This novel ocular translaminar autonomous system uses the human posterior segment to independently regulate intraocular and intracranial pressures to generate a translaminar pressure gradient. The TAS model allows the evaluation of human intracranial pressure in an ex vivo, preclinical manner that previously could not be studied. This model could potentially be used to study diseases such as glaucoma, traumatic brain injury, idiopathic intracranial hypertension, and spaceflight-associated neuro-ocular syndrome.
To set up the inflow syringes, load 30-milliliter syringe with 30 milliliters of the perfusion fluid of interest and attach a three-way stop cock to the syringe. Attach a 0.22-micrometer hydrophilic filter to the stop cock, and attach a 15-gauge luer stub adapter to the 0.22-micrometer hydrophilic filter. After removing air bubbles from the syringe setup, attach tubing to the luer stub adapter and close the side port of the stop cock with an unvented universal lock cap.
After setting up a second inflow syringe as just demonstrated, label one syringe channel one ICP, and label the other syringe channel two IOP. Then set up the outflow syringes as just demonstrated, but without loading the syringes with the infusion medium. To prepare a human whole eye globe sample, remove the optic nerve sheath and remove the vitreous humor from the posterior segment.
To ensure good fit on the round dome of the IOP chamber, trim additional sclera from the posterior segment as necessary, and use forceps to ensure that the retina is spread evenly over the human posterior of the segment. Then place the segment into the IOP chamber of the TAS over the round dome with the optic nerve facing up, and use an epoxy resin O-ring and four screws to seal the posterior segment. To set up the IOP chamber, insert the tubing into the in and out ports of the chamber and insert the IOP inflow syringe into the in port.
Insert the empty IOP outflow syringe setup into the out port and use the push/pull method to slowly infuse the perfusion medium into the inflow port to fill the posterior eye cup, while simultaneously slowly pulling the perfusion medium out through the outflow syringe to remove any air bubbles from the lines. Once both the in and out tubes are void of air bubbles, stop the infusion and lock the stop cocks in the off position. Remove the syringe from the IOP in port filter assembly and refill the syringe with 30 additional millimeters of medium, then reinsert the syringe setup into the filter assembly.
To set up the ICP chamber, place the chamber over the back of the posterior segment, taking care that the optic nerve is within the top chamber, and seal the top chamber with four screws then insert the tubing into the in and out ports of the ICP chamber, the ICP input syringe into the in port, and the empty ICP outflow syringe into the out port to flush the system with medium as just demonstrated. To set up the data recording system, turn on the eight-channel power source and the computer, and initiate the data acquisition software. Select File and New and select Setup and Channel Settings.
Select three channels, and rename the channels as indicated. Select two millivolts for the Range on all if the channels and in the Calculation column, select No Calculation for channels one and two. Select Arithmetic for channel three In the Formula window, under Channels, select Ch 2.
Under Function, select Math and Minus, and under Channels, Select Ch 1. To set up and calibrate the hydrostatic pressure transducers, connect the hydrostatic pressure transducers to the transducer lines attached to the multi-channel bridge amplifier, and attach an empty 30-milliliter syringe to the side port of the channel one ICP pressure transducer. Attach a sphygmomanometer to the bottom of the channel one ICP pressure transducer, and click the arrow next to the sampling time to set the sampling speed to 100.
Select Bridge Amp, Mains filter, and Zero, and wait for the system to zero out, taking care to not move the pressure transducer. Next, pinch the white tabs of the pressure transducer and push air through the transducer until 40 millimeters of mercury are obtained on the sphygmomanometer, then release the white tabs and remove the syringe and sphygmomanometer. On the Units Conversion page, select minus and highlight the highest plateau to indicate 40 millimeters of mercury.
Click the arrow for 0.1, enter 100, and highlight the lowest plateau to indicate zero millimeters of mercury. Click the arrow for 0.2, enter zero, select millimeters of mercury for the units, and click Okay. In the Bridge Amp window, click Okay, and calibrate the hydrostatic pressure transducers for channel two IOP as just demonstrated, using 100 millimeters of mercury for the highest plateau, and zero for the lowest plateau.
To connect the TAS posterior segment unit to the data acquisition system, place the TAS posterior segment unit into a 37 degree Celsius, 5%carbon dioxide incubator, and attach the ICP tubing from the out port of the channel one ICP pressure transducer. Attach the IOP tubing from the out port to the channel two IOP pressure transducer. On the Chart View page, select Start Sampling and set the sampling speed to Slow and one minute.
Then adjust the syringes on the ring stand up or down to regulate ICP and IOP pressures according to the protocol requirements, or place them on perfusion pumps for regulating pressure. In this representative analysis, the maintenance of the average normal pressure differentials in both chambers were tested through various parameters of IOP and ICP conditions. To ensure the viability of the tissue, the medium in the tissue was exchanged every 48 hours.
Minimal pressure increases occurred at the time of medium exchange, and did not affect the viability or morphology of the optic nerve head as observed by immunohistochemical analysis and collagen IV expression on days 14 and 30 of the experiment. Increasing the IOP or decreasing the ICP over various time points allows the maintenance of a range in elevated translaminar pressure gradient for seven days. At seven days of elevated translaminar pressure gradient, cupping and thickening were observed in posterior segments, and collagen IV expression demonstrates thickened beams with an increased signal expression over the experimental period.
Phase imaging reveals healthy retinal ganglion cells within the ganglion cell layer with no cupping in the control segments, while images from elevated translaminar pressure gradient cultures exhibit extensive cupping with no retinal ganglion cells remaining within the retinal nerve fiber layer, and increased remodeling of the extra cellular matrix. This technique will allow researchers to effectively study biomechanical disease paradigms, and discover molecular pathogenesis-targeting translaminar pressure in the human eye.
