological humor outflow. This entire device was fixed into saline to eliminate the refractive error in the measurement. Fluorescent particle solution was uniformly pumped to the root of PC to drive the intraocular flow with Micro-injection pump and this flow process was measured by PIV.
By comparing the intraocular flow fields obtained from these two conditions, proper inflow point was selected to drive the intraocular flow complying with physiological laws. In our study, inflow at the root of PC can better simulate the generation of aqueous humor.
Measurement of flow field in AC by PIV
Considering the symmetry of AC, we have decided to puncture two outflow points at the angle of AC, which are circumferentially 180°apart on one sagittal plane of the eyeball, to simulate the aqueous absorption process. Figure 2 (c) is the scheme of the experimental apparatus in this experimental condition.
As shown in Figure 2 (c), fluorescent particle solution was infused to the root of PC acting as the generation of aqueous. Two outflow points were punctured at the angle of AC, which are circumferentially 180° apart on one sagittal plane of the eyeball, to simulate the aqueous outflow on one sagittal plane. For the vitro eyeball with two outflow points in its AC, the AC is easier to collapse because of the instantaneously rapid outflow of intraocular liquid. And to prevent this phenomenon, we have elevated the saline column height to 17.69 cm to represent the episcleral venous pressure resistance on aqueous outflow. This entire device was fixed into saline to eliminate the refractive error in the measurement. Fluorescent particle solution was uniformly pumped to the root of PC to drive the intraocular flow with Micro-injection pump and the flow would be recorded and calculated with PIV.