Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Secondary degeneration of retinal ganglion cells occurs mostly in glaucoma. This paper illustrates an innovative operation approach model of partial optic nerve transection. This space-saving operative approach improves the investigator's ability to administer drugs or carriers or selective tracers of RGCs on the stump of the partially transected optic nerve and allows researchers to study the secondary injury mechanisms of RGCs in a new way.

Glaucoma is a chronic neurodegenerative disease, and the progressive neuropathology persists for a long time. Ocular hypertension is one clear cause for glaucoma, and decreasing the ocular hypertension has been shown to be the only confirmed effective therapy for glaucoma treatment. But the situation of some patients will further deteriorate, even after ocular hypertension have been controlled.

Therefore, secondary degeneration of RGCs believed to occur in glaucoma. However, the mechanism specifically related with primary or secondary degeneration are still not clear. The secondary degeneration of RGCs mechanism will guide the treatment of neurotrauma and CES neurodegenerative disease.

To assist with partial quantitative transection of the optic nerve, we present a newly designed, low-cost surgical instrument. The instrument comprises two parts, a hand-held pole and a grooved head. The grooved head is semicircular with a vertical depth of 200 micrometers, and the grooved surface enables the ventral optic nerve to lay within it and be stabilized for transection.

Lay the optic nerve in the groove of the instrument. The grooved surface of the instrument enables the optic nerve to be stabilized for transection. Then vertically transect the optic nerve with appropriate needle tip or knife point.

Please note that only the dorsal part of the optic nerve is cut off since the ventral part is protected by the instrument. Separate and dissociate the dura around the optic nerve, ensuring not cutting off the ophthalmic artery associated with the meningeal sheath. The primary injury was achieved in RGCs corresponding to the quantitatively transected optic nerve axons, dorsal side, while the secondary injury would be performed in RGCs corresponding to the untransected optic nerve axons, central and ventral side, without direct damage.

Rats were anesthetized, using a veterinary isoflurane vaporizer system. Anesthesia was monitored during surgery, and isoflurane dosage was adjusted accordingly. Constantly evaluate the depth and rate of breathing, and perform toe pinch evaluation in every five minutes to make sure the absence of deep pain.

Place the right side of the rat upwards on the surgical table with the head facing the surgery. Adjust the right orbit in the center of the surgical field of view. Then clean the incision area for several times along the lateral canthus to the external acoustic foramen of the right orbital skin with the application of 0.5%chlorhexidine and 75%ethanol.

Remove the fur between the lateral canthus to the external acoustic foramen, using iris scissors. Make a skin incision, using iris scissors, along the lateral canthus to the external acoustic foramen with a length of 0.5 to one centimeters. Then pinch the fascia and pull upward to create a triangular wedge with a 0.12 millimeter toothed forceps.

Insert the lower blade of Vannas spring scissors into the incision area and cut open the fascia with the same shear direction as scissors skin. Especially when cutting the subcutaneous fascia in the outer lateral canthus, use the sharp serrated forceps to pull upward the subcutaneous fascia on the surface of the fascia vertically, and cut the fascia with Vannas spring scissors to avoid damage of the orbital vein at the outer canthus to cause model failure by excessive bleeding. Cut the fascia with iris scissors, and expose the orbital vein.

Use blunt dissection to open both sides of the incision. The advantage of this procedure is that it can prevent bleeding when directly cutting off the blood vessels. Then use iris scissors to cut the right lateral canthus apart along to incision line to fully expose the surgical approach and fully expose the field of vision for the follow-up blunt dissection of orbital muscles.

Continue to clamp the folder of the subfascial orbital muscle, and blunt the separation vertically to the direction of skin and fascia incision. Then separate along sides gradually to reach the orbital depth until the appearance of orbital adipose tissue. This procedure will open up deeper portions of the orbital cavity, providing a larger surgical window to work in and allow unimpeded access to the tissues that overlie the optic nerve.

In the above procedures, if bleeding occurs, apply pressure, using sterile surgical swabs or cotton swabs. After exposure of the adipose tissue in the orbit, transform the rat head direction from facing the surgeon vertically to the right side of the surgeon. Cut off orbital adipose tissues covered on the orbital muscle cone around the optic nerve.

This action can ensure, expose the appropriate surgical approach. Please note to keep volume of resected fat tissue within a rational range so as to avoid sustained bleeding. After cutting the fat tissue, the lateral rectus is exposed.

Clamp the lateral rectus outward, and then cut it by Vannas spring scissors. The purpose of this procedure is to achieve better exposure of the optic nerve, as the lateral rectus is much wider and obviously block the view of the optic nerve. If there is still fat tissue under the lateral rectus, pull up the fat overlying the optic nerve, using a 0.12 millimeter toothed forceps, and cut with the Vannas spring scissors.

Then the tissue sheath around the optical nerve is visible. Continue to separate the tissue sheath along the direction of the optic nerve in the depth of orbit until the total exposure of the optic nerve. Keep the area clean by using surgical swabs to clean small amounts of blood that arise from the removal of the tissues.

Now the optic nerve should be visible. In order to access the nerve, remove the meningeal sheath that surrounds the nerve without damaging the ophthalmic artery. Gently rotate the sheath to examine the vascular pattern of dura with higher magnification of the operating microscope.

Look for an area that is devoid of blood vessels and permit a longitudinal cut on the dura. Rip the sheath parallel to the direction of the optic nerve with needle tip or knife point carefully in order to avoid damage of the vasculature with lateral cuts. The only left over covering the nerve was the arachnoid membrane.

It's very thin and transparent. Similarly to the previous step, rip the arachnoid membrane gently, with needle tip or knife point, parallel to the direction of the optic nerve. Lay the optic nerve within the groove of the instrument softly and carefully, resulting in the dorsal optic nerve that was slightly higher than the edge of the groove head.

Then transect the dorsal optic nerve above the platform edge of the groove head with the needle tip or the knife point to complete the partial optic nerve transection. Move the instrument a little deeper, towards the vertical direction of the optic nerve, to free the optic nerve. Then take out the groove head of the instrument gently.

Try not to scratch the ocular muscles or other tissues to avoid extra damage. Then the stump of partial optic nerve transection can be observed. Replace the lateral rectus, fascia, and other around the tissues of the eye to original position.

Then suture the muscle layer and the skin layer of the orbital in sequence. Apply antibiotic ointment to the wound to prevent infection after the suture. In the process of rat resuscitation, prepare thermal insulation by a heated mat, or cover with dry padding in the cage.

Blanket them to keep warm. Ensure rat airway patency during the recovery process. Animals were housed independently and monitored carefully after surgery.

In order to verify the success regarding the establishment of secondary injury model with new operative approach by using the self-designed surgical assistant instrument, the RGCs were retrogradely labeled immediately after the model established. The purpose of this procedure was to label the RGCs retrogradely by injecting a neural tracer dye into the superior colliculus. The dye would be retrogradely taken up by the RGCs in the retina and provides a marker for the living RGCs with the untransected axons in the right eye.

While RGCs corresponding to the optic nerve axons that were partially transected in the right eye were unable to be labeled with Fluoro-Gold, as a control eye, the left eye without any operation, the RGCs along the optic nerve on the retina were all marked with fluorescent gold dye in a retrograde manner from the superior colliculus. Results of the fluorescence-labeled RGCs with or without partial optic nerve transection were shown. Only the RGCs in the right retina corresponding the untransected portion of the optic nerve were labeled with fluorescent gold, and the clear boundary of the unlabeled and labeled RGCs were visualized, which demonstrated the partial transection of the optic nerve.

As the control eye, all RGCs of the left eye retina were labeled by fluorescent gold. These images show that the blood supply of the right eye was observed before and after operation. After the operation, the arteries were still fulfilling with blood, and no obstruction of the veins was observed.

This indicated that there was no damage to the blood supply system during the operation. The secondary degeneration model of RGCs was established successfully. Potential optic nerve transection model is a usable animal model for study the mechanism of secondary degeneration and screening for the neuroprotective drugs in combat against secondary degeneration.

The marriage of this model is the ability to separate primary from secondary degeneration accurately in location, both in the optic nerve and the retinas. The main advantage of our designed instrument is more reproducible and more easily to operate. With practice, all of the steps in the full surgical procedure can be accomplished in 10 to 15 minutes per eye once the initial entry cuts have been made.