The procedure begins with anesthetizing the mouse and the preparation of the surgical site. The mouse is then placed prone on the stereotaxic instrument. A sagittal incision of the skin was made inferior to the occiput and the subcutaneous tissue and neck muscles through the midline are bluntly separated.
Then the posterior part of the body is laid down so that the body forms a 135 degree angle with the fixed head, the dura mater of the CI sternum magna is penetrated with the glass capillary and the CSF will be collected. After CSF sampling, the muscles are realigned and skin is sutured about one milliliter of 0.9%NACL saline is injected subcutaneously to prevent dehydration and the mouse is kept warm until it recovers. Hi, my name is Liu from Dr.Karen Duff's lab at the The Top Institute of Columbia University.
Today I will show you how to collect cerebral spinal of fluid from the system mag of the mouse. With this technique, we can collect serial CSF samples for up to three times from the same mouse with two to three months interval. In our lab, we use this technique to determine the CSFA beta or tau levels at a different ages of our transgenic mouse models of Alzheimer's disease and we correlate these values with those of the brain.
At the end, we are interested in the potential of CSFA beta or tau levels as potential biological markers for the disease progression and also for the therapeutic trials in the mice. So let's get started. For this procedure, it is necessary to make capillaries with a micro pipette polar.
The capillaries used here are BOA silicate glass B 175 10 from Sutter Instruments pull the capillaries on a Sutter P 87 flaming micro pipette polar with the heat index set at 300 and the pressure index set at three 30. The glass capillary is five centimeters long and has a tapered tip. The tip of the capillary is trimmed with scissors so that its inner diameter is about 0.5 millimeters.
The surgical procedure begins with anesthetizing the mouse with ketamine 100 milligrams per kilogram and xylazine 10 milligrams per kilogram administered intraperitoneal during the anesthesia, keep the mouse warmed in a 37 degree Celsius incubator. After determining that the animal is fully anesthetized with a toe pinch withdrawal reflex, test shave the neck, then place the mouse on the stereotaxic instrument such as this one from David kink. In direct contact with the heating pad, insert a rectal temperature probe so the heat from the pad may be adjusted according to the mouse's body temperature.
Secure the head with the head adapters, swab the neck skin with 10%povidone iodine and 70%ethanol three times and make a sagittal incision inferior to the posterior portion of the head or occiput. The next steps should be performed under a dissection microscope using blunt forceps. Separate the subcutaneous tissue and muscles M by ventor services and M rectus capita dorsalis major so that the dura matter of the sternum magna is exposed Properly use a pair of micro retractors to hold the muscles apart.
Finally, lay the mouse down with the head at a 135 degree angle to the body. The mouse is now ready for CSF collection. This technique of collecting CSF from the sternum Magno was published previously.
C figure one A under the dissection microscope. The dura matter appears as a glistening and clear reverse triangle through which the medulla oblongata and a major blood vessel are visible. Arteria dural spinella C figure one B.The circulatory pulsation of the medulla and adjacent CSF space can be seen.
Blot dura matter dry with sterile cotton swab, penetrate the capillary into the CI sternum magna through the dura matter lateral to the arteria dorsalis spinels. There will be a noticeable change in resistance when the capillary is inserted correctly and CSF will then flow into the capillary. After the CSF has been collected carefully remove the capillary and connect it with a three milliliter syringe using polyethylene tubing that has a one millimeter internal diameter.
Inject the CSF into a 0.5 milliliter tube. Label the tube and freeze it immediately on dry ice. Once frozen, transfer the CSF to a minus 80 degree Celsius freezer.
The sampling technique just demonstrated is extremely gentle and allows subsequent CSF samples to be taken from the same mouse over two to three months of interval. Thus, after sampling the neck, muscles and skin should be realigned and closed with sutures. After the incision is sutured, inject about one milliliter of saline solution 0.9%NACL subcutaneously to rehydrate the mouse.
Keep the mouse warmed at about 37 degrees Celsius until it recovers. Monitor its weight at day one and day seven after surgery. I have just shown you how to collect cerebral spinal fluid from the system mag of the mouse.
When doing this procedure, there are three important things to remember. First, position the head and the body of the mouse properly on the stereotactic frame so that the ator of the Cy Magna can be exposed as much as possible. Second, carefully avoid the blood vessels when you penetrate the durometer with capillary.
Otherwise, your sample will get contaminated by the blood. Last but not least, please remember to keep your mice warm during the whole procedure to prevent hypothermia. Hypothermia is not only harmful to your mice, but also it can significantly affect your results by inducing a beta production or tall hypo phosphorylation relation.
Okay, that's it. Thank you for watching and good luck with your experiments.
