Fig. 4: AAV.ULK1.DN attenuates axonal degeneration and decreases the number of axonal LC3 puncta in a model of spinal cord injury in vivo.

a Scheme of experimental setup for spinal cord injury (SCI) model. DOI day of injection, RN red nucleus, RST rubrospinal tract, L left, R right. b Exemplary horizontal sections of thoracic spinal cord transduced with AAV.CTRL and AAV.ULK1.DN after SCI depict areas of lesion and RST up to 2000 µm rostral to lesion. White dashed lines and asterisks indicate lesion areas. White dashed boxes indicate areas shown at higher magnification in c. Scale bar: 200 µm. c Higher magnification of areas indicated in b. Red arrows show formation of bulbs in fragmented axons after lesion. Green arrows indicate intact axons after lesion. Scale bar: 100 µm. d Quantification of axon number index (a ratio of nonfragmented (intact) axon numbers to the total number of transduced axons) at the indicated distances from lesion after transduction with AAV.CTRL and AAV.ULK1.DN (n = 4–5 animals for each viral vector). e, f Exemplary horizontal sections of thoracic spinal cord showing the RST transduced with AAV.CTRL and AAV.ULK1.DN rostral to lesion. mCherry fluorescence is shown in red, immunohistochemical staining against LC3 appears in white. e White asterisks indicate lesion. Green arrows indicate axonal bulbs stained for LC3. Scale bar: 400 µm. f Higher magnification of axonal bulbs at a distance of 500–1000 µm rostral to the lesion. Green arrows indicate LC3-positive puncta in axonal bulbs. Scale bar: 10 µm. g Quantification of the number of axonal LC3 puncta/µm² at a distance of 500–1000 µm rostral to the lesion after transduction with AAV.CTRL and AAV.ULK1.DN (n = 3 animals for each viral vector). Data are presented as single data points and means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, according to two-tailed unpaired t-test.