4.6. Cerebrospinal Fluid (CSF) Collection Procedure

For CSF collection, the rats were anaesthetized with a combination of ketamine (70 mg/kg) and xylazine (6 mg/kg) i.p. and fixed in a stereotactic apparatus, with its body flexed downward, as described previously [70]. Briefly, the angle between the head of the rat and its body was set at 90°, as shown in Figure 6; punctured surfaces were positioned horizontally. The skin from the spinous process projecting at the initial part of the vertebral column and skull bones was cut along the median line (10–15 mm). The tissues were mechanically displaced from the midline of muscular fasciae on the neck until dura mater appeared. The dura mater looked like a stretched membrane and had a mat surface (rhombus). The parietal bone, vertebral column, and occipital protuberances formed the cerebral, caudal, and lateral corners of the rhombus, respectively. The membrane was perpendicularly punctured at the middle point of the midline (depth 1 mm). The maximum amount of the liquid was sampled. The needle was removed. A median longitudinal incision (1.5 mm) was made through the site of puncture in the dura mater. The residual liquid was removed from the cisterna magna (CM) with a cotton plug. The cavity of the CM was filled with freshly prepared acryl emulsion using a micropipette. After the induration of acryl, CM models were separated from surrounding tissues and examined in transmitted light. Profiles of the CM were visualized on the screen and sketched in the frontal and sagittal surfaces. The actual size of the CM was measured on models with a micrometer [71,72,73]. CSF sample volume ranged from 100–150 µL.

Fixation of rats in a stereotactic device during puncture of the great cerebral cistern. Adapted from ref. [70].