An improved method of transcutaneous cisterna magna puncture for cerebrospinal fluid sampling in rats.

A simple, reproducible and chronic technique of cerebrospinal fluid (CSF) collection in rats was developed by direct cisterna magna (CM) puncture utilizing stereotaxic apparatus. CSF collection apparatus was constructed using 1 mL syringe, silicone tubing, 21G disposable needle and water. Animal was placed on an elevated platform over stereotaxic apparatus base and puncture site was identified with the aid of stereotaxic co-ordinates. The volume of CSF collected varied from 100 to 180 μL with mean CSF volume of 150 μL. Neurological deficits were recorded according to the modified Bederson's scoring system 24h post CSF collection and differential cell count in CSF samples was performed. Animals continued to be normal with regular feed intake and gained body weight (∼24%) even after repeated sampling for four weeks and showed no severe neurological deficits (mean Bederson score<1 for four weeks). Neuropharmacokinetic data for Phenytoin sodium, MS 275 and Valproic acid (VPA) demonstrated CSF uptake with CSF(AUC)/plasma(AUC) ratio (K(p,CSF)) of 0.09, 0.01 and 0.33, respectively. This model exemplifies the 3R's of animal use and has been successfully implemented at Orchid Chemicals and Pharmaceuticals Limited for lead optimization of CNS penetrating HDAC inhibitors.