In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Hi, everyone. This is Yikui Zhang from Wenzhou Eye Hospital. The upper nerve lacks Exxons from the orexin ganglion cells, and then transmit the visual signal to the brain.

Larger model of upper nerve injury are essential to translate the novel treatment from robot model to lymph application. Here, we describe some in vivo methods to evaluate the structure and function of the orexin ganglion cells in the upper nerve in large animals. By presenting this method step-by-step, we hope to increase experimental reproductive disabilities and facilitate the usage of larger models of optic neurosis.

Shave the with electronic razor. And, prep it with 70%alcohol three times, to clean the skin and expose the subcutaneous vein. Insert a peripheral venous castor intravenously.

Then drip atropine and propofol. Intubate the goat with a six millimeter tracheal tube and connect to an artificial respirator. Anesthesia was maintained with 3.5%isoflurane in oxygen at a constant two liter managed flow rate.

Place the temperature sensor under the goat's tongue. Clip the oxygen saturation cleave to the proximal end of the goat ear. Tie the blood pressure cuff to the base of the thigh.

Clamp ECG cleaves on the limbs in order. Then, we can monitor the goat's vital indicators. Shave the hair and disinfect the skin with betadine and 70%alcohol three times at the center of the frontal bone.

Open the sterilizer bag containing scissors and the nails. Make skin incisions to expose the skull with a scissor. Steel screws for implanted at the center of the frontal bone.

Shave the hair and disinfect the skin with betadine and 70%alcohol three times at a middle point of two ears in occipital bone. Make skin incisions. Steel screws for implanted at the middle point of two ears in occipital bone.

The ground needle electrode is inserted subcutaneously beneath the frontal skull screw. The active electrode and the reference electrode are connected to a frontal and occipital screws respectively with alligator clips to reduce the electrode impotence. Patch one eye.

Apply topical anesthesia eye drops to the ocular surface. Pupils on both sides are dilated by mydriasis eye drops. Place the goat's head in a gas-filled stimulator aimed ambulant light.

Place the black blanket over the stimulator and the goat head for five minutes adaptation. Eyelid speculum is used to fully expose its pupil. Check the electrode tissue contact impotence.

Ensure that impotence is below 10 kilometers for each to avoid electromagnetic interference from other electrical devices in the same room. Check the baseline noise without light stimulation. Start FVEP recording.

Note that FVEP recording at each light intensity is performed twice. Note that moist the cornea with artificial tear if it appears dry. Repeat about steps for the contralateral eye.

Start FVEP recording at different light intensities consecutively. For each recording, 64 traces are averaged to yield one wave form. The first positive and active peak in the FVEP wave form was designated as P1 and N1.The sterilized ground electrode is positioned in the earlobe.

The sterilized active and reference electrodes are inserted subcutaneously along the midline and the frontal and occipital bone respectively. Eyelid speculum is used to fully expose its pupil. Patch one eye.

Record pattern VEP responses of the other eye at different spatial frequencies consecutively. Connect to the ground electrode with an alligator clip. Two sterilized needle reference electrodes are placed under the skin one centimeter behind the lateral canthus on both sides.

Eyelid speculum is used to fully expose its pupil. Two disinfected ERG recording electrodes are centered both cornea after topical application of artificial tear. Two stimulators are placed in front of each eye with a viewing distance of 50 centimeters.

Ensure that impotence is below 10 kilometer. Track the baseline noise without light stimulation. Aim the ambulant light.

Start pattern ERG recording from both eyes simultaneously at different spatial frequencies consecutively. Finally, return of the screen to record pattern ERG without visual stimulators and an active control. The bandpass filter is set to range from one to 75 Hertz for three CPD pattern ERG.

The bandpass filter is from one to 50 Hertz to smooth the shape without affecting its amplitude. The first positive and active peaks in the wave form are designated as P1 and N1.The pattern ERG amplitude measured from N1 to P1.To reduce electrode impotence, a sterilized needle reference is placed under the frontal skin. And a sterilized needle reference electrode is placed under the skin one centimeter behind the lateral canthus on one side.

A disinfected ERG recording electrode is placed on the cornea on a topical application of artificial tear. Patch one eye. A display is placed at viewing distance of 50 centimeters.

Eyelid speculum is used to fully expose its pupil. Then, the goat is placed on the chin rest. Align the goat with the camera to send his optic nerve head in the confocal scan laser ophthalmoscopy image by modifying its head position.

Click the start button to enter the detection end phase. Wait for the machine to finish loading. Press the yellow start button to start the formal imaging.

Adjust the joystick to evenly illuminate the infrared image to improve image quality. Slide the camera forward until the upright retinal OCT image is shown on upper right screen. Modify the joystick to have it evenly dance in a horizontal retinal OCT image.

Press the button on the joystick to catch your image automatically. Hold this joystick to maintain image quality on the live image screen until image execution is completed. Then press acquire.

Select a high quality initial photo. Right click and select set reference. Press the follow up button to allow program to automatically match the current scan to the baseline scan.

Once matched, pressed the button on the joystick to activate automatic real time tracking. Click the measurement button to enter the measurement platform. Choose the eraser a tool and wipe the RFL line, which is automatically labeled by machine.

Choose the line drawing tool to manually outline the boundary between IPL and INL. Between the red and blue lines is the GCC complex. Retina altered imaging in rhesus macaque is performed by using the same equipment and procedure as those used in goat.

Figure A shows representative FVEP in goat. Figure B shows representative pattern ERG wave form in goat. Figure C shows representative pattern FVEP in rhesus macaque.

And, figure D shows representative pattern ERG wave form in rhesus macaque. Figure two shows representative retinal OCT images around optic nerve head and the thickness of GCC complex in different peripheral regions in goat and the rhesus macaque. Large animal model plays an important role in translational research.

In many cases, promising therapeutic effects in small animal models failed to be translated to human patients. In this study, we presented video protocol of FVEP, PERG, and OCT in goat and the rhesus macaque. This in vivo method can be applied in larger model of various optic neurosis, such as glaucoma ischemic or traumatic optic neuropathy and optic neuritis.