The overall goal of this procedure is to improve collection of cerebrospinal fluid from mice without contamination from blood. This method provides an easy and reliable way to collect CSF for monitoring the biomarkers of neural diseases. The main advantage of this technique is that the addition with the micromanipulator to aid in CSF collection from mice decreases the contamination of blood during collection.
Begin by placing a pulled glass capillary into a capillary holder which is firmly mounted on a micromanipulator. Next, while viewing through a dissecting microscope, use straight forceps to break the tip of the sharpened capillary so that the inner diameter of the broken tip is about 10 to 20 microns. Then attach a thin tube and a syringe to the other end of the capillary holder, connect the thin tube and the syringe with a three-way valve and shift the three-way valve to open.
Then plunge the syringe to expel any contaminants and create positive pressure in the capillary tube. Repeat this a few times by disconnecting and reconnecting the syringe to the three-way valve. Then draw up air to 100 to 200 microliters before the final reconnection of the syringe with the three-way valve.
Move the micromanipulator with the glass capillary to the side to prevent damage during the surgical procedure. Before beginning the surgery, ensure that the mouse has been fully anesthetized by pinching all four limbs and observing the absence of a reaction. Then use dissecting scissors to cut and trim the hair at the back of the head of the mouse from above the eyes between the ears to the bottom of the neck.
Secure the head of the mouse using a stereotaxic frame mouse adapter. Make sure the head cannot move and is secured tightly in the adapter by gently pushing down onto the head of the mouse. Use scissors and curved forceps to cut through the mouse skin across the back of the neck and then down the middle of the skull between the ears and eyes of the mouse.
Pull the skin and tissue apart and out of the area over the cisterna magna. While viewing through the dissecting microscope, carefully dissect away the last thin layers of muscle over the base of the skull using forceps and move these layers of muscle to the side and out of the area of interest. Once the dura over the cisterna magna is exposed, use a wet cotton bud to wipe the membrane clean and then use a dry cotton bud to dry the area.
The cisterna magna appears triangular with one to two large blood vessels running through the area. Either side of or between the blood vessels is optimal for capillary insertion and CSF collection. Once the cisterna magna is exposed, use the micromanipulator to align the glass capillary to the back of the mouse head so that the sharpened point is just behind the membrane at a 30 to 45 degree angle.
Next, plunge the syringe once or a few times and then move the plunger back 100 to 200 microliters to make sure there are no contaminants and there is negative pressure in the glass capillary. Then move the capillary tip closer to the membrane until resistance can be seen, but without puncturing the membrane. Knock on the micromanipulator controls to gently tap the capillary tube through the membrane.
CSF should automatically be drawn into the capillary tube once the opening has been punctured. Leave the capillary in place to slowly collect CSF. If the CSF has stopped flowing, slowly draw the syringe up approximately 50 microliters to create more negative pressure in the capillary.
Ten to 15 microliters of CSF is collected in 10 to 30 minutes. Once the desired volume of CSF has been collected, close the tubing by shifting the three-way valve to close and remove the syringe connected to the tubing. Open and then close the tubing to create equalized pressure in the capillary, the tubing, and the area outside the capillary.
Then reconnect the syringe before gently removing the glass capillary from the mouse using micromanipulator. Next, place the collection tube containing one microliter of 20X protease inhibitor under the sharpened point of the glass capillary. Open the tubing and gently plunge the syringe.
The CSF should flow out of the capillary and into the collection tube. After collection, pulse centrifuge the CSF to mix the sample with the protease inhibitor at the bottom of the collection tube. CSF samples can be aliquoted and then stored for further analysis at minus 80 degrees Celsius.
CSF collected from APP/PS1 mice demonstrated a significant increase in levels of human A beta 42 at 12 months of age. In wild type mice, no human A beta 42 was detected. Once mastered, this procedure can be done in one hour if performed properly.
While attempting this procedure, it is important to remember to avoid blood vessels when inserting the capillary and to only make minor adjustments with the micromanipulator during CSF collection to avoid contamination with blood.
