Marcaine

The epidural space was accessed under radiographic guidance in the midline between the spinous processes of the eighth and ninth vertebrae, using a 20-gauge epidural needle (Braun, Melsungen, Germany) and the loss-of-resistance technique. The correct catheter position was verified by contrast-medium injection (Ultravist 300^®; Schering AG, Berlin, Germany) via a standard epidural catheter in all animals (Figure 1). With the aim of blocking segments T5 to T12, the animals received 0.75 mL per segment of 0.5% bupivacaine (Marcaine, Astra Zeneca, Sweden) as a bolus dose, followed by 0.15 mL/kg/h as a continuous epidural infusion.

AF-16, produced by organic solid phase synthesis, was purchased from Ross-Pedersen (K.J. Ross-Pedersen ApS, Klampenborg, Denmark). The peptide was purified by reversed-phase chromatography and analysed by amino acid analysis and mass spectrometry. The purity of AF-16 was about 97 %. The AF-16 peptide, VCHSKTRSNPENNVGL, was labelled with ¹⁴C in the N-terminal valine, specific activity 50 mC/mmol, 2 μM/ml (American Radiolabeled Chemicals, Saint Louis, Mo, USA). Marcaine® was purchased from AstraZeneca (Södertälje, Sweden), Isoflurane® from Baxter Medical (Kista, Sweden).

All surgical procedures were performed by one of three surgeons (IT, ST, and MW) using spinal anesthesia with 2.4 to 3.2 mL of 0.5% bupivacaine (Marcaine; AstraZeneca). We did not use the pneumatic tourniquet or drain for any patients. Anteromedial straight skin incision of 4 cm proximal to the superior patella to 1 cm distal to the tibial tuberosity was made, and subvastus approach were used in all surgeries. All patients received a cemented, posterior stabilized prosthesis (Scorpio NRG; Stryker Orthopedics, Mahwah, NJ, USA).
The postoperative rehabilitation regimens were the same for both groups and were started from 1 day after surgery in the afternoon.

Unilateral 6-OHDA lesions and adeno-associated virus (AAV) injections were performed as described previously [73 (link)]. In brief, mice were anesthetized with a mixture of 4% isoflurane in air (Isoba vet, Apoteksbolaget) and placed in a stereotaxic frame (Kopf Instruments, Model 923-B mouse Gas Anethesia Head Holder, with 922 Ear Bars for mouse). 6-OHDA hydrochloride was dissolved in 0.02% ascorbic acid (3.2 μg free-base per μL) and 0.7 μL was injected at 0.2 µL/min in the median forebrain bundle at following coordinates: AP = −0.7, ML = −1.2, DV = −4.7, tooth bar: −4.0 [73 (link)]. For marking iSPNs, we injected AAV5-hSyn-DIO-EGFP-WPRE into the striatum at two injection sites (0.5 μL each): AP = +1, ML = −2.1, DV = −2.9; and AP = +0.3, ML = −2.3, DV = −3.0 [73 (link)]. After the surgery, the wound was closed with tissue glue and the animal received an s.c. injection of an analgesic (Marcaine, AstraZeneca; 2.5 mg/mL, 1 µL/g b.w.). To prevent dehydration, mice received s.c. injections of sterile glucose-ringer acetate (0.6 mL) immediately after the surgery. Further injections and dietary supplementations were given as necessary in the days post-lesion. Control mice received a sham-lesion surgery (capillary was inserted without delivering any injection). The success of the lesion was verified by TH-immunohistochemistry after the experiments.

All TKAs were carried out in a unified manner. Surgeries were managed under lumbar analgesia with 2.0 to 2.8 mL of 0.5% bupivacaine (Marcaine; AstraZeneca). One of three surgeons (TI, ST and MW) conducted all surgical procedures. We did not use any pneumatic tourniquets. No drain was applied to any of the patients. We designed an anteromedial straight skin incision that began 4 cm proximal to the patella and ended 1 cm distal to the tibial tuberosity with the knee flexed. We lengthened the skin incision as necessary to adequately expose the knee joint during surgery. A subvastus approach was used for surgical approach. All patients received Scorpio NRG-PS (Stryker Orthopaedics, Mahwah, NJ), a cemented posterior stabilized prosthesis. The postoperative rehabilitation regimens were the same for both groups and started from the afternoon on the day following surgery.

Anesthesia was introduced with isoflurane (Piramal Healthcare, Morpeth Northumberland, UK) 3.5%/O₂ (1.5 L/min) and maintained with 2.0%/O₂. Bupivacain (Marcaine®; AstraZeneca, Copenhagen, Denmark) 2 mg/kg was administered subcutaneously (s.c.) at the incisional site. The preoperative and postoperative analgesia was provided by per os 0.4 mg/kg buprenorphine (Temgesic®; Rickitt Benckiser, Berkshire, UK) mixed in hazelnut butter at 12-hour intervals.