Cerebrospinal fluid (CSF) is a clear liquid that surrounds the brain and spinal cord, acting as a cushion and a medium for chemical communication. In biomedical research, this fluid offers a window into the health of the central nervous system. Analyzing its components can reveal biomarkers for various neurological diseases and brain injuries. Mice are frequently used as models in this research because their biological systems have similarities to humans.
Pre-Procedure Preparation
The process begins with anesthetizing the animal to ensure it remains immobile and does not experience pain. A common anesthetic is isoflurane, which is administered via inhalation and allows for rapid induction and recovery. The dosage is carefully monitored throughout the procedure to maintain an appropriate level of sedation, indicated by the animal’s respiratory rate.
Once anesthetized, the mouse is placed into a stereotaxic frame, a device that securely holds the head in a fixed position. The head is flexed downward to expose the collection site at the base of the skull. The fur over the neck and the back of the head is shaved, and the skin is sterilized with an antiseptic solution, such as 70% ethanol, to prevent contamination. Equipment includes fine-gauge needles or pulled glass capillaries, collection tubes, and a dissecting microscope for magnification.
Common CSF Collection Techniques
The most established method for terminal CSF collection in mice is the Cisterna Magna Puncture. This technique targets the cisterna magna, a small reservoir of CSF located at the base of the brain. After the mouse is secured and the area is prepped, the researcher makes a midline incision in the skin over the neck to expose the underlying muscles. These muscles are carefully dissected to reveal the transparent membrane covering the cisterna magna.
Using a micromanipulator attached to the stereotaxic frame allows for very fine and controlled movements of the collection instrument. A pulled glass capillary or a small-gauge needle is precisely guided toward the membrane. Researchers often use a dissecting microscope to visualize the triangular-shaped puncture site, which is outlined by specific anatomical landmarks on the medulla oblongata and cerebellum. The optimal insertion points are on either side of the large blood vessels that run through the area to minimize the risk of bleeding.
The needle or capillary is advanced slowly until resistance from the membrane is felt. A gentle movement punctures the membrane, and CSF, being under slight pressure, begins to flow into the capillary tube. A successful collection is indicated by the clear fluid drawing into the tube. Blood contamination is a primary concern; a pink or red tinge signifies a ruptured vessel. If blood appears, the sample is discarded.
For studies requiring repeated CSF sampling from the same animal over time, a different technique involving ventricular cannulation may be used. This survival surgery involves implanting a permanent cannula into one of the brain’s ventricles. While this method allows for chronic studies, the Cisterna Magna Puncture remains the standard for obtaining a terminal sample due to its relative simplicity and the larger volume of CSF that can be collected, around 10-15 microliters.
Post-Collection Sample Handling
Immediately following collection, the CSF sample undergoes a visual inspection. The clarity of the fluid is the first indicator of its purity. Any reddish discoloration suggests the presence of red blood cells. The collected volume is then measured by expelling the contents of the capillary into a pre-labeled microcentrifuge tube.
To preserve the integrity of the molecules within the CSF, such as proteins and biomarkers, prompt processing and storage are necessary. The sample is centrifuged at a low speed in a refrigerated centrifuge to pellet any contaminating cells. After centrifugation, the clear supernatant is carefully transferred to a new, clean tube.
For long-term preservation, the sample is snap-frozen. This process involves rapidly lowering the temperature of the sample, often by immersing the tube in liquid nitrogen. Once frozen, the CSF is stored at ultra-low temperatures, at -80°C. This immediate and deep freezing halts enzymatic activity and prevents the degradation of biological molecules.
Post-Procedure Animal Care
If the collection procedure is part of a survival surgery, such as with chronic cannulation, post-operative care is administered. The animal’s recovery is monitored closely as it emerges from anesthesia. This includes observing for any signs of neurological deficits, distress, or pain. The incision site is closed with sutures or surgical clips.
To manage pain, an analgesic is administered before the animal fully awakens. Ensuring the mouse has easy and immediate access to food and water is also a standard part of recovery protocols. Placing food pellets and a water source on the floor of the cage helps the animal, which may be temporarily groggy or sore, to eat and drink without extra effort.
Consistent monitoring continues for several days to ensure the animal is healing properly and does not exhibit signs of infection or other complications. Any adverse signs are addressed promptly in consultation with veterinary staff. These steps are part of the commitment to animal welfare in research.