Measurement of Brain Mn Levels

We used an inductively coupled plasma mass spectrometer (ICP-MS; model 7500a, Agilent Technologies Inc., CA, USA) to measure the Mn level in brain tissues. The cortical and hippocampal tissue samples (20 mg for each wet tissue sample) were digested by adding 500 μl of nitric acid; then the completely digested samples were diluted 20 times. Following the user manual, the instrument was operated at a radiofrequency power of 1,420 W, the argon carrier gas flow rate was 1.05 l/min, and the argon plasma gas flow rate was 15 l/min. Using the peak area mode, the data acquisition time was 2 s, and three measurements were repeated. ICP-MS measurements of Mn in blood and brain tissue samples were converted to concentrations based on the calibration curve and its linear fit equation and sample dilution (Figure 1B). The reliability of our measurements is confirmed by the fact that our baseline Mn levels were similar to the blood and brain Mn levels reported in the literature (Garcia et al., 2006; Moldovan et al., 2013; Jenkitkasemwong et al., 2018).

Manganese (Mn) treatment via Mn-containing drinking water increased Mn concentrations in sera and brains in 3×Tg-AD mice. (A) The body weights of the mice were decreased in the Mn group in the first and second month, and then regained their weights in the following 3 months. N = 10 male 3×Tg-AD mice for each group. *p < 0.05, repeated measure (RM) ANOVA. Additional male 3×Tg-AD mice were similarly treated, but their body weight was only measured before euthanization and was normal. (B) Inductively coupled plasma mass spectrometer (ICP-MS) calibration curve using Mn standards. (C,D) ICP-MS measurements of the Mn level in the sera (C), the cerebral cortex (D), and the hippocampus (E) in control 3×Tg-AD mice and 3 Mn-treated 3×Tg-AD mice. **p < 0.01, ***p < 0.001, t-test.