The overall goal of this technique is to create a circumscribed capsular infarct model with a persistent motor impairment using photothrombotic technique. Before creating the capsular infarct, it is necessary to identify the forelimb area in the internal capsule by introducing an adeno-associated virus expressing GFP to the forelimb area of the motor cortex. Begin by placing an anesthetized rat into a stereotaxic frame.
Then shave the head of the rat and sterilize the scalp using 70%alcohol and povidone and iodine solution. Inject 2%lidocaine hydro chloride under the scalp in the area of the intended scalp incision to reduce intra operative pain. Apply ophthalmic ointment to prevent drying of the eyes.
And place a sterile drape over the animal. After making a mid line incision of two centimeters in the scalp, position the stereotaxic arm to identify the forelimb area of the motor cortex according to the stereotaxic coordinates. Drill an entry hole at this position.
Move the needle to the drilled hole and lower the needle one millimeter through the dura and into the motor cortex. Use a high precision micro pump to slowly inject one micro liter of virus. Leave the needle in place for an additional 10 minutes to prevent back flow up the needle tract.
Once 10 minutes have elapsed, slowly withdraw the needle. After cleaning the operative site with saline irrigation, secure the wound with 3-0 nylon suture. Then release the rat from the stereotaxic frame and transfer it to a recovery chamber for monitoring.
Administer analgesic for post operative pain control. After two to three weeks, sacrifice the rat according to approved methods and section the cryoprotected brain. After staining for GFP, observe the AAV-GFP transduced axons in the internal capsule with a fluorescent microscope.
Compare the locations of transduced axons as seen on the fluorescent image with the rat brain atlas to accurately determine the stereotaxic coordinates of the transduced axons. To prepare the optical neural interface, first use a cutting drill to cut approximately four centimeters of a 27 gauge spinal needle with a stylet inside. Then strip 10 centimeters of the jacket of an optical fiber.
Insert the un-jacketed piece of the optical fiber into a metal tube. And clamp the tube around the fiber. Then use a presser to clamp the lower 1/2 of the metal tube twice.
The metal tube fills the space between the optical fiber and the hub of the spinal needle. Apply heat curable epoxy on the optical fiber. Apply additional epoxy to the empty space in the hub.
And insert the optical fiber into the spinal needle. Cure the epoxy for 20 minutes at 100 degrees Celsius for stable fixation. Once cured, cleave the optical fiber that protrudes out of the spinal needle and then use diamond lapping sheets to polish the optical fiber at the tip of the spinal needle.
Lastly, connect the FCPC connector part of the patch cord to the coupler of the green laser system. Then use a digital optical power and energy meter to measure the light intensity from the tip of the optical fiber to ensure accurate emission of light. Place a heat pad under the body of the animal to maintain body temperature at 37.5 degrees Celsius thought the surgery.
Begin by anesthetizing and preparing the rat for surgery as before. After making a mid line incision of two centimeters, adjust the height of the nose clamp until the Bregma and Lambda are aligned at the same level. The correct alignment is critical to correctly target the internal capsule.
Position the stereotaxic arm to locate the site for photothrombosis. Then drill a two millimeter hole at this point. Next, polish and clean the optical fiber tip of the ONI, then fix it to the stereotaxic frame without bending.
Check the tip of the ONI to ensure it is clean and intact. Measure the laser intensity from the tip of the optical fiber prior to the insertion of the optical interface to the target site of the rat brain. Adjust the laser intensity to 3.5 milliwatts at the tip of the optical fiber.
Now, lower the ONI into the target area of the internal capsule using the dorsoventral coordinate determined in the first section of the protocol. Then, inject two milligrams per kilogram of Rose Bengal through the tail vein and then start a countdown timer set to one minute. When the minute has elapsed, turn on the 532 nano-meter green laser for 90 seconds to create the infarct.
After a radiation, gently remove the ONI from the brain. After cleaning the operative site, secure the wound with 3-0 nylon suture. Then remove the rat from the stereotaxic frame and transfer it to a recovery chamber.
This image shows the extent of infarct lesions across varying intensities of laser light from two milliwatts to five milliwatts two weeks after photothrombotic lesioning. Arrows indicate the infarct lesion. The optical light intensity is considered to be between three milliwatts and four milliwatts in this experimental setting.
The next two images show the microscopic appearance of the capsular infarct three weeks after photothrombosis. In this coronal section of the rat brain, the arrow heads indicate the tract of the needle containing the optical fiber in the thalamus and up to the internal capsule. These serial nestled stained sections show the extent of the infarct lesion in the internal capsule.
The arrows indicate the infarct lesion.
