2.4. Compression/Contusion Injury, Transplantation of NPCs, and Growth Factor Application

We used a contusion/compression model with a 28-g modified aneurysm clip (Fehlings Laboratory, Canada) to induce SCI at the C6 level as previously described [30]. For all surgical procedures, animals were anesthetized with 1.5% isoflurane and a 1 : 1 mixture of O₂ and N₂O. Postoperatively, animals were subjected to intensive care and received buprenorphine (0.05 mg/kg s.c.; Bayer, Germany) as well as meloxicam (2 mg/kg s.c.; Boehringer-Ingelheim, Germany) for 3–5 days. Fluids and nutritional support were administered to all injured animals. An antibiotic prophylaxis (moxifloxacin, 4 mg/kg p.o.; Alcon, USA) was given for 7 days, and bladders were manually squeezed three times per day until bladder reflexive function had recovered. Animals were housed in a 12 h light-dark cycle at 26°C with food and water ad libitum.

For transplantation of NPCs, animals were anesthetized as described above, and the dura at the C6 level was microsurgically reexposed. 4 × 10⁵ NPCs diluted in 8 μl growth medium were injected into the spinal cord at four sites (i.e., 2 μl per site), bilaterally 2 mm rostral and caudal to the epicenter of the lesion at a depth of 1.5 mm, using a stereotactic injector with a Hamilton syringe (Hamilton Company, Switzerland) and 35 g microneedle (World Precision Instruments, Germany) at a rate of 5 nl/s. Animals in group 2 (vehicle) received the same amount of growth medium without NPCs. During the same surgery, a T1 laminectomy was performed and a rat intrathecal microcatheter (Alzet, USA), connected to a subcutaneous osmotic micropump (Alzet, USA) was subdurally placed with its open end over the epicenter of the lesion. This pump was used for continuous intrathecal administration of the most promising growth factor combination tested in the in vitro part of our study, with a concentration adapted to the in vivo use (30 μg/ml EGF, 30 μg/ml bFGF, and 1 μg/ml PDGF-AA) in all injured animals for 7 days.