Xylocain

Subjects reported to the laboratory in the morning after an overnight fast. After 15 min of rest in the supine position, a 3 mm incision was made over the lateral portion of the thigh under local anesthesia (2 ml lidocaine without epinephrine, 20 mg/ml Xylocain; AstraZeneca Pharmaceuticals), and a biopsy was obtained from m. vastus lateralis using a percutaneous Bergstrom needle with suction.

When the ongoing (unstimulated) neuronal activity of a unit was visibly stable, it was recorded for a control period of 30 min without any treatment (baseline activity). Subsequently, saline (0.9 % NaCl, 50 μl) was injected with a U-100 insulin syringe (BD, Franklin Lakes, NJ, USA) deep into the temporal muscle close to the crista temporalis within one minute. After another 30 min of recording, 50 μl of 10 mM α,β-meATP (α,β-methylene adenosine 5′-triphosphate disodium salt hydrate, Sigma-Aldrich, Taufkirchen, Germany) was injected into the temporal muscle and the recording continued for 60 min. After another 10 min without further treatment (new baseline), in most of the experiments 60 μl of 2 % lidocaine (Xylocain, Astra Zeneca, Wedel, Germany) was injected into the ipsilateral medial (semispinalis and rectus capitis) neck muscles followed by a recording period of 20 min. Next the temporal muscle was injected with 60 μl lidocaine and the activity recorded for another 20 min. Finally, 60 μl of lidocaine was applied onto the exposed cranial dura mater followed by a final recording period of 20 min. In some experiments 60 μl of lidocaine was injected only into the ipsilateral temporal muscle immediately followed by 50 μl of α,β-meATP, then the recording was continued for 60 min.

We primed micro-osmotic Alzet pumps (model 1007D) under sterile conditions one day before implantation and left them in an incubator at 37.0°C overnight. We anesthetized the animals with a ketamine (Ketavet, Pfizer, Germany, 150 mg/kg) and xylazine (Rompun, Bayer, Germany, 15 mg/kg) mixture dissolved in 8.5 ml of sterile 0.9% saline. Head skin incision was performed exposing the skull.
We implanted the cannula 0.2 mm to the right from Bregma corresponding to the location of the right ventricle. A drop of cyanoacrylate clay (Weicon, Germany) was introduced between the cannula cap and the skull. We slowly moved the cannula down until the cap touched the skull. We left the animals for 5 min to ensure drying of the clay. Then, we carefully sutured the skin above the cannula with a 5–0 polypropilen thread (Ethicon, USA).
Animals received lidocaine gel (Xylocain, AstraZeneca, Germany) locally and 0.5 ml of ringer lactate solution (B Braun Melsungen, Germany) intraperitoneally to substitute the liquid loss. We placed the cage with operated animals onto the 37°C warm bed for 2 hours.
The animals received a total amount of 84±16.8 µl of PEDF (20 µg/ml in CSF) or CSF at a pumping rate of 0.5±0.1 µl/hour.