Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

The goal of this procedure is to induce elevated intraocular pressure in the rat using injection of paramagnetic microspheres into the anterior chamber of the eye. First place a magnetic ring around the eye of an anesthetized animal, wash the magnetic microspheres, resuspend them in solution, and then load them into a syringe. Next, inject the magnetic microspheres into the anterior chamber of the eye.

Remove the magnetic ring during the recovery. The process data obtained from histological analyses like tunnel staining or optic nerve staining can measure the death of retinal ganglion cells that results from increased intraocular pressure. Wash the sterilized eight micron magnetic microsphere beads three times in one milliliter of hank's buffered saline solution by reus, suspending and centrifuging at 10, 000 times G for five minutes.

Then reus suspend in 0.5 milliliters of HBSS for a 30 milligrams per milliliter solution. Now using a rebound sonometer calibrated for use in the rat eye, take five baseline IOP measurements in the awake animals. After anesthetizing, the rats apply ocular ointment to the unrated contralateral eye to keep it moist.

During anesthesia apply 0.5%propane hydrochloride, local anesthetic to the operative eye. Next, wash this eye with 5%povidone iodine in water. After five minutes, W off the povidone iodine and wash the eye with 0.9%sterile saline solution.

Keep the eye moist during anesthesia with regular application of sterile saline. Now place a steroidal magnet around the eye to minimize the risk of iris trauma and minimize bead loss. Orient the needle tangential to the corneal surface as parallel to the iris as possible.

Proceed to inject 25 microliters of 30 milligrams per milliliter, gamma irradiated magnetic microspheres into the anterior chamber. Leave the needle in place for one minute post-injection to ensure that beads settle into the IOC corneal angle to impede aqueous drainage from the trabecular mesh. Work slightly angle the needle a few seconds after the beads have initially settled to allow some leakage of aqueous and to minimize transient increases in intraocular pressure.

To ensure continued use of the needle in separate procedures, flush the needle with PBS. Then 70%ethanol, followed by distilled water at this stage. If necessary, remove the magnet and use it to draw beads into areas of incomplete coverage.

Leave the magnet in place around the eye for a further 10 minutes post injection. To ensure beads settle well into the irid corneal angle. Reverse anesthesia using 0.25 milligrams per kilogram of ESOL hydrochloride.

Administer 0.5 propane hydrochloride for analgesia and chloramphenicol ointment topically to prevent infection. Leave animals to recover on a heat mat until they regain movement. Then transfer to a warm box.

Use the contralateral eye as an unrated control. Repeat IOP measurements every two to three days following bead administration and every two to three days thereafter, define the criteria for including eyes in studies, including the IOP is elevated above the contralateral control pressure by five millimeters of mercury, and the IOP does not exceed 60 millimeters of mercury eyes where the pressure returns to baseline by one week post-injection should not be included in the studies. However, it is possible to re-inject beads in eyes that fail to develop high IOP if desired.

Fix the dissected eyes and optic nerve in 4%paraldehyde, assessing the retinal neuron damage by tunnel staining and the optic nerve damage by toine blue.Staining. In this experiment, injection of magnetic beads into the irid corneal angle consistently induced a prolonged and robust rise in pressure. Furthermore, the increase in pressure was maintained throughout the duration of the experiment.

The mean IOP averaged over the full length of the experiment for control non bead injected eyes was 19.7 plus minus 0.3 millimeters of mercury and 40.5 plus minus 2.8 millimeters of mercury for bead injected eyes. Additionally, peak IOP increased from 22.8 plus minus 0.3 millimeters of mercury to 49.9 plus minus 2.3 millimeters of mercury to determine whether elevation in IOP leads to death of retinal ganglion cells. Tunnel staining was performed on retinas and histology on transverse optic nerve sections in the retina.

We observed an increase in tunnel staining in bead injected eyes with elevated IOP. The number of apoptotic nuclei rose approximately 15 fold from contralateral control hypertensive retinas as expected in eyes in which magnetic beads were injected without pressure, the number of tunnel positive cells were not significantly different from those of unin injected controls. Further investigation of the optic nerve pathology in the glaucoma model showed the accumulation of odine blue in many of the axons indicating degeneration of these cellular processes.