Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The overall goal of the following experiment is to monitor demyelination in the optic nerve in vivo. This is achieved by first implanting micro electrodes over the visual cortex. As a second step lyin is micro injected into the optic nerve, which induces a focal lesion of demyelination.

Then visually evoked potential recordings can be made. Ultimately, regression analysis shows a strong linear correlation between the visual evoked potential latency delay and the volume. This video will demonstrate our technique for recording visual evoked potentials from rats.

Using implanted skull electrodes, we'll also demonstrate a technique for focal demyelination of the optic nerve using a microinjection technique. All the experiments are conducted here at our laboratory at Macquarie University Faculty of Medicine. The technique will be demonstrated by Nitin Chichi, our PhD student To begin anesthetize an adult rat older than 12 weeks with an intraperitoneal injection of ketamine and meine before beginning the surgery.

Check for the absence of withdrawal reflexes. Be prepared to administer 10%doses of ketamine as needed to deal with any reflexes that should return to the animal during the surgery. Next, shave the skin at the surgical site and then place the animal on a heat pad to complete the preparations.

Scrub the skin with povidone iodine, drape the animal and apply ophthalmic ointment. Also, be sure to maintain asepsis by using sterile instruments. To begin the surgery, make a longitudinal skin incision on the midline of the head.

Then clear the connective tissue to get good exposure of the skull. Next, using a micro hand drill, carefully put small bur holes, seven millimeters behind the bgma and three millimeters lateral to the midline into the holes. Implant screw electrodes through the skull and about 0.5 millimeters into the cortex.

Then implant a reference screw electrode on the midline, three millimeters rostral to the bgma as needed. Apply dental cement to encase and fix the screw electrodes. Lastly, suture the skin closed.

The electrodes can be left exposed so that skin doesn't need to be reopened For each recording. Now, quickly administer a non-steroidal anti-inflammatory drug or nsaid, or an opioid analgesic before the animal recovers from the anesthesia. To finish, treat the skin with an antibiotic ointment.

Put the animal in a recovery cage with a warming pad and monitor it constantly until it is fully ambulatory. Then allow it to recover for at least a week. Before making VEP recordings, prepare the animal for surgery as previously described, and make a one to 1.5 centimeter skin incision above the orbit of eye, their eye with fine iris scissors.

Open the subcutaneous tissue and access the orbital cavity. Then under a stereoscope, open the conjunctiva and anterior tendons capsule. Next, retract the extraocular muscles and intra orbital lacrimal glands.

To expose about three millimeters of the optic nerve around the optic nerve, use an ophthalmic blade to open the dura and arachnoid matter layers longitudinally. Then insert a glass micro pipette attached to a Hamilton syringe into the nerve, two millimeters posterior to the globe. Over 30 seconds, inject a bolus of lyin with dye to complete the procedure.

Suture the skin incision. Apply antibiotic ointment to prevent infection. Move the animal to a warming pad and monitor it until it recovers.

After anesthetizing the animal and preparing the skin as before, transfer it to a dark room there. Maintain its body temperature within half a degree of 37 degrees Celsius, using a homeo themic blanket system and a temperature probe while being monitored dark. Adapt the animal for five to 30 minutes.

After half an hour, dilate the pupils with 1%tropic IDE eyedrops. Then using scissors and forceps. Open the skin over the skull and access the pre placed NC two screw electrodes.

Connect the screws to the impedance meter. Then measure and maintain the electrode impedance below five kilo ohms. Now connect the screw over the contralateral visual cortex of the stimulated eye.

Next, insert a needle electrode into the tail to surface the ground. Now place a calibrated mini ganzfeld stimulator onto the skin around the eyelids to improve the eye isolation. Then deliver photic stimulation using 100 flashes at one hertz with low pass filter at one hertz and the high-band pass.

Filter at 100 hertz. Once completed, suture the skin closed and keep the animal on a warming pad to recover from the anesthesia. Repeated recordings from a few per week to one every few weeks are feasible.

Intercession, VEP traces show that there is a significant delay in the N one latency. After the optic nerve injection, recordings were taken daily to demonstrate the reproducibility. Partial optic nerve lesions of demyelination could be observed in tissue sections stained with luxal fast blue.

After measuring the lesion volume, correlation between the latency delay and the size of the lesion became apparent. This technique can be used to induce lesions in the optic nerve and to correlate these lesions with changes in VP, it can be used as a model of focal demyelination. Since the lesion size induced is quite variable, it's important to correlate the changes histologically.