Retinal ganglion cells or RG Cs are central nervous system neurons that transmit visual information from the retina to the brain via the optic nerve Optic nerve. Transection is a reproducible model of apoptotic neuronal cell death in the adult central nervous system. Here, optic nerve transection is performed by opening the superior orbit of the eye, gaining access to the structures that overly the optic nerve.
The lacrimal gland and the extraocular muscles are retracted and the meninge coverings of the optic nerve are opened. The optic nerve is then lifted out of the meningeal coverings and cut. Ultimately 90%of injured retinal ganglion cells whose axons make up the optic nerve die by programmed cell death within two weeks of injury.
The main advantage of this technique over other brain injury models is that optic nerve transection is more reproducible and the injured neurons can be targeted more easily by experimental manipulations. The implications of this technique extend towards therapies for central nervous system injury and disease because the neuro retina is part of the adult central nervous system. Demonstrating this procedure will be Philippe Donofrio and Mark Magus to graduate students from my laboratory.
Begin this procedure by placing a rat in the anesthesia box and dialing in an isof fluorine concentration of 4%Once the animal's breathing has slowed, switch the gas flow to the gas mask attachment for the stereotaxic frame and place the animal in the stereotaxic apparatus. Then turn the isof fluorine concentration down to 2%Place the nose cone and monitor anesthesia. Wet the fur on top of the head with 70%ethanol to make the fur easier to cut.
Then using clippers, remove the fur from between the eyes using sterile cotton gauze pads. Clean the incision area with three alternating applications of Betadine, followed by 70%ethanol. Next, apply ophthalmic eye ointment to the cornea to keep it from drying out during surgery and spread the ointment over the surface by opening and closing the eyelids.
Using a number 11 blade, make an incision along the midline of the head from approximately 0.5 centimeters in front of the eyes to one centimeter behind the eyes. Using forceps, retract the flap of skin over the eye. Use the back of the scalpel to gently tease away the underlying connective tissue and free the overlying skin.
Then retract the flap of skin laterally and downward and hold it in place with a surgical retractor. Tape the retractor to the base of the stereotaxic instrument while pulling on the overlying fascia with sharp forceps, make an incision along a superior rim of the orbital bone. This will withdraw the fascia overlying the orbit of the eye.
Use forceps to push down on the fascia overlying the orbit, and using a small cautery device, clearly demarcate the rim of the orbital bone. Use of the cautery device will prevent bleeding from the underlying vessels that communicate with the venous sinuses. Use cold sterile PBS to clean the surgical area of any blood.
The surgical area should be periodically cleaned this way to allow better visualization of the structures in the orbit of the eye. Once the incision has been cleaned, use forceps and the back of the scalpel to clean the connective tissue at the back of the eye that overlies the orbital contents. This will open up the orbital cavity providing a larger surgical window to work in.
Next, using Dumont number seven B sharp curve serrated forceps, remove the connective tissue that surrounds the ophthalmic division of the trigeminal nerve, which sits near the midline and remove the nerve using forceps. Although this step is not necessary, removing the nerve will provide a larger window of access to the optic nerve Later on following removal of the nerve. Use forceps to retract the blood vessel below and completely cauterize the vessel.
This step is also not necessary, however, cautery of the vessel allows it to be moved anteriorly, thereby creating a larger window. Once the optic nerve is reached, use a pair of forceps that have had their tip spent inward. To carefully pick and remove the thin layer of connective tissue over the extraocular muscles and the lacrimal gland.
Retract the extraocular muscles from the anterior to the posterior of the orbit. Grip the proximal part of the muscle with a pair of forceps and use a second pair of curves, serrated eye dressing, forceps oriented in the same direction as the muscle to apply outward traction. Remove the most anterior muscle of the orbit, the superior oblique.
Again, using the same technique, remove the medial rectus located between the lobes of the lacrimal gland and the hard gland near the midline of the dorsal surface of the eye. Using forceps, gently remove any remaining connective tissue over the surface of the lacrimal gland and lift the gland upward. Do not compress or squeeze the gland to retract the gland.
Lift the posterior end of the gland upward and then cauterize the vessel at the posterior pole. Next, flap the lacrimal gland forward to open the posterior part of the orbit, allowing unimpeded access to the muscles that overly the optic nerve. Again, apply sterile PBS to keep the area moist and clean any blood with surgical swabs using sharp forceps.
Once again, remove the thin connective tissue that surrounds the muscles of the posterior orbit and separate the underlying bundles of muscle using the curve serrated eye dressing forceps. Pull in line with the muscle fibers to retract the muscles independently or in unison. Finally, attach the remaining muscle lens to the retractor along with the retracted muscles and tape down the retractor to apply traction.
This will rotate the eye forward and outward in order to reveal the fat containing sheath that surrounds the optic nerve. Using fine tip dumont, pull upward on the connective tissue that covers the fatty sheath of the optic nerve using small venous spring scissors. Make it longitudinal cut.
Expand the cut as necessary. The fat contained by the sheath will begin to bulge out. Next, carefully pull upward from the edge and cut off the crescent shaped flaps of tissue using forceps to pull on the fat.
Cut the fat overlying the optic nerve with the Venice spring scissors. With sterile PBS and surgical swabs, clean the small amounts of blood that arise from the removal of the tissue. The optic nerve is now visible to access the optic nerve.
The meningeal sheath that surrounds the nerve must be removed without damaging the ophthalmic artery, which feeds the inner retina. Using forceps to gently rotate it, examine the vascular pattern of the meningeal sheath using fine tip dumont forceps. Pinch the dura and pull upward, creating a triangular shaped wedge near the base of the wedge.
Use the scissors to make a small longitudinal incision in the meningeal sheath. Taking care not to damage the vasculature. Insert the lower blade of the scissors into the incision and cut the sheath parallel to the direction of the optic nerve.
Use the forceps and scissors to drape the dura to either side of the optic nerve. The only remaining covering of the nerve is the thin transparent arachnoid membrane To determine if the membrane is still present, use sharp forceps to pinch the surface of the nerve and pull away from the surface. If meningeal coverings are still present, they'll be visible.
If the arachnoid is present, pinch the membrane and pull upward. To create a triangular wedge of tissue, make a small incision with the tip of the scissors. Then insert the lower blade of the scissors and make a longitudinal cut in the arachnoid.
Next, use scissors and forceps to drape the arachnoid to either side of the optic nerve. Using a microsurgical hook, elevate the optic nerve out of the meningeal sheath. Pass the tip of the hook around the outer edge of the nerve and make sure that the hook stays in contact with the nerve.
Avoid catching the meningeal membranes with the hook and accidentally transecting them. Gently lift the optic nerve out of the meningeal sheath and completely transect the nerve behind the point supported by the hook. The transected optic nerve stump will now have a free end.
Remove the hook if the RG Cs are going to be retrograde labeled. In order to quantify survival, place a small piece of gel foam soaked in 3%fluoro gold or another retrograde tracer over the transected optic nerve stump. Relieve the traction on the extraocular muscles and push on the superior ocular surface towards the midline to return the eye to a neutral position.
At the same time, push the gel phone down into the orbit of the eye. This ensures that the eye is rotated back into place and the gelfoam remains around the optic nerve.Stump. Return the lacrimal gland and the extraocular muscles to their natural return the flap of skin to the midline and suture the wound.
Apply ophthalmic eye ointment to both eyes. Once the surgery is complete, turn off the isof fluorine and allow the animal to breathe oxygen for several minutes. Remove the animal from the stereotaxic device.
Place the animal in a cage underneath a heat lamp to recover, to eliminate the chance of the rat aspirating bedding during recovery. Ensure that there is no bedding in the recovery cage. Administer post-surgical analgesics.
According to institutional guidelines, monitor the animal carefully after surgery. Transection of the optic nerve results in the loss of 90%of injured RGC within 14 days Post taxotomy. The main mechanism of RGC death is apoptosis.
The normal density of R GCs is approximately 2, 500 cells per square millimeter. Epi, fluorescence or confocal imaging can be used to visualize retrograde labeled RG cs. After taxotomy.
RGC apoptosis is delayed by approximately four days after OX otomy leaving a time window for experimental manipulations at one day after ox otomy and retrograde labeling with Flora Gold RGC cell bodies in the ganglion cell layer of the retina and axon fales in the nerve fiber layer of the retina are clearly visible. When imaging a flat mounted preparation by 14 days after OX otomy, the majority of R GCs have died and a few remaining rgs are interspersed between retinal microglia. When RGC undergo apoptosis, microglia phagocytosis the dead cells and as a result become cellularly labeled with a fluorescent tracer that was used to label the RG CS indicated here by the red arrowheads.
The appearance of the tracer in the phagosomes of microglia is different from that in surviving rgc. Microglia contain the tracer in highly concentrated, extremely bright phagosomes that are relatively large and scattered throughout their cytoplasm. RG CS have a more diffuse pattern of staining with small punctate vesicles that have been retrograde, transported down their axons filling the cell cytoplasm.
These vesicles indicated by the white arrows are much smaller and have less intense fluorescence, allowing one to differentiate surviving RGC for microglia. Furthermore, microglia have much smaller cell bodies and tend to have a ST or AME board morphology as opposed to rgc that have relatively large and rounded cell bodies. Once masked, this technique can be successfully completed in 15 minutes if performed properly while attempting this procedure, it's important to prevent damage to the retinal blood supply because the retina will degenerate if the vasculature is compromised Following this procedure.
Other methods like intraocular injections or injections into the transected optic nerve stump can be performed in order to answer additional questions like What are the mechanisms involved in retinal ganglion cell apoptosis.
