Minocycline selectively inhibits M1 polarization of microglia methods
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Materials and Methods
The animal experiments described in this article were performed in accordance with protocols approved by the institutional animal committee. All animals were treated and cared for in accordance with the Nagoya University School of Medicine Guidelines pertaining to the treatment of experimental animals. SOD1 G93A transgenic mice, which carry the G93A mutant form of the human SOD1 (B6.Cg-Tg [SOD1-G93A] 1Gur/J line), were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The protocols for the maintenance and genotyping of these mice were described previously. **The transgenic mice were randomly divided into minocycline hydrochloride (Nichi-iko Pharmacceutical Co. Ltd., Toyama, Japan)-treated and untreated groups. Minocycline (33 mg/kg) was administered intraperitoneally five times a week from 8 weeks after birth to end stage.
Quantitative RT-PCR
Total RNA was extracted from the lumbar spinal cords using an RNeasy Lipid Tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturer's recommendations. The cDNA was prepared from 1 µ g of total RNA by using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) following the standard protocols. Quantitative PCR was performed on a M × 3000P (Agilent Technologies, Santa Clara, CA, USA) using the synthetic primers and SYBR Green (Agilent Technologies). Samples were subjected to 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min, after holding at 50 °C for 2 min and 95 °C for 10 min. Relative expression was calculated using the 2- (Ct experimental sample - Ct internal control sample (GAPDH)) method. The sequences of primers used are listed in.
Statistical analysis
The Kaplan-Meier method (log-rank test) was used for comparing the lifespan and disease onset of mice of each genotype. Mann-Whitney's U -test was used for analyzing disease duration. Two-way ANOVA was used to evaluate the difference in the temporal expression profile of M1 and M2 markers between two groups. We subsequently performed an unpaired Student's two-tailed t -test at each week. In all statistical analyses, a value of P <0.05 was considered to indicate significance. The statistical analysis was performed using SAS 9.3 (SAS Institute Inc., Cary, NS, USA) and SPSS (SPSS Inc., Chicago, IL, USA) software. The investigators performing the statistical analyses were blinded to the group assignments in all procedures.
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The animal experiments described in this article were performed in accordance with protocols approved by the institutional animal committee. All animals were treated and cared for in accordance with the Nagoya University School of Medicine Guidelines pertaining to the treatment of experimental animals. SOD1 G93A transgenic mice, which carry the G93A mutant form of the human SOD1 (B6.Cg-Tg [SOD1-G93A] 1Gur/J line), were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The protocols for the maintenance and genotyping of these mice were described previously. **The transgenic mice were randomly divided into minocycline hydrochloride (Nichi-iko Pharmacceutical Co. Ltd., Toyama, Japan)-treated and untreated groups. Minocycline (33 mg/kg) was administered intraperitoneally five times a week from 8 weeks after birth to end stage.
Total RNA was extracted from the lumbar spinal cords using an RNeasy Lipid Tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturer's recommendations. The cDNA was prepared from 1 µ g of total RNA by using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) following the standard protocols. Quantitative PCR was performed on a M × 3000P (Agilent Technologies, Santa Clara, CA, USA) using the synthetic primers and SYBR Green (Agilent Technologies). Samples were subjected to 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min, after holding at 50 °C for 2 min and 95 °C for 10 min. Relative expression was calculated using the 2- (Ct experimental sample - Ct internal control sample (GAPDH)) method. The sequences of primers used are listed in.
The Kaplan-Meier method (log-rank test) was used for comparing the lifespan and disease onset of mice of each genotype. Mann-Whitney's U -test was used for analyzing disease duration. Two-way ANOVA was used to evaluate the difference in the temporal expression profile of M1 and M2 markers between two groups. We subsequently performed an unpaired Student's two-tailed t -test at each week. In all statistical analyses, a value of P <0.05 was considered to indicate significance. The statistical analysis was performed using SAS 9.3 (SAS Institute Inc., Cary, NS, USA) and SPSS (SPSS Inc., Chicago, IL, USA) software. The investigators performing the statistical analyses were blinded to the group assignments in all procedures.
Machine-readable layer
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