Additional file 1 of Hearing modulation affects Alzheimer’s disease progression linked to brain inflammation: a study in mouse models
Creators: name: Ko, Yoo-Seung
name: Ryu, Young-Kyoung
name: Han, Sujin
name: Park, Hyung Joon
name: Choi, Munyoung
name: Kim, Byeong C.
name: Jeong, Han-Seong
name: Jang, Sujeong
name: Jo, Jihoon
name: Lee, Sungsu
name: Choi, Won-Seok
name: Cho, Hyong-Ho
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
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: Mental Health

description: Supplemetary Material 1. Figure S1. Auditory threshold evaluations of 5xFAD and Tg2576 mice. The ABR thresholds were measured for click and tone bursts in (A) 5xFAD and (B) Tg2576 mice. Figure S2. Establishment of a DIHL model induced by a combination of kanamycin and furosemide injections in Tg2576 mice. (A) Experimental timeline for Tg2576 mice. (B) The ABR thresholds were measured for click and tone burst stimuli in Tg2576 mice (n = 6–8). Data are presented as the mean ± SEM. Statistical significance was evaluated using ANOVA with Tukey’s post hoc test, supplemented by Student’s t-test where appropriate. Significance levels are indicated as *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Figure S3. Hearing impairment exacerbates cognitive dysfunction in both WT and Tg2576 mice. (A) Experimental timeline of Tg2576 mice. (B) Novel object recognition performance was assessed by calculating object preference using the formula: [100 (TNO-TFO)/(TNO + TFO)] in both the WT and Tg2576 groups. T - contact time; FO - familiar object; NO - novel object. Learning and memory deficits in the DIHL group of WT and Tg2576 mice were examined. In the Y-maze test: (C) The DIHL group exhibited reduced spontaneous alternation performance (SAP). (D) The DIHL group showed increased same-arm return (SAR), indicating memory impairment (n = 5). Data are presented as the mean ± SEM. Statistical significance was evaluated using ANOVA with Tukey’s post hoc test, supplemented by Student’s t-test where appropriate. Significance levels are indicated as *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Figure S4. Hearing impairment reduces LTP in both WT and Tg2576 mice. (A) The experimental timeline for Tg2576 mice. (B) Brain slices were collected at either 4.5 months or 5.5 months (n = 5) for the LTP test. Compared with WT mice, Tg2576 mice exhibited reduced LTP. Both the WT and Tg2576 DIHL groups showed a significant decrease in LTP. Data are presented as the mean ± SEM. Statistical significance was evaluated using ANOVA with Tukey’s post hoc test, supplemented by Student’s t-test where appropriate. Significance levels are indicated as *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Figure S5. Localization of the cortex and hippocampus regions for immunohistochemical staining of Aβ plaque. The regions of the cortex and hippocampus were delineated in the Aβ plaque-stained images. Figure S6. Hearing impairment results in a significant increase in APP expression in the brain tissue of Tg2576 mice. Western blotting was performed to analyze APP protein expression levels. DIHL significantly increased APP expression in both the cortex and hippocampus of Tg2576 mice. Data are presented as the mean ± SEM. Statistical significance was evaluated using ANOVA with Tukey’s post hoc test, supplemented by Student’s t-test where appropriate. Significance levels are indicated as *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Figure S7. Hearing impairments increased tau phosphorylation in the brain tissue of 5xFAD mice. (A) Representative immunofluorescence images show p-tau (AT8) in the cortex (left) and hippocampus (right) of WT and 5xFAD mice. Data are presented as the intensity of p-tau (pThr205/pSer202)-positive cells per 1 mm² (n = 7–8). Scale bars represent 100 μm. (B) Western blotting was performed to analyze p-tau (AT8) protein expression levels. Quantitative analysis demonstrated that DIHL significantly elevated p-tau (AT8) expression levels in both the cortex and hippocampus of 5xFAD mice (n = 6–8). Data are presented as the mean ± SEM. Statistical significance was evaluated using ANOVA with Tukey’s post hoc test, supplemented by Student’s t-test where appropriate. Significance levels are indicated as *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Figure S8. Localization of the cortex and hippocampus regions for immunohistochemical staining for S6 phosphorylation. The regions of the cortex and hippocampus were delineated on the p-S6-stained images. Figure S9. Localization of the cortex and hippocampus for immunohistochemical staining of Iba1 and GFAP. The regions of the cortex and hippocampus were delineated on the (A) Iba1- and (B) GFAP-stained images. Figure S10. Hearing impairment also activates microglia and astrocytes in the brain tissue of Tg2576 mice. DIHL increases the protein expression levels of Iba1 and GFAP. Representative immunofluorescence images show (A) Iba1 and (B) GFAP in the cortex (left) and hippocampus (right) of WT and Tg2576 western blotting was performed to analyze the protein expression levels of GFAP and Iba1.