Apoptosis of motor neurons with induction of caspases in the spinal cord after ischemia.
OBJECTIVE
Some neuronal subpopulations are especially vulnerable to ischemic injury. In the spinal cord, large motor neurons are vulnerable to ischemia and are selectively lost after transient ischemia. However, the mechanisms of the neuronal loss have been uncertain. We hypothesized that spinal motor neurons might be lost by apoptosis and investigated a possible mechanism of neuronal death by detection of double-strand breaks in genomic DNA and immunohistochemical analysis for caspases, ie, interleukin-1beta converting enzyme (ICE), Nedd-2, and CPP32.


METHODS
We used a rabbit spinal cord ischemia model created with a balloon catheter. The spinal cord was removed at 8 hours, 1, 2, or 7 days after 15 minutes of transient ischemia, and histological changes were studied with hematoxylin-eosin staining. To detect double-strand breaks in DNA, a staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed. Furthermore, expression of ICE, Nedd-2, and CPP32 was investigated by Western blotting and immunohistochemical analysis.


RESULTS
Motor neurons were selectively lost at 7 days after transient ischemia. TUNEL study demonstrated that no cells were positively labeled until 1 day after ischemia, but nuclei of some motor neurons were positively labeled at 2 days. Western blot analysis revealed no immunoreactivity for ICE and slight immunoreactivities for Nedd-2 and CPP32 in the sham-operated spinal cords. However, immunoreactivity became apparent at 8 hours after transient ischemia, decreased at 1 day, and returned to baseline level at 2 days. Immunohistochemical analysis demonstrated that motor neurons were responsible for induction of those caspases.


CONCLUSIONS
Double-strand breaks in genomic DNA and induction of three caspases were demonstrated. These results indicate that motor neuron death in the spinal cord after transient ischemia is profoundly associated with activation of apoptotic processes.
Hayashi
T
T
Department of Neurology, Tohoku University School of Medicine, Sendai, Japan. hayashi@neurol.med.tohoku.ac.jp
Sakurai
M
M
Abe
K
K
Sadahiro
M
M
Tabayashi
K
K
Itoyama
Y
Y
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D020170:Caspase 1; D053143:Caspase 2; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D053938:DNA Fragmentation; D004790:Enzyme Induction; D005821:Genetic Techniques; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007511:Ischemia / Q000503:physiopathology*; D009046:Motor Neurons / Q000737:chemistry / Q000201:enzymology / Q000473:pathology*; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D013116:Spinal Cord / Q000737:chemistry / Q000201:enzymology* / Q000503:physiopathology
D011506:Proteins; D004247:DNA; D053143:Caspase 2; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1