Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

The goal of this video is to demonstrate a photochemical induced model of posterior ischemic optic neuropathy and evaluate its effects with retrograde loin of retinal ganglion cells In patients over 50 years old. Ischemic optic neuropathy is the most prevalent type of acute optic neuropathy. The condition can present as one of two subtypes according to the source of specific blood supply affected and clinical presentation, anterior ischemic optic neuropathy or posterior ischemic optic neuropathy.

While the pathogenesis of anterior ischemic optic neuropathy has been extensively studied, posterior ischemic optic neuropathy has remained poorly understood owing to its low prevalence, variable presentation and ill-defined diagnostic criteria. Furthermore, no treatments have been proven to effectively prevent or reverse vision loss from anterior or posterior ischemic optic neuropathy. Therefore, a reproducible and reliable animal model of posterior ischemic optic neuropathy is of great value to study the disease process in vivo and test new therapeutic regimens for neuroprotection and axon Regeneration Photo chemically induced ischemic injury to the microvasculature resulting in vasogenic edema and thrombosis effectively creates regional tissue ischemia.

Ischemic damage is spread to neighboring areas and further exacerbated by microvascular compression owing to a concomitant vasogenic edema. We selectively induced this form of ischemia through the director of Bbar optic nerve to mirror the damage caused by posterior ischemic optic neuropathy. Here y Wang and Dale Brown provide a step-by-step demonstration of the procedure.

To our knowledge, the model of ischemic injury in the posterior optic nerve has been developed so far as this model produces ischemia while avoiding physical trauma. So physiological processes of posterior ischemic optic neuropathy may be better mimicked. In this video, we will demonstrate the entire procedure of our rep model of posterior ischemic optic neuropathy Following surgical exposure of the posterior optic nerve eryri bee.

A photos sensitizing dye is intravenously injected and a laser beam is focused on the optic nerve surface. Photochemical interaction between eryri B and the laser during I radiation damages the vascular endothelium prompting microvascular occlusion mediated by platelet thrombosis and aous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback owing to a loss of axonal input, a remote injury induced and clinically relevant outcome.

YA laser operating at 532 nanometers and 300 milliwatts maximum power. Next is a beam chopper operating at 250 hertz, which is used to reduce the average power to 7.5%of its peak power. We have a normally ENC closed beam shutter with a small 100 micron diameter hole drilled into it to produce a weak aiming beam when closed.

Next is a 25 centimeter focal length plano convex lens, and finally, a red angle prism to direct beam downward onto the optic nerve. After originating from its source, the laser beam passes through the chopper, reducing the average power to 7.5%of its peak. The chop beam is then intercepted by the shutter, which when closed produces a very weak aiming beam by spatially filtering the beam through the 100 micron diameter hole.

This weak beam is then focused by the lens and redirected downward through the right angle. Prism onto the exposed posterior optic nerve I Radiation is begun by switching a safety filter into the optical path of an operating microscope, followed by opening the beam shutter after one second delay. At this point, the full power chop beam is focused and directed downward on the exposed optic nerve.

Upon an interaction between the beam and ethin bee dissolved in the bloodstream, that 5 32 nanometer green laser light is efficiently absorbed by the circulating ery and B dye and fluoresce is yellow molecular oxygen colliding with a triplet state dye molecule, accept its energy of excitation becoming singlet state oxygen, which is highly reactive. The CT oxygen then directly per oxidizes the vascular endothelium and causes punctate damage to the barrier, which specifically attracts platelets and also facilitates leakage of plasma into the parenchyma. This damage process thus leads to vascular occlusion by thrombosis as well as by vasogenic edema owing to microvascular compression, the ladder causes expansion of the lesion beyond the penetration depth of the laser light.

If the technique is done correctly, the platelet thrombin will contain no fiber. Thus distinguishing this method in the much less efficient heat dependent foot coagulation process. Operation of high peak power enhances the effective penetration depth into the opalescent optic nerve while reducing the average power to 7.5%via beam.

Chopping helps to ensure that a foot chemical process and not a thermal one is inducing Tissue injury peri to our Anesthetized rats with an intraperitoneal injection of the ketamine Diaz in cocktail. Once anesthetized gently puts a tongue forward to prevent asphyxia, shave the surgical sites and apply replicating, augment to both eyes to prevent drawing of cornea during surgery. Assess steps of anesthesia to ensure the animal will feel no pain during surgery.

Begin with catheterization of the femoral vein. Prior to surgical procedures, attach a sterilized and saline flushed length of tubing to a one ML syringe containing a premeasured 2%eryri B solution. At a dose of 20 milligrams per kilogram body weight, mount the syringe into an infusion pump set to a rate of 600 microliters per minute.

Clean the surgical site by wiping the area with 10%povidone iodine, followed by 70%ethanol. Make an incision at the base of the right thigh using a number 15 blade. Cut and spread through the membrane with scissors and separate the muscle with forceps until the branch of the femoral vein becomes visible.

Continue dissecting downward with scissors and forceps as necessary to separate the vessel from the surrounding tissues as necessary. Clean the surgical area with cold sterile BSS to ease visualization of the vein. Carefully separate the sheath surrounding the vein, artery, and nerve from the underlying tissue.

Then isolate the vein with blunt dissection along its sides. Pass two needle lists nylon sutures under the vein by carefully elevating the vessel, placing fine tip forceps underneath and grabbing and pulling them across. Ligate the distal vein tightly and make a loose knot around the proximal vein.

Make a partial cut of the vein near the distal ligation and expand as necessary With forceps holding the vein wall at the cut, carefully insert the saline flush tubing into the vessel. Then tighten the loose proximal knot around the tubing. Anchor the tubing in place by tying it to the distal suture.

After checking the quality of the catheterization and making sure it is free from obstructions or leaks temporarily close the incision with sutures to protect the catheterization and tissue. Prepare the surgical site with 10%Poon iodine detergent solution and 70%ethanol. Make an incision along the skin behind the eye with a number 15 blade, then cuts the connective tissue Using Venice sprain scissors.

Continue dissecting through the connective tissue along the superior ream of the optical bone. Take care to avoid disrupting the local blood Vessels. Dissect Through optical tissue until the superior rectus muscle is visualized.

The optic nerve lies Beneath. It retract the flap of skin and the connective tissue laterally and downward. The eye will rotate forward and outward.

Carefully insert a pair of sharp forceps and expand them parallel to the nerve in order to expose it. The network of micro vessels feeding it will be targeted during laser irradiation. Make sure to wear orange colored safety glasses at all times while operating the laser irradiation apparatus to protect yourself from the laser light.

Power up the laser and adjust peak and average powers several minutes. Before all irradiation procedures. Gently place the animal on the apparatus and position it so that the aiming beam shines near the site of optic nerve exposure.

Then carefully re-expose the length of the optic nerve with fine tip forceps. Make small adjustments in positioning so that the aiming beam strikes the optic nerve between three and four millimeters behind the optic nerve head. Inject the solution of 2%rosin B via activation of the infusion pump and allow it to circulate for a few seconds during this time at a small drop of BSS to moisten the surface of the optic nerve.

After verifying the position of the aiming beam, initiate the foot switch controlled safety filter followed by laser irradiation and irradiate for 90 seconds. Yellow fluorescence appearing as orange through the filter should be observed from the superior surface of the nerve, and the fluorescence should be made uniform across the optic nerve. With slight positional adjustments of the rat, the safety filter will open automatically following irradiation.

In some cases, micro hemorrhage can be observed, relieve traction on the extra ocular muscles, return the eye to a neutral position and close the incision with interrupted sutures. Cut the sutures holding the femoral vein catheterization and carefully withdraw the tubing. Tie off the proximal femoral vein tightly to prevent leakage and close the incision with interrupted sutures.

Apply antibiotic ointment to both incisions and check the fundus for vascular integrity of the central retinal vein and artery. Return the animal to a clean heated cage for recovery, administer post-surgical analgesics and care for the animal. Following the institutional animal care guidelines.

Frequently examine the postoperative animal to ensure the incision site is clean and free from infection, assess for pain and provide adequate pain medication and monitor for appropriate feeding and other behaviors. Confocal imaging Of retinal flat months can be used to quantify survival of retinal gaming cells after posterior ischemic optic neuropathy induction using retro lipin of flared compared to control animals. Significantly fewer flower goat cells are present in animals two weeks after PI one induction.

No significant difference between the number of retinal ganglion cells in controlling the sham animals is observed. This indicates that the retinal gang cell loss is elicited by the combination of a B and laser irradiation rather than the thermal energy from the Laser alone. The most critical part of the procedure is exposure and irradiation of the optic nerve.

Careful dissection is necessary in order to prevent damage to the nerve by the shark fine tip forceps or by stretching At the ophthalmic artery. Enter the optic nerve around one millimeter from the optic nerve head. If radiation three to four millimeter away from the optic nerve head is important to restrict the ischemia to the capillary supp supplying the nerve rather than the arteries and any of its branches feeding the inner retina Endoscopic examination is necessary to ensure the vascular integrity of the central retinal artery and vein following the procedure With preset parameters for laser radiation.

This model has a reproducible time course of cell death as peak intensity and irradiation duration are two determinants of ischemic damage. Severity modification of these parameters can be made to adjust the severity of the resulting of retinal ganglio cell damage for different study aims With further optimization. This model provides a novel platform for research on pathogenesis and molecular changes of posterior ischemic optic neuropathy and can be used to screen treatment drugs for this and other central nervous system ischemic diseases.