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 ex...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.展开更多
Objective： To investigate the biochemical characteristic of the neurons associated Zusanli （ST 36） in the rat by using Alexa Fluor 594 conjugated cholera toxin subunit B （AF594-CTB） neural tracing and calcitonin ...Objective： To investigate the biochemical characteristic of the neurons associated Zusanli （ST 36） in the rat by using Alexa Fluor 594 conjugated cholera toxin subunit B （AF594-CTB） neural tracing and calcitonin gene-related peptide （CGRP） fluorescent immunohistochemical techniques. Methods： Four male Sprague Dawley rats were injected with AF594-CTB into the corresponding area of the Zusanli in the human body. After 3 surviving days, the rat＇s spinal cord and dorsal root ganglia （DRGs） at lumbar segments were dissected following perfusion with 4% paraformaldehyde, cut into sections, and then stained with CGRP- fluorescent immunohistochemical method. Results： AF594-CTB labeled sensory neurons were detected in the L3-L6 DRGs with high concentration in L4 DRG, and the labeled motor neurons located in the dorsolateral and intermediate regions of lamina IX from L3-L5 segments with high concentration at L4. Meanwhile, CGRP- positive neural labeling distributed symmetrically on both sides of DRGs, anterior and dorsal horns of spinal cord. In the AF594-CTB labeled neurons, 37% sensory neurons and 100% motor neurons expressed CGRP- positive. Conclusion： These findings present the morphological evidence to demonstrate that the sensory and motor neurons associated Zusanli in the rat distributed with segmental and regional patterns, and contained CGRP-expression.展开更多
基金supported by the CACMS Innovation Fund,No.CI2021A03407(to WZB)the Project of National Key R&D Program of China,No.2019YFC1709103(to WZB)+1 种基金the National Natural Science Foundation of China,Nos.81774432(to JJC),81774211(to WZB),82004492(to JW),81801561(to DSX)the Fundamental Research Funds for the Central Public Welfare Research Institutes of China,Nos.ZZ13-YQ-068(to JJC),ZZ14-YQ-032(to JW),ZZ14-YQ-034(to DSX).
文摘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.
基金Supported by the National Natural Science Foundation of China(No.81072759)the National Basic Research Program of China(973 program,No.2011 CB505201)the Self-selected Research Program from China Academy of Chinese Medical Sciences(No.2009Z02024)
文摘Objective： To investigate the biochemical characteristic of the neurons associated Zusanli （ST 36） in the rat by using Alexa Fluor 594 conjugated cholera toxin subunit B （AF594-CTB） neural tracing and calcitonin gene-related peptide （CGRP） fluorescent immunohistochemical techniques. Methods： Four male Sprague Dawley rats were injected with AF594-CTB into the corresponding area of the Zusanli in the human body. After 3 surviving days, the rat＇s spinal cord and dorsal root ganglia （DRGs） at lumbar segments were dissected following perfusion with 4% paraformaldehyde, cut into sections, and then stained with CGRP- fluorescent immunohistochemical method. Results： AF594-CTB labeled sensory neurons were detected in the L3-L6 DRGs with high concentration in L4 DRG, and the labeled motor neurons located in the dorsolateral and intermediate regions of lamina IX from L3-L5 segments with high concentration at L4. Meanwhile, CGRP- positive neural labeling distributed symmetrically on both sides of DRGs, anterior and dorsal horns of spinal cord. In the AF594-CTB labeled neurons, 37% sensory neurons and 100% motor neurons expressed CGRP- positive. Conclusion： These findings present the morphological evidence to demonstrate that the sensory and motor neurons associated Zusanli in the rat distributed with segmental and regional patterns, and contained CGRP-expression.
