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. Because we previouly reported that spinal motor neurons were lost probably by programmed cell death, and it ha been appreciated that several cell death-inducing pathways are set in motion subsequent to the cellular stress that affects ER, collectively known as ER stress. Therefore, we investigated a possible mechanism of neuronal death by immunohistochemical analysis for Grp78 and caspase12.
Methods
We used a rabbit spinal cord ischemia model with use of a balloon catheter. The spinal cord was removed at 8 hours, 1, 2, or 7 days after 15 min of transient ischemia (n = 5, each time point), and histological changes were studied with hematoxylin-eosin staining. Western blot analysis for Grp78 and caspase12, temporal profiles of Grp78 and caspase12 immunoreactivity, and double-label fluorescence immunocytochemical studies were performed.
Results
The majority of motor neurons were preserved until 2 days, but were selectively lost (about 70%) at 7 days of reperfusion. Western blot analysis revealed scarce immunoreactivity for Grp78 and caspase12 in the sham-operated spinal cords. However, they became apparent at 8 hours after transient ischemia (p < 0.0001), which returned to the baseline level at 1 day (p < 0.0001). Double-label fluorescence immunocytochemical study revealed that both Grp78 and caspase12 were positive at 8 hours of reperfusion in the same motor neurons which eventually die.
Conclusion
This study demonstrated that immunoreactivities for both Grp78 and caspase12 were induced in the same motor neuron which eventually die. These results suggest that endoplasmic reticulum stress were induced in motor neurons by transient spinal cord ischemia in rabbits.