Studying Axonal Outgrowth and Regeneration of the Corticospinal Tract in Organotypic Slice Cultures.
Studies of axonal outgrowth and regeneration after spinal cord injury are hampered by the complexity of the events involved. Here, we present a simple and improved in vitro approach to investigate outgrowth, regeneration of the corticospinal tract, and intrinsic parenchymal responses. We prepared organotypic co-cultures using explants from the motor cortex of postnatal donor mice ubiquitously expressing green fluorescent protein and cervical spinal cord from wild type pups of the same age. Our data show that: a) motor-cortical outgrowth is already detectable after 1 d in culture and is source specific; b) treatment with neurotrophin-3 and C3 transferase from Clostridium botulinum significantly enhances axonal outgrowth during the course of cultivation; c) outgrowing axons form synaptic connections, as demonstrated by immunohistochemistry and calcium imaging; and d) migrating cells of motor-cortical origin can be reliably identified without previous tracing and are mostly neural precursors that survive and mature in the spinal cord parenchyma. Thus, our model is suitable for screening for candidate substances that enhance outgrowth and regeneration of the corticospinal tract and for studying the role of endogenous neural precursors after lesion induction.
1 Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin , Berlin, Germany .
Martin
M
Pohland
2 NeuroCure Clinical Research Center and Clinical and Experimental Research Center for Multiple Sclerosis, Charité-Universitätsmedizin Berlin , Berlin, Germany .
Robert
R
Glumm
1 Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin , Berlin, Germany .
Luminita
L
Stoenica
3 Institute for Integrative Neuroanatomy, Charité-Universitätsmedizin Berlin , Berlin, Germany .
Markus
M
Höltje
4 Department of Neurosurgery, HELIOS Klinikum Berlin Buch , Berlin, Germany .
Jürgen
J
Kiwit
3 Institute for Integrative Neuroanatomy, Charité-Universitätsmedizin Berlin , Berlin, Germany .
Gudrun
G
Ahnert-Hilger
1 Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin , Berlin, Germany .
Ulf
U
Strauss
1 Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin , Berlin, Germany .
Anja U
AU
Bräuer
2 NeuroCure Clinical Research Center and Clinical and Experimental Research Center for Multiple Sclerosis, Charité-Universitätsmedizin Berlin , Berlin, Germany .
Friedemann
F
Paul
1 Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin , Berlin, Germany .|4 Department of Neurosurgery, HELIOS Klinikum Berlin Buch , Berlin, Germany .
Jana
J
Glumm
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000199:Actins / Q000235:genetics; D000818:Animals; D001369:Axons / Q000187:drug effects*; D001905:Botulinum Toxins / Q000494:pharmacology; D002465:Cell Movement; D002540:Cerebral Cortex / Q000166:cytology / Q000254:growth & development; D049452:Green Fluorescent Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009044:Motor Cortex / Q000254:growth & development; D009414:Nerve Growth Factors / Q000494:pharmacology; D009416:Nerve Regeneration / Q000187:drug effects*; D009924:Organ Culture Techniques; D011712:Pyramidal Tracts / Q000166:cytology / Q000254:growth & development*; D013116:Spinal Cord / Q000254:growth & development; D013569:Synapses / Q000187:drug effects
D000199:Actins; D009414:Nerve Growth Factors; C518911:neurotropin 3, mouse; D049452:Green Fluorescent Proteins; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins
GFP; axonal outgrowth; motor cortex; organotypic slice cultures; spinal cord