In other experiments, CNO challenges were conducted under anesthesia to collect cerebrospinal fluid (CSF) samples. Animals received a subcutaneous injection of CNO (10 or 3 mg/kg) and were then sedated with tiletamine HCl and zolazepam HCl (Telazol, 3–5 mg/kg, i.m.). Once the animals were anesthetized, the cervical spine area was shaved and disinfected with betadine solution and alcohol. Each CSF sample was obtained from the cisterna magna, using a sterile 23-ga bevel-tipped needle by pressure difference and collected by gravity.31 (link) CSF samples were collected in prechilled sterile Eppendorf tubes, immediately frozen on dry ice, and stored at −80 °C until the time of the assay.
Serial CSF taps were performed in sessions separated by 2 weeks. To reduce the risks associated with repeated spinal taps, and given the time restrictions imposed by the anesthetized state, in each session, only 2–3 serial CSF taps were conducted per animal, so that not all time points were collected for all monkeys.
Thus, after subcutaneous injection of CNO 10 mg/kg, five individual CSF samples were available for analyses at 120 min postinjection, four at 60 min postinjection, three at 10, 30, and 45 min postinjection, and two at 90, 160, and 1440 min. One 90 min postinjection CSF sample was discarded due to blood contamination. Six months later, serial CSF taps were again performed after a subcutaneous injection of CNO, 3 mg/kg, in sessions separated by 2 weeks. After the 3 mg/kg CNO injection, five individual CSF samples were available for analyses at 1440 and 160 min postinjection, four at 90 min postinjection, and three at 30 min postinjection.
Blood samples were collected from the femoral vein immediately following each CSF tap. All blood samples were collected in prechilled 2 mL tubes containing EDTA (3.5 mg) and immediately placed on ice. Samples were centrifuged at 3000 rpm for 15 min in a refrigerated centrifuge (at 4 °C). Plasma was pipetted off and stored at −80 °C until assayed.
Serial CSF taps were performed in sessions separated by 2 weeks. To reduce the risks associated with repeated spinal taps, and given the time restrictions imposed by the anesthetized state, in each session, only 2–3 serial CSF taps were conducted per animal, so that not all time points were collected for all monkeys.
Thus, after subcutaneous injection of CNO 10 mg/kg, five individual CSF samples were available for analyses at 120 min postinjection, four at 60 min postinjection, three at 10, 30, and 45 min postinjection, and two at 90, 160, and 1440 min. One 90 min postinjection CSF sample was discarded due to blood contamination. Six months later, serial CSF taps were again performed after a subcutaneous injection of CNO, 3 mg/kg, in sessions separated by 2 weeks. After the 3 mg/kg CNO injection, five individual CSF samples were available for analyses at 1440 and 160 min postinjection, four at 90 min postinjection, and three at 30 min postinjection.
Blood samples were collected from the femoral vein immediately following each CSF tap. All blood samples were collected in prechilled 2 mL tubes containing EDTA (3.5 mg) and immediately placed on ice. Samples were centrifuged at 3000 rpm for 15 min in a refrigerated centrifuge (at 4 °C). Plasma was pipetted off and stored at −80 °C until assayed.
