Objective To explore the innervation and biochemical characteristics associated with acupoint"Sanyinjiao"(SP 6)in the rat by using neural tracing and immunofluorescence techniques.Methods Five male SD rats were used in the present study.Alexa Fluor 488 conjugated cholera toxin subunit B(AF 488-CTB

5L

1%)was injected into the site superior to the prominence of the medial malleolus

a corresponding site to acupoint SP 6in the human body.After 3surviving days

the rat's local tissue of SP 6region

spinal cord and dorsal root ganglia(DRGs)at lumbar(L)segments(L 1-S 1)were dissected following perfusion with 4% paraformaldehyde

cut into sections(20μm for DRG and SP 6

and 40μm for spinal cord)and observed under light microscope.In addition

substance P(SP)immunoreaction in the spinal cord

DRG and SP 6region tissues was examined with immunofluorescence.Results All the labeling of AF 488-CTB appeared on the ipsilateral side of the injection.The labeled sensory and motor neurons distributed in L 3-L 6DRGs and spinal ventral horns with high concentration in the L 4and L 5segments.In addition

SP-positive neural labeling was found in small sensory neurons in DRG

axonal terminals in laminaⅠ-Ⅱof spinal dorsal horns

and free nerve fibers in local subcutaneous tissues.Additionally

24.1% of AF 488-CTB labeled sensory neurons simultaneously presented a SP-positive expression.Conclusion These findings reveal that the sensory and motor neurons associated with acupoint SP 6distribute in L 3-L 6DRGs and spinal ventral horns of rats

concentrating in the L 4and L 5segments

and 24.1% sensory neurons show SP-positive

suggesting apossible involvement of the primary SP-positive sensory neurons in the transmission of acupuncture stimulation signals.