Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3-7 days after injection into the rat gastrocnemius muscle

Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B(AF488-CTB).This was injected into the gastrocnemius muscle of rats,and it was found that motor,sensory,and sympathetic neurons were labeled in the spinal ventral horn,dorsal root ganglia,and sympathetic chain,respectively.Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle.The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection.It was found that labeled motor and sensory neurons could be observed 12 hours post-injection.The intensity was found to increase over time,and the morphology appeared clear and complete 3-7 days post-injection,with clearly distinguishable motor neuron axons and dendrites.However,14 days after the injection,the quality of the images decreased and the neurons appeared blurred and incomplete.Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features,and the surrounding microglia were also found to be unaltered.Overall,these results imply that the cholera toxin subunit B,whether unconjugated or conjugated with Alexa Fluor,is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.

大鼠“承山”穴和坐骨神经之间的神经解剖学联系

Objective:The aim of this study is to assess the neural interconnection between the acupoint"Chéngshān(承山BL57)"and sciatic nerve from the peripheral nervous system(PNS)to the central nervous system(CNS).Methods:SD rats were systematically examined from the hip to the hind limb along the route of sciatic nerve with regional anatomical technique,and further traced from the BL57 and the trunk of sciatic nerve by the neural tracing technique with cholera toxin subunit B(CTB)and CTB conjugated Alexa Fluor 488 and 594(CTB-AF488/594).Results:After regional anatomy,it was found that the sciatic nerve ran down from the hip to the hind limb and sequentially sent out the tibial and sural branches respectively to the deep and superficial layers of BL57.The CTB labeling associated with both BL57 and sciatic nerve distributed in the same spinal segments and definite regions,including the sensory neurons in lumber 3(L3)-lumber 6(L6)dorsal root ganglia(DRGs),transganglionic axons mainly in the L3-L6 spinal dorsal horn,Clarke’s nucleus,and gracile nucleus,as well as motor neurons mainly in the L3-L6 spinal ventral horn.However,the amount of neural labeling was significantly less in the cases of BL57 than that of sciatic nerve.Conclusions:These results indicate that there is distinct sensory and motor interconnection between the BL57 and the sciatic nerve,which may potentially serve for the active role of the BL57 playing in meliorating the disorders of sciatic nerve.