Intrathecal injection means the introduction of a substance by injection into the subarachnoid space of the spinal cord. This is a strategy to bypass the blood-brain barrier by using an alternate route of delivery. In this protocol, we here present the experimental procedure of intrathecal injection in the mouse as follows. STEP 1
Shave the fur on mouse’s back and sterilize the skin surface with 70 % ethanol.
STEP 2
Fix the animal by gripping appropriately with the left hand and expose the back of the iliac crest, then hold the 29gauge injection needle in the right hand and insert it into the iliac crest after keeping the mouse calm.
Note: In ICR mice: The L5 intervertebral space is almost parallel to the iliac spine, the horizontal distance of the L6 intervertebral space to the iliac crest is about 3 mm;
In C57BL/6 mice: The horizontal distance of the L5 and L6 intervertebral space to the iliac crest is about 1.5 mm and 4 mm respectively.
STEP 3
Insert the needle gently at an angle of 30°-45° into the midline skin.
In ICR mice and C57BL/6 mice, the injection site is located at a horizontal distance of about 3 mm and 4 mm from the iliac crest to the tail respectively. If the needle cannot enter the L6 intervertebral space, reduce the angle slowly when it connects the bone, and move the needle tip forward along the groove between the spinous process and the mastoid process until it enters into the L5 intervertebral space, the depth of the needle tip to enter the spinal canal is about 5 mm.
STEP 4
Push the needle tip to insert 10 μL of solution into the spinal canal. When the tail of the mouse shakes or trembles suddenly, it indicates a successful injection. Retain the needle approximately 1 min after finishing delivery. Withdraw the needle with gentle rotation to avoid leakage.
STEP 5
Move the mouse back to the cage for recovery from paralysis.
Note: No anesthesia and less irritation to mice make the method feasible and commonly used. it is beneficial to minimize the impact on the experimental results such as behavioral tests.

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