Fig. 2: AAV.ULK1.DN reduces the number of axonal degeneration bulbs after axotomy in vitro and attenuates acute axonal degeneration after optic nerve crush in vivo.

a Scheme of experimental setup for axonal degeneration assays in microfluidic chambers. DOP day of preparation of embryonic day 18 rat cortical neurons, DIV day in vitro, AAV transduction with adeno-associated viral vectors, MC medium change. b Representative images of axons growing in microfluidic chambers transduced with viral vectors. Exemplary photomicrographs were taken directly before and 360 min after axotomy. Red arrow: example of axonal bulb used for quantification. Dotted line: area of lesion. Scale bar: 50 µm. c, d Detailed quantification of the number of newly formed bulbs within different distances (indicated by colors) proximal to the area of lesion at the indicated time points after axotomy and transduction only with AAV.CTRL (c, n = 4 independent cultures) or AAV.ULK1.DN (d, n = 4 independent cultures). e Quantification of the number of newly formed bulbs within 400 µm distance proximal to the area of lesion at the indicated time points after axotomy and transduction with AAV.CTRL and AAV.ULK1.DN (n = 4 independent cultures). f Scheme of experimental setup for optic nerve crush (ONC) model. DOI day of intravitreal injection of AAV. g Representative images of axons in the optic nerve expressing mCherry after transduction with viral vectors, taken proximal to lesion 5–360 min after axotomy. Scale bar: 20 µm. h Quantification of the axonal integrity ratio (ratio of the sum of the length of remaining axonal fragments to the initial axon length) at the indicated time points after ONC and transduction with AAV.CTRL and AAV.ULK1.DN (n = 3–4 animals for each viral vector). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, according to two-way repeated measurements (RM) ANOVA and Dunnett’s (c, d) or Sidak’s multiple comparison’s test (e, h).