Tracing Brainstem Connections in Spinal Cord Injury

Three weeks after injury, animals were anesthetized as described in cervical injuries and subjected to intra-brainstem injections of AAV2-mCherry, as described previously (Goulao et al., 2019 (link)). A midline incision was made at the base of the cranium using a sterile #11 blade. After deflection of the muscle and the C1/cranium ligament, the bone covering a portion of the brainstem was removed. Using a Hamilton Gastight Syringe (Hamilton, Reno, NV, United States) with a 33-gauge needle, 0.3 μl of virus was injected 2 mm lateral to the right (ipsilateral rVRG tracing), 1 mm rostral and 2.6 mm ventral to the brainstem obex, using a stereotaxic apparatus (Kopf Instruments, Tujunga, CA, United States) and an UltraMicroPump (World Precision Instruments, Sarasota, FL, United States). The needle was left in place for 5 min before careful retrieval from the medulla. Postoperative care was given as described for C2 hemisection injuries. Spinal cord sections were later analyzed for mCherry labeling using a Zeiss Imager M2 upright microscope with Metamorph Software.