This procedure demonstrates the optic nerve crush injury and subsequent analysis of retinal ganglion cell survival in a mouse model. This is achieved by injection of a fluorescent dye into the superior colliculus. On day one, the DI is retrograde, transported to the retinal ganglion cells to label this cell population as a second step.
An optic nerve crush injury is made on day four to cause optic nerve degeneration, which will lead to a gradual death of the retinal ganglion cells. On day 11, the retina is dissected in order to investigate retinal ganglion cell survival. The result of this procedure shows the number of remaining viable retinal ganglion cells based on a account of flora Gold labeled retinal ganglion cells performed using fluorescence microscopy.
Hi, I'm from Dr.Lab in National Eye Institute, NIH. Now I'm going to show you how to perform the mouse optical nerve crash injury model. The main advantage of this technique is that it'll provide a simple, stable, and efficient way to label retina gangling cells, and to investigate the survival or degeneration of the optical crush injury.
This measure can help answer key questions in the optical neuro field, such as glaucoma. The implications of this technique extend towards therapy if we apply some treatment such as growth factors or some receptor agonists or inhibitors before or at the same time as optic nerve clash. Generally, individuals new to this method will struggle Bleeding often happens in the beginning as the optical is visual blood vessels.
Thus, visual demonstration of this method is critical as the optic nerve garage part is difficult to learn. However, practice would make it easier After deeply anesthetizing the mouse with an approved anesthetic. Shave the hair on the head around the area of the incision site.
Carefully place the mouse into the stereotaxic apparatus and ensure that the head is stable. Disinfect the scalp three times each with iodine and a 70%alcohol solution alternating between each solution. Using a sterile scalpel, make a sagittal incision in the scalp to expose the skull.
Clean the skull with 3%hydrogen peroxide. Then identify bgma where the sagittal suture intersects the coronal suture on the skull. Measure 2.9 millimeters posterior and 0.5 millimeters lateral torema.
And mark this point carefully drill a small hole at the point of the second mark. During drilling, apply saline to the site where the hole is drilled to prevent secondary heat injury. Once a suitable hole has been drilled, position a Hamilton syringe over the hole.
Ensure that the needle has clear passage through the meninges and lower the tip of the syringe to a depth of 1.6 millimeters from the surface of the brain. Then very slowly as a flow rate of 0.2 microliters per minute, inject one microliter of fluorgold neural tracer dye into the superior colliculus. Allow the needle to sit in place for five minutes to prevent the fluorgold from diffusing up along the needle track.
Next, carefully withdraw the needle and close the incision site with sutures. Then release the mouse from the stereotaxic apparatus. Administer a subcutaneous injection of buprenorphine for analgesia.
Move the mouse to a warm, dry area. Monitor the animal until it is able to maintain an upright posture. Then return it to the home cage for the first three days after the labeling procedure.
Systemic analgesics and topical antibiotic ointments are given twice daily and the mouse is closely monitored. Three days after the tracer dye injection, the mouse is deeply anesthetized with an approved anesthetic. Place the mouse under the dissecting microscope and send to the mouse to visualize the eye.
Use a pair of spring scissors to incise the conjunctiva of the left eye at the four o'clock position. Next, gently dissect the orbital muscles and move them to one side. Taking care not to damage any blood vessels.
Expose the white optic nerve at the level of its exit from the eye globe. Then using cross action forceps. Crush the optic nerve at a distance of two millimeters from the eyeball, applying pressure for three seconds.
After completion of the crush injury, cover the optic nerve with the displaced tissues and conjunctiva and suture using 10 zero size silk suture material. After suturing and before the mouse recovers from anesthesia, apply some antibiotic ophthalmic ointment to the eyes and administer an injection of buprenorphine for analgesia. Finally, move the mouse to a warm and dry recovery area.
Monitor the animal until it has recovered sufficiently from anesthesia to maintain an upright posture. Then return the animal to the home cage. Ensure that the animal is monitored closely and administer systemic analgesics and topical antibiotics twice daily.
For the first three days following the optic nerve crush procedure, the retina harvested seven days after the optic nerve crush procedure. After euthanizing the mouse by carbon dioxide, asphyxiation, and cervical dislocation, use a pair of forceps to a nucleate the eyes by applying pressure to the orbit. Immerse the eyes in 4%para formaldehyde for two hours to fix.
Then remove the 4%para formaldehyde and wash the eyes three times in PBS. After fixation, dissect the retina. According to the previously published protocol image, the surviving viable retinal ganglion cells in defined areas of the retina using a fluorescent microscope, retinal ganglion cell density in each region can be calculated from these images.
This microscope image shows a fixed, flattened and mounted retina. This image shows the flora gold labeled retinal ganglion cells before optic nerve crush injury in an uninjured normal retina. The retinal ganglion cells have been retrograde labeled with green flora, gold neural tracer dye injected into the superior colliculus.
This image shows the flora gold labeled retinal ganglion cells seven days after the optic nerve crush injury. Compared with the previous image of retinal ganglion cells in the uninjured normal retina, the number of viable retinal ganglion cells is significantly lower in the retina with an optic nerve crush injury. Once oid, the first part, labeling retinal ganglion cells can be done in 20 minutes.
The second part, optical nerve crash can be done in five minutes while attempting this procedure, it's essential not to use too much force and not to crash for too long because they may lead to damage to the ophthalmic artery under the fall. Subsequently, retina, the optical nerve crash injury mirror model is useful to study the process of retina ganglion, cell death and survival. This model is also often used to investigate the effect of different regions and the genes on retinal ganglion, cell and proptosis and the survival.
One advantage of this model is that it has a high degree of reproducibility, so good luck to work.
