To begin, prepare a 23G butterfly needle by cutting its plastic sleeve protection, such that the end of the bare needle is exposed by 7 millimeters. Then, connect the butterfly needle with polymer tubing to a 1 milliliter syringe. Place an anesthetized rat on a stereotaxic frame and maintain anesthesia through a face mask.
Next, remove the fur on the rear head and neck of the rat. Fix the head of the rat with ear bars. Move the nose bar of the stereotaxic frame to lower the animal's head by approximately 45 degrees vertically.
Find a slightly depressed surface on the rear head with the aspect of a rhombus between the occipital protuberance and the atlas spine. Rub the surface with 70%ethanol. For CSF collection, insert the butterfly needle vertically into the center of the rhombus-shaped depressed surface into the cisterna magna til the movement is blocked by the plastic sleeve protection of the needle.
Gently draw the syringe piston in order to let the CSF slowly flow through the needle. Collect about 100 microliters of the CSF into polymer tubing. Pinch the polymer tubing very close to the butterfly needle and cut the tubing at this point.
Draw the clear sample into the syringe. Expel the sample into the sterile 0.2 milliliter microtube and store on ice for up to one hour. Disinfect the site of CSF withdrawal on the animal's head.
Remove the rat from stereotaxic frame and put it back in its cage. Put aside 2 microliters of the sample for quality control and store the rest at 80 degrees Celsius for further analysis. Repeated CSF withdrawal was performed across five days in three experimental groups of rats and expressed as a percentage of successful withdrawals that resulted in clear CSF.
In the double head implant endowed rats cannulated for CSF withdrawal, the success rate was 71.1%indicating that the cannula on the animal's head may interfere with repeated CSF sampling. In contrast, when CSF collection was carried out by cisterna magna puncture in only tethered or telemetry electrodes implanted animals, the success rate was 86.7%and 88.9%These results suggest that the puncture technique is more suitable for multiple CSF withdrawals in electrode implanted animals.
