A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

1Pulling the glass capillary tube

2The glass capillary tube is purchased from The Sutter Instrument Inc (Borosilicate glass, B100-75-10).

3Pull the capillary tubes on a Sutter P-87 Flaming micropipette puller, with the heat index set at 300 and the pressure index set at 330.

4Trim the tip of the glass capillary tube with scissors, so that the tapered tip has an inner diameter of about 0.5 mm.

5Cisterna magna puncture technique for CSF

6CSF samples are taken from the cisterna magna (Figure 1) using a method that was published previously 1.Figure 1

71. The mice are anesthesized by Ketamine (100mg/kg) and xylazine (10mg/kg), administered intraperitoneally. During the time of anesthesia induction, the mice are kept in a 37oC incubator.

82. The skin of the neck is shaved, and the mouse is then placed prone on the stereotaxic instrument with direct contact of a heating pad. A rectal temperature probe is inserted into the rectum so that the heat produced by the heating pad is adjusted in response to the changes of body temperature. The head is secured with the head adaptors. The surgical site is swabbed with 10% povidone iodine, followed by 70% ethanol (repeat 3 times), and a sagittal incision of the skin is made inferior to the occiput.

93. Under the dissection microscope, the subcutaneous tissue and muscles (m. biventer cervicis and m. rectus capitis dorsalis major) are separated by blunt dissection with forceps. A pair of microretractors is used to hold the muscles apart.

104. The mouse is laid down so that the head forms a nearly 135o angle with the body.

115. Under the dissection microscope, the dura mater of the cisterna magna appears as a glistening and clear reverse triangle through which the medulla oblongata and a major blood vessel (arteria dorsalis spinalis), and the CSF space are visible (Figure 2).Figure 2

126. The dura mater is blotted dry with sterile cotton swab. Penetrate the capillary tube into the citerna magna through the dura mater, lateral to the arteria dorsalis spinalis (Figure 2). Following a noticeable change in resistance to the capillary tube insertion, the CSF flows into the capillary tube.

137. Carefully remove the capillary tube, and connect it to a 3 ml syringe through a polyethylene tubing that has a 1 mm internal diameter. Inject the CSF into a pre-marked 0.5 ml eppendorf tube, and freeze the tube immediately on dry ice and then transfer it into a -80°C freezer.

148. After CSF sampling, the muscles are re-aligned, and skin is sutured (4-0, Ethicon, Johnson & Johnson). About 1 ml of 0.9% NaCl is injected subcutaneously to prevent de-hydration. The mouse is kept at the incubator to maintain body temperature until it recovers; the weight of the mouse is monitored 1 day, and 1 week after the surgery.

15Results and Discussion

16We described a highly reliable protocol for serial sampling of CSF from mice without detectable plasma contamination.

171. The whole procedure usually takes 10 min per mouse (including anesthesia). The volume of CSF obtained is dependent on the mouse strains. In the PS/APP double Tg mice we used 2, the average volume is about 5 μl (3 to 7 μl), while the P301L (JNPL3) mice 3 give a yield of about 10 μl- 15 μl. For serial sampling, a maximum of 7-8 μl can be safely taken each time at an interval of 2-3 months.

182. During the surgery, it's important to position the head and the body of the mouse properly on the stereotaxic frame so that the dura mater of cisterna magna can be exposed sufficiently. Carefully avoid the blood vessels when penetrating the dura mater with the capillary tube to prevent contamination from plasma proteins, which can be monitored in the CSF sample by immunoblotting for ApoB 4.

193. Minimizing the tissue damage during each surgery is important, as tissue conglutination can increase the difficulty by predisposing bleeding for the following sampling.

204. The body temperature needs to be well maintained during the surgery because hypothermia caused by anesthesia can significantly affect the phosphorylated tau levels in the brain very rapidly 5.

215. The Ab ELISA protocol and antibodies that we used were described in detail in Refolo et al. 6. Two μl of CSF from the PS/APP mice will give satisfactory results when diluted 1:50-1:60. For the tau ELISA, 4-5 μl CSF from P301L (JNPL3) mice will be sufficient for the detection of total tau, or tauphosphorylated at threonine231 (Kits are purchased from Bio-Source).

226. This technique can be applied to other mouse models of neurological diseases, for which a small volume of CSF is satisfactory for desired assays.

23We described a highly reliable protocol for serial sampling of CSF from mice without detectable plasma contamination.

241. The whole procedure usually takes 10 min per mouse (including anesthesia). The volume of CSF obtained is dependent on the mouse strains. In the PS/APP double Tg mice we used2, the average volume is about 5 μl (3 to 7 μl), while the P301L (JNPL3) mice 3 give a yield of about 10 μl- 15 μl. For serial sampling, a maximum of 7-8 μl can be safely taken each time at an interval of 2-3 months.

252. During the surgery, it is important to position the head and the body of the mouse properly on the stereotaxic frame so that the dura mater of cisterna magna can be exposed sufficiently. Carefully avoid the blood vessels when penetrating the dura mater with the capillary tube to prevent contamination from plasma proteins, which can be monitored in the CSF sample by immunoblotting for ApoB 4.

263. Minimizing the tissue damage during each surgery is important, as tissue conglutination can increase the difficulty by predisposing bleeding for the following sampling.

274. The body temperature needs to be well maintained during the surgery because hypothermia caused by anesthesia can significantly affect the phosphorylated tau levels in the brain very rapidly 5.

285. The Ab ELISA protocol and antibodies that we used were described in detail in Refolo et al. 6. Two μl of CSF from the PS/APP mice will give satisfactory results when diluted 1:50-1:60. For the tau ELISA, 4-5 μl CSF from P301L (JNPL3) mice will be sufficient for the detection of total tau, or tau phosphorylated at threonine 231 (Kits are purchased from Bio-Source).

296. This technique can be applied to other mouse models of neurological diseases, for which a small volume of CSF is satisfactory for desired assays.