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Oxidative damage and reduction of redox factor-1 expression after transient spinal cord ischemia in rabbits
Masahiro Sakurai1, Tetsuya Nagata, Koji Abe
1Departments of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan.
Objective:
The mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. However, the mechanisms of such vulnerability are not fully understood. We previously reported that spinal motor neurons may be lost by programmed cell death and thus now investigate a possible mechanism of neuronal death with immunohistochemical analysis for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and redox factor-1 (Ref-1).
Methods:
We used a rabbit spinal cord ischemia model with a balloon catheter. The spinal cord was removed at 8 hours, 1, 2, or 7 days after 15 minutes of transient ischemia, and histologic changes were studied with hematoxylin-eosin staining. Western blot analysis for Ref-1, temporal profiles of 8-OHdG and Ref-1 immunoreactivity, and double-label fluorescence immunocytochemical studies were performed.
Results:
Most motor neurons were preserved until 2 days but were selectively lost at 7 days of reperfusion. Western blot analysis of a sample from sham control spinal cord showed a characteristic 37-kDa band that was reduced after ischemia. Immunohistochemistry showed the nuclear expression of Ref-1 in motor neurons of control spinal cords, and immunoreactivity was decreased 1 day after ischemia. On the other hand, no nuclear expression was seen of 8-OHdG in motor neurons of control spinal cords, and immunoreactivity was increased 1 day after ischemia. Double-label fluorescence immunocytochemical study revealed that both 8-OHdG and Ref-1 were positive at 8 hours of reperfusion in the same motor neurons, which eventually die.
Conclusion:
These results suggest that Ref-1 decreased in motor neurons after transient spinal cord ischemia and that this reduction preceded oxidative DNA damage. The reduction of Ref-1 protein at the moderately late stage of reperfusion may be one of the factors responsible for the delay in neuronal death after spinal cord ischemia.
Insights
Spinal cord injury involves motor neuron vulnerability to ischemia. Reduced redox factor-1 (Ref-1) precedes oxidative DNA damage, potentially delaying neuronal death after spinal cord ischemia.
Area of Science:
- Neuroscience
- Cell Biology
- Ischemia Research
Background:
- Spinal cord injury (SCI) mechanisms are linked to motor neuron vulnerability during ischemia.
- Previous research suggests programmed cell death contributes to motor neuron loss in SCI.
- The precise mechanisms underlying this vulnerability remain incompletely understood.
Purpose of the Study:
- To investigate the role of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and redox factor-1 (Ref-1) in motor neuron death following spinal cord ischemia.
- To elucidate the temporal relationship between Ref-1 expression, oxidative DNA damage, and neuronal survival after SCI.
Main Methods:
- A rabbit model of transient spinal cord ischemia was induced using a balloon catheter.
- Histological analysis (hematoxylin-eosin staining) was performed on spinal cord tissue collected at various reperfusion times (8 hours to 7 days).
- Western blot analysis, immunohistochemistry for 8-OHdG and Ref-1, and double-label fluorescence immunocytochemistry were employed to assess protein levels and cellular localization.
Main Results:
- Motor neurons were preserved until 2 days post-ischemia but selectively lost by 7 days.
- Ref-1 nuclear expression in motor neurons decreased one day after ischemia, while 8-OHdG immunoreactivity increased.
- Both 8-OHdG and Ref-1 were detected in the same motor neurons exhibiting eventual cell death at 8 hours of reperfusion.
Conclusions:
- A reduction in Ref-1 protein occurs in motor neurons following transient spinal cord ischemia.
- This decrease in Ref-1 precedes the onset of oxidative DNA damage (8-OHdG accumulation).
- The reduction of Ref-1 may be a contributing factor to the delayed neuronal death observed in spinal cord ischemia.