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. In the companion video, a method was demonstrated for ex sodomizing or cutting the axons of the entire RGC population as a model of apoptotic neural cell death in the adult central nervous system. Here this method is coupled with procedures that directly target neurotrophic factors, short interfering RNAs, plasmids, or viral vectors that encode anti ATO proteins to injured retinal ganglion cells or the retina to target the retina globally.
An intraocular injection apparatus is assembled and used to inject substances into the vitreous chamber of the eye to specifically target retinal ganglion cells. Either Gelfoam is placed or a Hamilton syringes used to inject substances into the optic nerve. Stump imaging results indicate that retinal ganglion cell survival can be increased by delivery of anti-apoptotic factors.
Using these techniques, This method can be used to identify proteins that are involved in adult neuron apoptosis in the central nervous system. The implications of this technique extend towards therapy or diagnosis of central nervous system disease because optic nerve transection is a reproducible model for neuronal apoptosis in the adult CNS Generally, those new to this technique struggle because they damage the lens when inserting the glass pipette into the vitreous chamber of the eye. To prepare the syringe system for intraocular injections begin by removing any needle or RN nut.
Present on a 10 microliter rn gast tight 1701 Hamilton syringe. Insert the PFA feral into the peak cup feral of the compression fitting. Then insert the complete fitting into the end of the syringe and loosely screw the RN nut over the top.
Insert one end of one 16th inch peak tubing into the compression fitting through the opening in the RN nut. Make sure the peak tubing is fully seated. Tighten the RN nut to compress the feral, which will seal the peak tubing.
Repeat this process on both ends of the dual RN glass coupler. Attach the free end of the peak tubing to one end of the dual RN glass coupler by tightening the RN nut uphold. The glass micro pipette will form the needle and barrel for the injection system.
Insert the glass pipette into the free end of the dual RN glass coupler. Tighten the RN nut to fix the pipette in place. Under a dissecting microscope, create a tip with the appropriate diameter by holding a pulled micro pipette at an angle and rubbing the tip along the glass frosting of a glass slide to create a break.
Ideally the final tip should be slightly beveled. Next, using a priming kit from Hamilton Syringe Company filled with syringe, peak tubing linker, dual RN glass coupler, and the glass micro pipette with mineral oil for use. With the hydraulic injection system, replace the needle with a Hamilton 9 0 0 3 0 needle.
Next place a rubber septum over the Hamilton 9 0 0 3 0 needle to provide a seal while injecting the mineral oil. Insert the Hamilton 9 0 0 3 0 needle of the priming syringe into the back of the barrel of the 10 microliter Hamilton syringe of the injection system. Press the septum tightly against the end of the syringe to create a seal.
Slowly depress the plunger mineral oil will pass through the glass elements of the injection system. Filling the glass pipette. Push all of the oil through the injection system in order to remove any air bubbles along the tract.
If the filling syringe is running out of mineral oil, slowly withdraw the needle while constantly dispensing oil. To prevent air bubble formation, refill the priming syringe and inject again. When the entire system is filled with mineral oil, insert the original plunger of the 10 microliter.
Hamilton syringe completely depress the plunger to remove additional mineral oil from the system. Wipe the glass micro pipette and the end of the syringe clean. With 70%ethanol, withdraw the plunger to the two microliter mark on the barrel.
The end of the glass micro pipette will fill with air, providing a buffer zone between the mineral oil and the fluid to be injected. Fill the pipette barrel by placing the end of the micro pipette in the desired solution and withdrawing the plunger of the Hamilton syringe. No more than four to five microliters of solution will be injected into an adult rat eye to perform the intraocular injection.
Begin by switching the gas flow to the gas mask attachment for the stereotaxic frame. Then place an isof fluorine anesthetized rat in the stereotaxic apparatus. Turn the isof fluorine concentration down to 2%and monitor anesthesia.
Cover the animal with a surgical blanket. To maintain body temperature throughout the surgery, open the eyelids and place one drop of acan anesthetic solution. On the surface of the cornea injection requires two people, one to insert the glass pipette into the vitreous chamber and maintain the pipette position.
And another to depress the plunger on the syringe, which delivers the desired solution. Under a surgical microscope, grip the glass micro pipette and dual RN glass coupler, spread the eyelids forming a V-shape opposite the injection site With the fingers, the eye will elevate out of the orbit. Gently insert the tip of the glass micro pipette through the in an area devoid of blood vessels and through the sclera at a downward angle.
When the pipette penetrates the sclera, a small pop will be felt. The person holding the syringe should now inject four microliters of solution by depressing the plunger to the two microliter mark on the syringe. The injection should take approximately one to two seconds.
The successfully injected solution will flush through the vitreous chamber, hold the micro pipette steady for approximately five seconds, and then withdraw in the same direction As the needle was inserted, the conjunctiva will flap back over the small opening, helping to seal the scleral puncture. Once the surgery is complete, turn off the isof fluorine and allow the animal to breathe oxygen for several minutes. Cover the surface of the cornea with ophthalmic eye ointment In order to prevent corneal drying during recovery, then remove the animal from the stereotaxic device and return it to the recovery cage.
To manipulate retinal ganglion cells through retrograde transport following ex otomy. First perform an optic nerve transection according to the instructions in the companion video optic nerve transection protocol, 2, 2 4, 1 tracers drugs. Plasmids, siRNAs, or viral vectors can be applied to the optic nerve stump.
There are two main methods to accomplish this procedure using gel foam or direct injection into the optic nerve. If the gel foam method is used, ensure that the retinal ganglion cells have been pre-labeled by injecting retrograde tracers into the superior colliculus one week prior to taxonomy. Immediately after the optic nerve is cut, placed a small piece of gel foam soaked in the experimental solution over the transected optic nerve stump, the orbital contents are then returned to the previous location.
When the eye is returned to a neutral position, use forceps to push the piece of gel foam down into the orbit, ensuring that it remains over the end of the optic nerve. The injection technique is more effective for the delivery of substances that must gain access to the cytoplasm of the axon in order to be retro greatly transported by op plasmic flow, A gast tight 10 microliter Hamilton syringe is used to inject the desired solution into the transected optic nerve. A needle with a short bevel is desirable.
Load the syringe with 10 microliters of the solution to be injected. Typically irna plasmids or viral vectors to inject grip the edge of the optic nerve with fine tip dumont forceps. Insert the end of the needle into the optic nerve, parallel with the nerve.
Until the bevel is no longer visible, the inserted needle will be encased on all sides by the nerve. Once the needle is inserted, gently squeezed the sides of the nerve with the fine tip forceps and inject one quarter of the solution while rotating the syringe. One quarter turn clockwise or counterclockwise.
Continue repeating these steps until a full rotation of the needle has been completed and the entire contents of the syringe have been injected. Remove the tip of the syringe from the nerve. Leave any injected fluid that has reflux from the nerve within the orbit as this will form an additional pool for uptake by the ends of the axons.
Return the orbital contents to their original position, close the wound and allow the animal to recover. Animals should be housed independently after surgery. Post-surgical analgesics should be administered according to the guidelines of your animal care.
Authorities and animals should be carefully monitored after surgery. As demonstrated in this video, RGC can be directly targeted by delivering peptides, drugs, vectors, plasmids, or siRNAs to the transected optic nerve stump. In this representative example, fluoro gold retrograde labeling was performed and SI three labeled peptides were immediately injected into the optic nerve.
Live imaging of the retina was then performed in order to prevent the peptides from leaching out of the tissue during fixation, as can be seen here, SI three labeled peptides injected into the optic nerve were retro, greatly transported back to the RGC somato in the retina. In this example, SI three labeled siRNAs were injected into the optic nerve stump. Immediately after taxotomy and live imaging of the retina was performed again, SI three labeled siRNAs injected into the optic nerve were retrograde, transported by RG CS as indicated by their colocalization.
With fluorgold retrograde labeling Once mastered, this technique can be performed in seconds if done properly. While attempting this procedure, it's important to take care to avoid damaging the lens. Don't forget that working with viral particles can be extremely hazardous, and institutional guidelines for working with biohazards should always be followed while performing this procedure.
