Additional file 1 of Complement C1q-mediated microglial synaptic elimination by enhancing desialylation underlies sevoflurane-induced developmental neurotoxicity
Creators: name: Wang, Gang
name: Liu, Hua-yue
name: Meng, Xiao-wen
name: Chen, Ying
name: Zhao, Wei-ming
name: Li, Wen-ting
name: Xu, Han-bing
name: Peng, Ke
name: Ji, Fu-hai
Publisher: name: figshare
Year: 2024
Subjects: subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Mental Health
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Fig. S1. Neonatal sevoflurane exposures induced activation and phagocytosis of microglia in the mouse hippocampus. (A) Representative confocal images of PSD95+ puncta (green), GFAP+ astrocytes (gray), and IBA1+ microglia (red) in the hippocampus of sevoflurane-treated mice. Scale bars = 20 μm. (B) Quantification analysis showed that the sevoflurane group had increased PSD95+ puncta in microglia. n = 6. Unpaired t-test. (C) Representative confocal images of the fluorescent latex beads phagocyted by BV2 cells in two groups. Scale bars = 5 μm. (D) Representative confocal images of IBA1+ microglia in the two groups. Scale bars = 20 μm. (E) Quantification analysis showed that sevoflurane led to increased IBA1 intensity. n = 6. Unpaired t-test. (F) Representative confocal images showing the morphology of IBA1+ microglia in two groups. Scale bars = 5 μm. (G) Quantification analysis showed that sevoflurane led to increased soma size and reduced total branch points and total process length. n = 20. Unpaired t-test. Fig. S2. Inhibition of microglial phagocytosis with minocycline reduced synapse loss after neonatal sevoflurane exposures. (A) Representative confocal images of IBA1+ microglia (red) containing PSD95+ puncta (green) in control and sevoflurane-treated mice with PBS or minocycline treatment. Orthographic view and 3D rendering are shown. Scale bars = 5 μm. (B) Quantification analysis showed that minocycline had no impact on the control mice and decreased the engulfment index compared to PBS in the sevoflurane-treated mice. n = 20. One-way ANOVA followed by a post hoc Tukey’s test. (C) Representative confocal images of Vglut2 (green) and PSD95 (red) in the four groups. Scale bars = 5 μm. (D) Quantification analysis showed that minocycline had no impact on the control mice and increased synapse density compared to PBS in the sevoflurane-treated mice. n = 6. One-way ANOVA followed by a post hoc Tukey’s test. Fig. S3. Sevoflurane increased C1q expression in BV2 cells. (A) Representative Western blot bands of C1q in BV2 cells in the control and sevoflurane groups. (B) Quantification of Western blot showed that the expression of C1q was increased in sevoflurane-treated BV2 cells. n = 3. Unpaired t-test. Fig. S4. C1q was mainly expressed in microglia in the hippocampus of mice exposed to neonatal sevoflurane. (A) Representative confocal images of GFAP+ astrocytes (red), IBA1+ microglia (red), and C1q (green) in the hippocampus of sevoflurane-treated mice. Scale bars = 20 μm. (B) Colocalization area analysis revealed that C1q was mainly expressed in microglia. n = 6. Unpaired t-test. Fig. S5. Sevoflurane increased the protein expression of C3, while inhibition of C1q decreased the levels of C3 in sevoflurane-treated mice. (A) Representative Western blot bands of C3 in the hippocampus of control and sevoflurane-treated mice. (B) Quantification of Western blot showed that the expression of C3 was increased in the hippocampus of sevoflurane-treated mice. n = 3. Unpaired t-test. (C) Representative Western blot bands of C3 in the hippocampus of control and sevoflurane-treated mice with IgG or anti-C1q (ANX005) treatment. (D) Compared to treatment with sevoflurane and IgG, ANX005 downregulated C3 expression. n = 3. One-way ANOVA followed by a post hoc Tukey’s test. Fig. S6. Inhibition of C1q by ANX005 decreased the phagocytosis of BV2 cells after sevoflurane treatment. Representative confocal images of the fluorescent latex beads phagocyted by BV2 cells in control and sevoflurane-treated BV2 cells with IgG or ANX005 treatment. Scale bars = 5 μm. Fig. S7. Sevoflurane or ANX005 treatment did not affect the vision or swimming ability of mice. (A-B) The visible platform trial results showed no differences among groups in time to find the platform and swimming speed. n = 8. One-way ANOVA followed by a post hoc Tukey’s test. Fig. S8. Inhibition of sialidase by NADNA decreased C1q tagging to synapses after neonatal sevoflurane exposures. (A) Representative confocal images of PSA-NCAM in the CA1 region of control and sevoflurane-treated mice with PBS or NADNA treatment. Scale bars = 100 μm, Scale bars = 20 μm. (B) Quantitative analysis of PSA-NCAM immunofluorescence indicated that NADNA increased PSA-NCAM in sevoflurane-treated mice. n = 6. One-way ANOVA followed by a post hoc Tukey’s test. (C) Representative confocal images of colocalization of C1q (green) and PSD95 (red) in the four groups. Scale bars = 5 μm. (D) Quantitative analysis of C1q and PSD95 colocalization revealed that NADNA reduced C1q tagging to synapses in sevoflurane-treated mice. n = 6. One-way ANOVA followed by a post hoc Tukey’s test. Fig. S9. Inhibition of sialidase by NADNA decreased microglial-mediated synapse engulfment after neonatal sevoflurane exposures. (A) Representative confocal images of IBA1+ (red) containing PSD95+ puncta (green) in the hippocampus of control and sevoflurane-treated mice with PBS or NADNA treatment. Orthographic view and 3D rendering are shown. Scale bars = 5 μm. (B) Quantification analysis showed that NADNA treatment decreased the engulfment index compared to treatment with sevoflurane and PBS. n = 20. One-way ANOVA followed by a post hoc Tukey’s test. Fig. S10. Sevoflurane or NADNA treatment did not affect the vision or swimming ability of mice. (A-B) The visible platform trial results showed no differences among groups in time to find the platform and swimming speed. n = 8. One-way ANOVA followed by a post hoc Tukey’s test.