This method facilitates the efficient intrathecal delivery of adeno-associated viral transgenes in awake small animals for the experimental treatment of central nervous system diseases such as ALS. The main advantage of this method is that the viral solution includes 1%lidocaine hydrochloride which induces a transient paralysis after a successful injection. Although this method provide a visual indicator for judging the success of the intrathecal automated situation, sufficient practice is essential for improving the success rate of the delivery.
Before beginning the procedure, attach a 27 gauge needle to a 25 microliter Hamilton syringe and align the beveled tip of the needle with the volumetric scale on the syringe. Then carefully load 8 microliters of 4 x 10 to the 10th genome copies of the virus solution into the syringe, taking care to avoid bubbles. Next, place an awake 30 to 70 day old 12 to 30 gram mouse on a bed piece in the prone position in a biosafety hood and cover the upper body with sterile gauze to calm the mouse and to avoid being bitten.
Firmly grip the mouse on its pelvic girdle with the thumb on one side and the forefinger and middle finger on the other side keeping the skin between the bilateral pelvic girdles taut with a thumb and forefinger. Then shave the fur between the bilateral pelvic girdles and sterilize the exposed skin with an iodine based scrub and 70%ethanol. For direct intrathecal delivery of the adeno-associated virus solution, palpate the intervertebral space along the midline between the bilateral pelvic girdles and use a fingernail to depress an indentation into the skin to indicate the L5 to L6 intervertebral space.
Rotate the base of the tail slightly and gently to reveal the midline of the spine and adjust the bevel of the needle toward the head of the animal. With the mouse fixed firmly into position, align the needle along the midline of the spine and insert the needle gently and vertically into the center of the indentation keeping the syringe in a central sagittal plane. When the needle comes into contact with the bone, slowly decrease the angle to approximately 30 degrees and slip the needle into the intervertebral space.
A sudden tail flick is a sign of a successful entry into the intradural space. When the needle enters the intervertebral space, the tip will feel firmly clamped. Inject the vector solution, and maintain the needle within the intradural space for one minute.
Then slowly withdraw the needle with rotation to minimize leakage. Immediately score the transient weakness of the mouse limbs to evaluate the injection quality and return the mouse to its cage for recovery from the paralysis. At the appropriate experimental endpoint, fix the limbs and head of the injected mouse in the prone position on a foam box cover and use scissors to strip and remove the skin from the head to the sacrum.
Clip the skull between the eyes, cut along the midline of the skull and the horizontal line above the cerebellum and open the skull on each side. Use tweezers to remove the occipital bone and use ophthalmic scissors to open the spinal canal bilaterally. Cut the ribs on both sides and carefully remove the upper section of vertebrae.
Use curved tweezers to lift the brain and sever the nerves of the skull base. Then carefully extract the whole brain and spinal cord and fix the tissues in 4%paraformaldehyde for 24 hours. At the end of the fixation period, cryoprotect the brain and cervical and lumbar spinal cord in 30%sucrose solution overnight at 4 degrees Celsius followed by embedding in optimum cutting temperature compound before snap freezing in liquid nitrogen.
Then use a cryostat to obtain 25 micrometer thick sections of each tissue, storing the frozen sections in 0.01 molar PBS at 4 degrees Celsius as they are obtained. For immunohistochemical analysis of the tissues, pretreat the free floating sections with 1%hydrogen peroxide for 10 minutes followed by a 10 minute wash in PBS. Next incubate the samples in blocking solution containing 5%serum and 0.3%non-ionic detergent in PBS for 1 hour followed by overnight labeling at 4 degrees Celsius with the appropriate primary antibodies of interest.
The next day, wash the sections with 3 10 minute washes in PBS plus Tween and incubate the slides with the appropriate corresponding biotinylated secondary antibodies at room temperature for 1 hour after the last wash. At the end of the incubation, wash the sections 3 times in fresh PBST as just demonstrated and incubate the samples with infinity biotin peroxidase complex for 40 minutes followed by staining with an appropriate chromogenic agent. Mount the labeled sections onto glass microscope slides and soak the slides in anhydrous ethanol for 5 minutes once the mounted sections have fully dried.
Next soak the slides in xylene for 10 minutes and seal them with an appropriate mounting medium. Then image the slides with a light microscope equipped with a charge coupled device at 100, 200, and 400 times magnifications. eGFP immunostaining of cervical and lumbar spinal cord tissue sections reveals little or no transduction in the lumbar spinal cord of mice with a weakness score of 0, slightly enhanced transduction in mice with a weakness score of 1, and strong and widespread transductions in mice with a weakness score of 4 or 5.
Quantification of the GFP staining intensity of spinal cord tissue sections from mice displaying various degrees of transient limb weakness indicates that the severity of the weakness after the virus solution injection closely correlates with the extent of the spinal cord transduction. In the brain, robust eGFP signals are detected in the olfactory bulb, dorsolateral prefrontal cortex, dentate gyrus, and the CA3 zone of the hippocampus, cerebellar cortex, and marginal areas of the brainstem, including the facial nucleus, choroid plexus, and the ependymal epithelial cells. Motor neurons in the anterior horns are also strongly transduced in different levels of the spinal cord.
Moreover, in the cortex, GFP positive neurons including pyramidal cells are detected as well as various glial cell types including microglia, astrocytes, and oligodendrocytes. This technique enables researchers in the neurology field to explore the therapeutic effects of direct intrathecal delivery in small awake animals on central nervous system diseases.
