Objective:To discover a novel and intuitive anterograde tracing method for observing the course of sensory nerve fibers in dorsal root ganglia (DRG).Methods:From July 2022 to August 2023, studies were conducted in the Department of Plastic Surgery of the First Affiliated Hospital of Sun Yat-sen University. Three healthy 1-month old female SD rats, weighed approximately 180-220 g, were randomly selected. An adeno-associated virus carrying a neuron-specific promoter and red fluorescent protein (AAV- syp-mCherry) was prepared. The virus was injected into the spinal roots outside L 4-L 6 DRG through sterile surgery. After 1 month of breeding, L 4-L 6 spinal cord tissue of immunofluorescent staining of the side injected with viral tracer, DRG, sciatic nerve, and plantar skin of the SD rats were collected for immunofluorescent staining of neurofilament protein (NF) and microtubule associated protein 2 (MAP2). Distribution characteristics of AAV-positive nerve fibers were observed using a confocal microscope by average optical density (AOD) . Results:One month after injection of the viral tracer, red markers were visible in the dorsal horn of the L 4-L 6 spinal cord (AOD=0.5419±0.0245), DRG (AOD=0.4917±0.0087), sciatic nerve (AOD=0.6237±0.0634), dermis of plantar skin and skin appendages on the injected side (AOD=0.4943±0.0165). The red fluorescent clusters were found co-labeled with NF-positive nerve fibers or MAP2-positive neurons, it suggested that AAV- syp-mCherry could be transported anterogradely within sensory axons to distant target organs (skin) and offered a long-term image. Conclusion:The anterograde tracing method by adeno-associated virus can effectively show the complete trace of the central and peripheral processes of primary sensory neurons, and can serve as an effective methodology in observation of the pathways of peripheral sensory nerve fibers.

Objective:To discover a novel and intuitive anterograde tracing method for observing the course of sensory nerve fibers in dorsal root ganglia (DRG).Methods:From July 2022 to August 2023, studies were conducted in the Department of Plastic Surgery of the First Affiliated Hospital of Sun Yat-sen University. Three healthy 1-month old female SD rats, weighed approximately 180-220 g, were randomly selected. An adeno-associated virus carrying a neuron-specific promoter and red fluorescent protein (AAV- syp-mCherry) was prepared. The virus was injected into the spinal roots outside L 4-L 6 DRG through sterile surgery. After 1 month of breeding, L 4-L 6 spinal cord tissue of immunofluorescent staining of the side injected with viral tracer, DRG, sciatic nerve, and plantar skin of the SD rats were collected for immunofluorescent staining of neurofilament protein (NF) and microtubule associated protein 2 (MAP2). Distribution characteristics of AAV-positive nerve fibers were observed using a confocal microscope by average optical density (AOD) . Results:One month after injection of the viral tracer, red markers were visible in the dorsal horn of the L 4-L 6 spinal cord (AOD=0.5419±0.0245), DRG (AOD=0.4917±0.0087), sciatic nerve (AOD=0.6237±0.0634), dermis of plantar skin and skin appendages on the injected side (AOD=0.4943±0.0165). The red fluorescent clusters were found co-labeled with NF-positive nerve fibers or MAP2-positive neurons, it suggested that AAV- syp-mCherry could be transported anterogradely within sensory axons to distant target organs (skin) and offered a long-term image. Conclusion:The anterograde tracing method by adeno-associated virus can effectively show the complete trace of the central and peripheral processes of primary sensory neurons, and can serve as an effective methodology in observation of the pathways of peripheral sensory nerve fibers.