This article describes a method for studying retinal ganglion cell signal transmission to the optic nerve under controlled pressure conditions. The procedure involves the preparation of a human eye sample and the use of a translaminar autonomous system to simulate intraocular and intracranial pressures.
Begin with a human eye. Remove the sheath to expose the optic nerve.
Bisect the eye, then remove the vitreous humor from the posterior segment.
Trim the sclera, the outer layer, and spread the retina containing the retinal ganglion cells connected to the optic nerve via the lamina cribrosa, a mesh-like structure.
Place this segment over the dome of the lower chamber of the translaminar autonomous system, with the optic nerve facing upwards then seal it.
Connect the tubing and flow a perfusion medium.
The liquid's movement builds pressure inside the eye cup, resembling intraocular pressure.
Next, position the upper chamber covering the optic nerve and seal it.
Connect the tubing to flow the medium around the nerve, resembling intracranial pressure.
The pressure difference between these chambers establishes a translaminar pressure across the lamina cribrosa, enabling the study of retinal ganglion cell signal transmission to the optic nerve under this pressure condition.
To prepare a human whole eye globe sample, remove the optic nerve sheath and remove the vitreous humor from the posterior segment. To ensure a 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 4 screws to seal the posterior segment.
To set up the IOP chamber, insert the tubing into 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 in and out tubes are void of air bubbles, stop the infusion and lock the stopcocks in the off position.
Remove the syringe from the IOP import filter assembly and refill the syringe with 30 additional milliliters 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 4 screws.
Then insert the tubing into 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.
繁體中文
