Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons
Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins, representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord. Our recently developed method of in vivo electroporation of...
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Wolters Kluwer Medknow Publications
2020-01-01
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doaj-476613f466394fc6b2850d718673d3a52020-11-25T02:47:40ZengWolters Kluwer Medknow PublicationsNeural Regeneration Research1673-53742020-01-011561160116510.4103/1673-5374.270315Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axonsYan GaoYi-Wen HuRun-Shan DuanShu-Guang YangFeng-Quan ZhouRui-Ying WangMost current studies quantify axon regeneration by immunostaining regeneration-associated proteins, representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord. Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves. A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers. Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury. Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy. At 12 and 18 hours, and 1, 2, 3, 4, 5, and 6 days of injury, lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images. Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay. We found that in vivo electroporation did not affect caspase-3 expression in dorsal root ganglia. Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique. Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing. The results facilitate direct time course analysis of peripheral nerve axon regeneration. This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University, China (approval No. GLMC201503010) on March 7, 2014.http://www.nrronline.org/article.asp?issn=1673-5374;year=2020;volume=15;issue=6;spage=1160;epage=1165;aulast=Gaoaxon regeneration; cell apoptosis; dorsal root ganglion; in vivo electroporation; micrornas; peripheral nervous system; sciatic nerve; tissue clearing |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yan Gao Yi-Wen Hu Run-Shan Duan Shu-Guang Yang Feng-Quan Zhou Rui-Ying Wang |
spellingShingle |
Yan Gao Yi-Wen Hu Run-Shan Duan Shu-Guang Yang Feng-Quan Zhou Rui-Ying Wang Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons Neural Regeneration Research axon regeneration; cell apoptosis; dorsal root ganglion; in vivo electroporation; micrornas; peripheral nervous system; sciatic nerve; tissue clearing |
author_facet |
Yan Gao Yi-Wen Hu Run-Shan Duan Shu-Guang Yang Feng-Quan Zhou Rui-Ying Wang |
author_sort |
Yan Gao |
title |
Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons |
title_short |
Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons |
title_full |
Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons |
title_fullStr |
Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons |
title_full_unstemmed |
Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons |
title_sort |
time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons |
publisher |
Wolters Kluwer Medknow Publications |
series |
Neural Regeneration Research |
issn |
1673-5374 |
publishDate |
2020-01-01 |
description |
Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins, representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord. Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves. A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers. Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury. Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy. At 12 and 18 hours, and 1, 2, 3, 4, 5, and 6 days of injury, lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images. Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay. We found that in vivo electroporation did not affect caspase-3 expression in dorsal root ganglia. Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique. Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing. The results facilitate direct time course analysis of peripheral nerve axon regeneration. This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University, China (approval No. GLMC201503010) on March 7, 2014. |
topic |
axon regeneration; cell apoptosis; dorsal root ganglion; in vivo electroporation; micrornas; peripheral nervous system; sciatic nerve; tissue clearing |
url |
http://www.nrronline.org/article.asp?issn=1673-5374;year=2020;volume=15;issue=6;spage=1160;epage=1165;aulast=Gao |
work_keys_str_mv |
AT yangao timecourseanalysisofsensoryaxonregenerationinvivobydirectlytracingregeneratingaxons AT yiwenhu timecourseanalysisofsensoryaxonregenerationinvivobydirectlytracingregeneratingaxons AT runshanduan timecourseanalysisofsensoryaxonregenerationinvivobydirectlytracingregeneratingaxons AT shuguangyang timecourseanalysisofsensoryaxonregenerationinvivobydirectlytracingregeneratingaxons AT fengquanzhou timecourseanalysisofsensoryaxonregenerationinvivobydirectlytracingregeneratingaxons AT ruiyingwang timecourseanalysisofsensoryaxonregenerationinvivobydirectlytracingregeneratingaxons |
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