We developed a simple method to induce robust and progressive glaucoma degeneration based on temporary intraocular pressure elevation. This represents a unique opportunity to study key points of glaucoma pathophysiology. This is an easy-learning, non-invasive, low-cost, and highly-reproducible model that enables in vivo function analysis and short-term experimental designs using a reduced number of animals for each experiment.
The retina and optic nerve are assessable parts of the central nervous system, so modeling the progressive impairment of these structures can provide valuable insights into other neurodegenerative disorders. To measure baseline intraocular pressure, or IOP, of both experimental and contralateral control eyes, place the anesthetized rat in a ventral decubitus position on the bench top, such that the corneal surface is easily accessible to the tip of the tonometer. Position the tonometer tip such that it slightly touches the central corneal zone perpendicularly.
Then, acquire the measurement by pressing the measurement button. Next, place the rat in a slight lateral decubitus position under a stereo microscope and inspect the experimental eye at 40 times magnification. During the procedure, keep the contralateral control eye lubricated by instilling a drop of carmellose sodium or sodium hyaluronate.
Next, using curved forceps, gently push the experimental eyeball forward to expose the vasculature surrounding 360 degrees of the limbus. Then, with the other hand, using a low-temperature ophthalmic cautery, gently cauterize the vessels all around the cornea. Be careful not to cauterize the corneal periphery.
Confirm the success of the surgical procedure by observing the emergence of small circular marks of cauterization on the scleral limbus, the obliteration of limbal vasculature, and pupil dilation in the operated eye. After the surgery, check the immediate postoperative IOP in both eyes as demonstrated earlier. Then, apply a drop of ophthalmic prednisolone acetate to the anterior surface of the experimental eye.
After 40 seconds, replace it with an ophthalmic antibiotic ointment. Apply a daily clinical follow-up of the experimental eye with topical medications composed of a non-steroidal anti-inflammatory drug and antibiotic ointment. For optomotor response analysis, place the rat for habituation on a platform built with four computer monitors in a quadrangle.
Maintain the red cross-hair cursor on the video frame between the eyes of the freely moving rat, as it indicates the center of the virtual cylinder. Observe the optomotor response consisting of a reflexive tracking of the rat head and neck elicited by the rotating gradings. Initially, introduce a stimulus with low spatial frequency, then increase the frequency progressively until tracking movement is not noticed.
To record the pattern electroretinogram, after anesthetizing the rat, carefully insert the active electrode at the temporal periphery of the cornea. Additionally, insert the reference electrode into the subcutaneous tissue of the ipsilateral temporal canthus and the ground electrode into one of the hind limbs. Then position the rat 20 centimeters from the stimulus screen, monitor the signal baseline, and start the acquisition by pressing the Analysis button on the acquisition system.
After isolating the retinas, transfer them into a 24-well culture plate containing one-milliliter PBS, keeping the inner retina facing up. Permeabilize the tissue by washing with a 0.5%non-ionic surfactant diluted in PBS. Then, incubate the tissue and blocking solution for one hour at room temperature with gentle shaking.
During the incubation, prepare Brn-3a primary antibody solution and store at four degree Celsius. After tissue blocking, incubate the retinas in 200 microliters of primary antibody solution at four degrees Celsius for 72 hours with gentle shaking. Next, wash the tissue with PBS.
Incubate the tissue in 200 microliters of the secondary antibody solution for two hours at room temperature and then with nuclear counter stain solution for 10 minutes for fluorescent nuclei staining. After three PBS washes, transfer the retina onto a glass microscope slide using two small brushes maintaining the vitreous side up. Position the dorsal retinal quadrant up on the microscope slide.
Finally, apply 200 microliters of antifade mounting medium on a glass cover slip and place it onto the flat mounted retina for microscopic analysis. Under a confocal epifluorescence microscope, using a 40 times magnifying objective, examine the flat mounts to estimate retinal ganglion cells. After eyeball enucleation, remove the proximal segment of the intraorbital portion of the optic nerve, including part of the intraocular portion.
Then, immediately place the samples into vials or tubes containing 200 to 300 microliters of cold fixative. After two hours, wash the tissue with cold 100 micromolar sodium cacodylate buffer. Then, post-fixate the tissue for one hour in 1%osmium tetroxide and five nanomolar calcium chloride at four degrees Celsius under gentle shaking.
After three washes with cold sodium cacodylate buffer, wash the optic nerve fragments with cold distilled water before incubating overnight and 1%urinal acetate at four degrees Celsius. The following day, after three washes with water, progressively dehydrate the tissue in increasing acetone concentrations. Then, perform three rounds of embedding and epoxy resin acetone solutions before embedding the tissue in pure epoxy resin for 24 hours.
The next day, remove the samples from the specimen carrier and transfer them to embedding molds for 48 hours at 60 degrees Celsius. After polymerization, use an ultra microtome to cut transversal semi-thin sections of the optic nerve fragments. Then, collect and transfer the sections onto a microscope glass slide.
Stain the sections with toluidine blue and image them using an optic microscope at 100 times magnification. For ultra structural analysis, perform ultra thin cross-sections and collect them on a copper grid. Then, stain the sections with urinal acetate and lead citrate for transmission electron microscopic examination.
Full-circle cauterization induced IOP elevation immediately after surgery compared to control eyes. The peak for postoperative IOP was observed on day one and decreased gradually to the baseline levels on day six. Further, retinal function was evaluated both behaviorally and electrophysiologically using optomotor reflex and pattern electroretinogram, respectively.
After the surgery, both parameters showed two distinct phases of impairment:ocular hypertension acute phase on day three and a secondary degeneration phase on day 30. Compared to control optic nerves, axonal counts and semi-thin transversal optic nerve sections decreased progressively after surgery. Transmission electron micrographs of optic nerves from the control eye demonstrated densely-packed myelinated fibers separated by thin glial cell processes and evident axonal microtubules and neurofilaments.
In contrast, after three days of ocular hypertension, a few degenerated fibers, cytoplasmic vacuolation in glial cell processes, and condensed chromatin in glial cell nuclei were observed. After seven days, there was an increase in degenerated axonal fibers and hypertonic glial cell processes. And after 14 days, more disarrangement of the optic nerve fibers associated with the invasion of glial cell processes among the axons were observed.
Furthermore, the density of retinal ganglion cells decreased with time mainly in the dorsal and temporal retinal quadrants. While performing this procedure, try to gently touch limbal vessels with a cautery tip, sparing the cord periphery and ensuring continuous cauterization marks are observed all around the scleral limbus. Following this procedure, a variety of methods can be applied, such as functional biochemical and immunohistochemical evaluation of eye structures and other methods that may acknowledge on glaucoma physiopathology.
