Retrograde labeling

Retrograde labeling [8] of brainstem neurons was performed on 23 mice (uninfected = 3, rHIgM12 = 9, saline = 11) at 9 weeks after antibody treatment. Briefly, dorsal laminectomy of the lower thoracic vertebrae was performed on anesthetized mice. The spinal cord was hemisected on the right side using microdissecting blades. The retrograde tracer, Fluoro-Gold, was applied and the surgery site was closed. One week post-surgery (i.e., 10 weeks post-treatment), mice were terminally anesthetized, perfused with 4 % paraformaldehyde, and brains and spinal cords were collected. Serial vibratome (Lancer Series 1200) sections (40 μm thick) of the brainstem were collected and mounted under Vectashield (Vector Laboratories Inc., Burlingame, CA). Cell bodies containing retrograde tracer (Fluoro-Gold), visualized by UV illumination (360–370-nm excitation, 420–460-nm emission) were counted at 200× from 16 brainstem slices per mouse. A cell was counted as positive if a large cross-section of the cell body was labeled with Fluoro-Gold. All analyses were performed without knowledge of the experimental groups.