The overall goal of this procedure is to induce chronic ocular hypertension using laser photocoagulation of the trabecular meshwork in mouse size. After anesthetizing the mouse, the pupil of the targeted eyes dilated, and then the anterior chamber is flattened. The second step is to induce ocular hypertension by delivering a laser beam around the circumference of the trabecular meshwork perpendicular to the surface.
Next, the intraocular pressure is tracked in the mouse over time using a tonometer. The final step is to perform automotive tests to show changes of visual acuity. Ultimately, visual defects caused by hypertension can be monitored over time.
After obtaining an approved I-A-C-U-C protocol, anesthetize a 40 to 60 day old C 57 black six mouse by an intraperitoneal injection of ketamine and xylazine. Next carefully add eye ointment to the left control eye, and then dilate the pupil of the right eye by topical treatment with one or two drops of 1%atropine sulfate solution. After the pupil is fully dilated, insert a sharp glass micro pipette into the anterior space of the eye to drain out the fluid in the anterior chamber.
This will flatten out the anterior chamber and enhance laser induction. Then position the mouse under the slit lamp, restrain the mouse in a plastic cone holder and secured into place on a homemade platform. Next, prepare the Argonne laser source by setting the wavelength to 514 nanometers the power to 100, milliwatts the pulse length to 50 milliseconds, and the spot size to 200 micrometers.
With the laser prepared, hold the mouse with the restrainer and expose the right eye of the mouse to the light source behind the slit lamp in order to align the laser. Then apply the laser illumination to the corneal limbus using the argon laser deliver 80 to 100 laser spots perpendicularly around the circumference of the trabecular meshwork. See 57 black six mice.
Have a pigmented iris, which serves as a barrier for any potential stray energy. Following the procedure, instill topical 0.5%moxifloxacin on the ocular surface to disinfect the laser treated area and 0.5%propane to relieve the pain. Keep the animal on a heating pad for recovery for about an hour until it is fully awake.
Place an awake mouse into a tube and then load them into the plastic cone holder. Next, restrain it on the platform. Take care not to over restrain the mouse as this may cause artificially increased intraocular pressure measurements.
Allow five to 10 minutes to let the mouse get adapted to the holder position. Then slowly approach the rebound chronometer towards the control eye until the probe tip is two to three millimeters away from the surface of the cornea. The untreated control eye is always measured first to get a baseline reading for the laser treated eye that is measured next.
Once the rebound chronometer is in position, press the measurement button causing the probe tip to gently hit the center surface of the cornea. Take three consecutive sets of six measurements of intraocular pressure of the same eye. These measurements are then averaged for each eye test the visual acuity and contrast sensitivity using an motor test.
This is accomplished by first placing the mouse on an elevated platform surrounded by four computer monitors and allowing it to move freely. Set up the monitors for the optometry apparatus so that they display horizontally drifting sinusoidal gradings as visual stimuli with mean luminance of 39 canula per meter squared. The moving direction of the grading should alternate consecutively between clockwise and counterclockwise.
During the test. Record the mouse's movements using a camera placed above the mouse. Use a clockwise drifting grading to identify the visual function of the left eye and a counterclockwise drifting grading for the right eye.
Each test takes about 15 minutes and is repeated by two observers independently during the test count movements that are in concert with the drifting gradings as positive movements. If they occur within 15 seconds of initiation after 15 seconds, increase the visual stimulus to find the highest response, eliciting visual stimulus as the animal's visual acuity. Next, examine the contrast sensitivity of each animal at three preselected Spatial frequencies define the contrast threshold for each eye as being the lowest contrast that elicits visual responses at the prefixed frequencies.
The intraocular pressures tended to be elevated in nearly every laser treated eye. However, less than 5%of mice exhibited physical signs of severe damage, such as deflated eyeballs, severe cataract, significant pigment, detachment, or bleeding. One example of intraocular pressure change is shown here.
One week after laser treatment. The intraocular pressure of the treated eye was almost twice as high as the untreated eye and remained elevated for about 16 weeks. The elevated pressure returned to near normal.
At around 20 weeks, a decrease of visual acuity occurred in the treated eye. Following the procedure. This persisted for at least six months and lasted longer than the increase intraocular pressure.
Similarly, a lower contrast sensitivity of the treated eye with elevated intraocular pressure was observed.
