Autophagy-mediated stress response in motor neuron after transient ischemia in rabbits

Histological findings in the spinal cord after 15 minutes of ischemia stained with hematoxylin-eosin (HE). The spinal cord of the sham group (A) and the ischemia group at 2 days (2d) after ischemia (B) showed no histologic changes. At 7 days (7d) after ischemia (C), there was selective loss of motor neurons in the ischemia group, without apparent gliosis or cellular infiltration. Bar = 50 μm.

More than 70% of the motor neurons in the spinal cord were damaged at 7 days (7d) after reperfusion. 2d, 2 days. *P < .001 compared with sham group.

Representative Western blot for LC3 and GABARAP. A, LC3-I is cytosolic form of LC3 and LC3-II is a lipidated form of LC3. Immunoreactivity was only weakly (LC3 and GABARAP) detected in the sham group. In the ischemia group, strong bands for LC3 and GABARAP were observed at the expected sizes (16kD and 14kD, respectively) at 8 hours (8h) after reperfusion, and LC3 expression was preserved until 2 days (2d) after reperfusion. There was no change in actin expression in either group. B, Quantitative analysis showed that 15 minutes ischemia significantly increased LC3 and GABARAP expression at 8 hours after reperfusion. S, sham; 1d, 1 day. *P < .01. **P < .001 compared with sham group.

Immunostaining against LC3 and GABARAP in motor neurons in a sham group spinal cord (A, E) and in an ischemia group spinal cord at 8 hours (8h) (B, F), 1 day (1d) (C, G) and 2 day (2d) (D, H) after reperfusion. Arrows show motor neurons that express immunoreactive LC3 (B, C, and D) and GABARAP (F, G), respectively. Bar = 100 μm.

Co-localization of LC3 and GABARAP in motor neurons at 8 hours after ischemia. LC3 was detected by green-fluorescent Alexa Fluor 488 (green) (A), and GABARAP by orange-fluorescent Alexa Fluor 555 (red) (B). The merged image is shown in (C) with double positive (arrow) as yellow color. Bar = 20 μm.