Additional file 2 of Brain injury drives optic glioma formation through neuron-glia signaling
Creators: name: Chatterjee, Jit
name: Koleske, Joshua P.
name: Chao, Astoria
name: Sauerbeck, Andrew D.
name: Chen, Ji-Kang
name: Qi, Xuanhe
name: Ouyang, Megan
name: Boggs, Lucy G.
name: Idate, Rujuta
name: Marco Y Marquez, Lara Isabel
name: Kummer, Terrence T.
name: Gutmann, David H.
Publisher: name: figshare
Year: 2024
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 2. Supplementary Figures. Fig. S1. Following unilateral optic nerve crush (u-ON-CR) performed at 6 weeks of age, the prechiasmatic region of Nf1flox/flox; hGFAP-Cre mice ipsilateral to the u-ON-CR exhibits (a) greater cellularity and (b) GFAP expression at 12 weeks of age. (c) Optic gliomas persist at 24 weeks of age in Nf1flox/flox; hGFAP-Cre mice following optic nerve crush performed at 6 weeks of age, as evidenced by increased (d) optic nerve volume and (e) proliferation (%Ki67+ cells) (n=5). (f) Following optic nerve crush (ON-CR) at 6 weeks of age, wild type (Nf1flox/flox) mice exhibit no change in (g) optic nerve volume (n = 5) or (h) proliferation (%Ki67+ cells; sham, n = 7; ON-CR, n = 6) when analyzed at 12 weeks of age. Scale bars: a, b Upper panel scale bar, 200 μm; lower panel scale bar, 50 μm d, g 100 μm; e, h 50µm. Two-tailed Student’s t test (ns, not significant). Asterisks denote the age at euthanasia and analysis. Fig. S2. Representative images of Nf1OPG mouse optic nerves following ON-CR at 6 weeks of age have increased (a) cellularity and (b) GFAP expression relative to the sham operation group. Optic nerves from Nf1f/R1809C; hGFAP-Cre mice following optic nerve crush at 6 weeks of age have increased (c) cellularity and (d) GFAP expression compared to the sham operation group when analyzed at 12 weeks of age. Scale bars: a-d upper panel scale bar, 200 μm; lower panel scale bar, 50 μm. Fig. S3. Following ON-CR at 6 weeks of age, RNAscope reveals increased numbers of (a) Tmem119+ cells and (b) Ccl5+ cells in the optic nerves of Nf1flox/flox; hGFAP-Cre mice at 12 weeks of age. PLX3397 (275mg/kg PLX) treatment reduces ON-CR-induced increases (c) in Blbp+ and Olig2+ cell content (%Blbp+, %Olig2+ cells; n=5), as well as (d) Ccl5 expression, in the optic nerves at 12 weeks of age relative to those fed the control (CTL) diet (n=3). (e) TAMs (Iba1+ cells) in 12-week-old Nf1flox/flox; hGFAP-Cre mice express p65-NFκB following ON-CR at 6 weeks of age. Nf1flox/flox; hGFAP-Cre mice that underwent ON-CR at 6 weeks of age exhibit reduced Blbp+ and Olig2+ (%Blbp+, %Olig2+ cells; n=5) cell content at 12 weeks of age following (f) Caffeic acid phenethyl (10mg/kg CAPE, NFκB inhibitor, NFκB-IN) treatment relative to PBS-treated controls. Data are presented as the means ± SEM. Scale bars: a, b, e, f, g 50 μm; Two-tailed Student’s t test. Fig. S4. (a) Analysis of differential gene expression by bulk RNA sequencing of optic nerves from 12-week-old Nf1flox/flox; hGFAP-Cre mice following optic nerve crush (ON-CR; n=3) or sham surgery (n=3) at 6 weeks of age. (b) R studio version 2023.03.0 was used to identify enriched pathways and to calculate the mean and p-value for ON-CR relative to sham mouse groups, as well as to determine the FDR for each shared pathway. The 40 most significant pathways were identified using the mean FDR ≤ 0.05, and log fold changes greater or equal to ±5. (c, d) αIL1β treatment after ON-CR at 6 weeks of age results in reduced optic nerve Blbp+ and Olig2+ cell content in Nf1flox/flox; hGFAP-Cre mice at 12 weeks of age compared to IgG controls (%Blbp+, %Olig2+ cells; n=5). Data are presented as the means ± SEM. Scale bars: c, d 50μm; Two-tailed Student’s t test. Fig. S5. (a) Increased β-APP and SMI-32 expression is seen in the optic nerves of Nf1flox/flox; hGFAP-Cre mice 7 days after TBI (modCHIMERA). Following TBI at 6 weeks of age, increased (b) optic nerve cellularity and (c) GFAP expression is observed by immunohistochemistry in Nf1flox/flox; hGFAP-Cre mice at 12 weeks of age relative to sham controls. Immunofluorescence reveals increased (d) %Blbp+ (n = 5) and (e) %Olig2+ (n = 5) cells in Nf1flox/flox; hGFAP-Cre mice at 3 months of age following TBI at 6 weeks of age compared to sham injury controls. Data are presented as the means ± SEM. Scale bars: a 50 μm, b, c upper panel bar, 200 μm; lower panel scale bar, 50 μm, d, e 40μm. Two-tailed Student’s t test.