The overall goal of this procedure, is to produce a rodent model of anterior ischemic optic neuropathy, for use in characterizing stroke response mechanisms. And for analyzing the ethicacy of neuroprotective agents in the treatment of optic nerve stroke. This measure can help answer key questions in the field of optic nerve stroke.
Including, mechanisms of edema, post-stroke inflammation, and pass-ways of protection and repair. The main advantage of this technique is that the ischemic damage is isolated to the anterior portion of optic nerve. Without surgery, the technique is painless.
And the severity of the damage can be reproducibly varied at will. This method avoids the pain associated with many nerve damage models. Including, crushing, or cutting the optic nerve.
It also enables detailed analysis of the effects of isolated exo-ischemia, on a single neuron class, in vivo. Start by obtaining a custom made contact lens designed according to the specifications shown. Also, prepare 2.5 milimaler rose bengal, in pH 7.4 phosphate buffered saline.
Fills are sterilized with a 0.5 micron syringe filter. And prepare one millimeter aliquots, place the aliquots in a light, tight, container and store at negative 20 degrees Celsius for up to six months. On the day of the procedure, set up the frequency doubled neodymium yttrium aluminum garnet ophthalmic medical laser.
Which generates a 532 nanometer laser light. Mount the laser on a Haag-Streit ophthalmic slit lamp, using a standard ophthalmic laser adapter, to enable simultaneous visualization of the animals eye, and laser spot application. Turn on the laser, and allow it to warm for at least five minutes, before use.
Set the appropriate laser power parameters for the experimental animal used. Mount a high ASA speed digital camera on one of the eye pieces of the slit lamp, with a custom made adapter. After appropriately, anesthetizing the experimental animal, and ensuring the appropriate level of anesthesia, by the absence of the response to a toe pinch.
Dilate the animals pupils with one percent tropicamide. And anesthetize the surface of the eye, with 0.5 percent proparacaine. Then, use the scissors to cut the whiskers close to the muzzle on the side of the induction, to allow a clear view of the eye.
Next, put a drop of one percent methylcellulose, or other ophthalmic coupling drop, onto the inside of the custom made contact lens. And then apply the lens onto the rats eye. Place the animal on a platform, adjusted to the height of the slit lamp.
Set the animals head at a 45 degree angle, so that the eye is perpendicular to the slit lamp and laser beam. Then, visualize the eye through the ophthalmic slit lamp. In a healthy eye, the optic nerve head has a clear border and a reddish surrounding Q.And the retinal vessels are thin and evenly distributed.
Make sure the aiming beam is the correct size. As well as focused and centered directly on the visualized optic nerve. Then photograph the retinal fundus, using the digital camera.
Next, inject one milligram per kilogram, of rose bengal intravenously through the tail vein. And wait for 30 seconds. A smooth, continuous, intervaneous injection of rose bengal, must be followed after 30 seconds, by laser exposure, of the optic nerve head.
Variation in time, between these two steps, result in increase variability of degree of optic nerve stroke damage. Which can bias your results. After 30 seconds, activate the laser power.
The optic nerve, shows a golden glow within the vessels, emerging from the disc at the laser spot sight. Indicating rose bengal in the blood stream, illuminated by the green laser light. Immediately after induction, remove the contact lens, cover both eyes, with ophthalmic triple antibiotic ointment, dexamethasone.
Single house the animal, in a cage on a 37 degree Celsius warming pad, under close observation until full recovery. This colored fundus picture, shows optic nerve swelling and optic disc pallor. With loss of the surrounding choroidal flush.
The veins are enlarged, and curve. This n-face SDOCT picture, post-induction, shows disc edema, and venous dilation. The scan direction is indicated with a green arrow, the cross section in the next image, is generated from within the green box.
Here, a cross section, shows optic nerve disc edema, as evidenced by reduced gray intensity, and increased thickness of the retinal nerve fiber layer. Consistent with higher water content. The diameter of the intraretinal optic nerve, indicated between the black arrows, is increased.
While attempting this procedure, it is important to remember to have a clean, polished, contact lens. Or cover slip, that enables good visualization of the nerve, and precise focusing, of the laser light over the nerve. Following this procedure, other methods like, immunohistochemistry, cytokine analysis, or gene expression analysis, can be performed in order to answer additional questions.
Like inflammatory responses after stroke, or changes in the target tissues.
