TY - JOUR
T1 - Isolation of mature spinal motor neurons and single-cell analysis using the comet assay of early low-level DNA damage induced in vitro and in vivo
AU - Liu, Z.
AU - Martin, L. J.
PY - 2001
Y1 - 2001
N2 - We developed an isolation technique for motor neurons from adult rat spinal cord. Spinal cord enlargements were discretely microdissected into ventral horn tissue columns that were trypsin-digested and subjected to differential low-speed centrifugation to fractionate ventral horn cell types. A fraction enriched in α-motor neurons was isolated. Motor neuron enrichment was verified by immunofluorescence for choline acetyltransferase and prelabeling axon projections to skeletal muscle. Adult motor neurons were isolated from naïve rats and were exposed to oxidative agents or were isolated from rats with sciatic nerve lesions (avulsions). We tested the hypothesis, using single-cell gel electrophoresis (comet assay), that hydrogen peroxide, nitric oxide, and peroxynitrite exposure in vitro and axotomy in vivo induce DNA damage in adult motor neurons early during their degeneration. This study contributes three important developments in the study of motor neurons. It demonstrates that mature spinal motor neurons can be isolated and used for in vitro models of motor neuron degeneration. It shows that adult motor neurons can be isolated from in vivo models of motor neuron degeneration and evaluated on a single-cell basis. This study also demonstrates that the comet assay is a feasible method for measuring DNA damage in individual motor neurons. Using these methods, we conclude that motor neurons undergoing oxidative stress from reactive oxygen species and axotomy accumulate DNA damage early in their degeneration.
AB - We developed an isolation technique for motor neurons from adult rat spinal cord. Spinal cord enlargements were discretely microdissected into ventral horn tissue columns that were trypsin-digested and subjected to differential low-speed centrifugation to fractionate ventral horn cell types. A fraction enriched in α-motor neurons was isolated. Motor neuron enrichment was verified by immunofluorescence for choline acetyltransferase and prelabeling axon projections to skeletal muscle. Adult motor neurons were isolated from naïve rats and were exposed to oxidative agents or were isolated from rats with sciatic nerve lesions (avulsions). We tested the hypothesis, using single-cell gel electrophoresis (comet assay), that hydrogen peroxide, nitric oxide, and peroxynitrite exposure in vitro and axotomy in vivo induce DNA damage in adult motor neurons early during their degeneration. This study contributes three important developments in the study of motor neurons. It demonstrates that mature spinal motor neurons can be isolated and used for in vitro models of motor neuron degeneration. It shows that adult motor neurons can be isolated from in vivo models of motor neuron degeneration and evaluated on a single-cell basis. This study also demonstrates that the comet assay is a feasible method for measuring DNA damage in individual motor neurons. Using these methods, we conclude that motor neurons undergoing oxidative stress from reactive oxygen species and axotomy accumulate DNA damage early in their degeneration.
KW - Amyotrophic lateral sclerosis
KW - Apoptosis
KW - Axotomy
KW - DNA single-strand breaks
KW - Single-cell gel electrophoresis
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U2 - 10.1177/002215540104900804
DO - 10.1177/002215540104900804
M3 - Article
C2 - 11457924
AN - SCOPUS:0034927126
SN - 0022-1554
VL - 49
SP - 957
EP - 972
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
IS - 8
ER -