We used the RNeasy Minikit from Qiagen to extract mRNA, after which 200 ng of purified mRNA was reverse-transcribed into cDNA using Superscript II (Invitrogen, Carlsbad, selleck compound CA, USA). The mRNA levels for GAD65, GAD67, and β-actin were quantified with a Realplex2 real-time PCR system (Eppendorf, Hamburg, Germany) using SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK). Ratios of GAD65 and GDA67 to β-actin mRNA were compared and analyzed by a two-way ANOVA followed by a Bonferroni post hoc test. Asterisks (∗) indicate statistically significant
differences between groups, with ∗ = p < 0.05 and ∗∗ = p < 0.01. Mechanical sensitivity was assessed by placing animals on an elevated wire mesh grid and
stimulating the hindpaw with von Frey hairs. We used the up-down paradigm (Chaplan et al., 1994) to define threshold. Animals were tested three times, once every other day before surgery to determine baseline threshold and once 2 days after surgery, to assess the magnitude of the mechanical allodynia. Only animals that displayed at least a 50% drop of the mechanical withdrawal threshold were included in the MGE SP600125 transplantation (transplanted group) or the medium injection (control group) groups. Behavioral testing took place on days 7, 14, 21, and 28 after MGE/medium injections. For the behavioral tests, the investigator was blind to treatment (cell medium or MGE injection). Prior ADAMTS5 to analyzing the behavioral results, MGE-transplanted animals were killed, and then the spinal cord was immunostained for the presence of GFP+ cells in the spinal cord. Only successfully transplanted animals (defined as containing at least one GFP+ cell per section) were included in the MGE-transplanted group and their behavior subsequently analyzed. Importantly, the investigator performing this anatomical analysis was not the investigator who performed the behavior analysis and
thus was blind to the behavior results. Transplanted animals from which no GFP-immunoreactive cells were detected were included in a “failed transplant” group. As the mechanical thresholds of the “failed transplant” and the medium-injected control groups were not significantly different, results from the “failed transplant” animals were not included in the statistical analysis. In another set of experiments, we injected 10 μl of a 1% formalin solution (Sigma-Aldrich, St. Louis, MO, USA) into the hindpaw of medium or MGE-cell-transplanted mice, ipsilateral to the transplanted side. We scored the mice for the total time spent flinching or licking the injected hindpaw (in 5 min bins). The behavioral scores were made by an experimenter blinded to treatment group. Only after the animals were defined as belonging to the MGE transplant, “failed transplant,” or medium transplant group were the behavioral results analyzed.