2.5 mRNA Analysis
Intact rats and rats exposed to sham injury (n = 10 / group) were sacrificed by decapitation and the lumbar spinal cords were dissected into the left and right halves. RNA purification, quality evaluation, cDNA synthesis and quantitative RT-PCR were described elsewhere (Kononenko et al., 2018). mRNA levels of three opioid receptor genes were normalized to geometric mean of expression levels of two control genes Actb and Gapdh(Kononenko et al., 2017). In each experiment, the internal control gene-stability measure M did not exceed the established limit of 0.5.

2.6 Experimental time line / drug treatment design (Fig. 2).

Design 1. Animals were subjected to CCI or sham injury on Day 0. HL-PA was analyzed on Day 1 immediately before and 30 and 60 min after spinal cord transection.
Design 2. Rats were subjected to the CCI or sham injury on Day 0. HL-PA was analyzed on Day 3 immediately before and 30 and 60 min after spinal cord transection. A test compound was administered on Day 2 (Treatment 1) or Day 3 (Treatment 2 or 3).
Design 3. U50,488H or saline was administered to intact rats immediately after spinal transection on Day 1 (Treatment 2); HL-PA was analyzed immediately before the injection and transection, and 30 and 60 min afterwards.
nor-Binaltorphimine (nor-BNI; 6 mg/kg; subcutaneously, S.C.) was administered on Day 2 (Design 2 / Treatment 1) or Day 0 (Design 3 / Treatment 1). β-Funaltrexamine (β-FNA; 3 mg/kg; S.C.) was injected on Day 2 (Design 2 / Treatment 1); and naloxone (10 mg/kg; intraperitoneally, I.P.) or naltrindole (5 mg/kg; I.P.) on Day 3 before (Design 2 / Treatment 2: the -50 min time point) or after spinal transection (Design 2 / Treatment 3: the 40 min time point).
[(S)-3-fluoro-4-(4-((2-(3-fluorophenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide]) (LY2444296 also known as FP3FBZ) (0.3 mg/kg; I.P.) was administered 90 min before the transection on Day 3 (Design 2 / Treatment 2: the -90 min time point). (2)-(trans )-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide (U50,488H; 1 microgram in 5 microliters of saline / rat; intrathecally, I.T.) was injected on Day 1 (Design 3 / Treatment 2), respectively.
Doses and timeline for naloxone (Norris, Perez-Acosta, Ortega & Papini, 2009), naltrindole (Nizhnikov, Pautassi, Truxell & Spear, 2009; Petrillo et al., 2003; Rutten, Schroder, Christoph, Koch & Tzschentke, 2018), nor-BNI (Horan, Taylor, Yamamura & Porreca, 1992; Patkar et al., 2013; Rutten, Schroder, Christoph, Koch & Tzschentke, 2018) and β-FNA (Petrillo et al., 2003) were robustly established in previous studies to block all or selective opioid receptors. nor-BNI, a selective κ-opioid antagonist exerts long-lasting antagonistic effects that persist for at least 1 month. Selective blockage of κ-receptors by nor-BNI gradually increases in time reaching a plateau 2 days after S.C. administration. The 0.3 mg/kg dose of LY2444296 was selected in a pilot experiment as the minimal dose that produced effects lasted at least for 2.5 h; no effect at the 0.1 mg / kg dose (n = 8) was evident. U50,488H (369 g/mol) was injected at the 1 microgram in 5 microliters / rat dose similar to those of bremazocine (315 g/mol), another -agonist that produced HL-PA in a dose range from 10 nanograms to 1 microgram per rat (Bakalkin & Kobylyansky, 1989).

2.7 Data analysis

The data and statistical analysis comply with the recommendations ofthe British Journal of Pharmacology on experimental design and analysis in pharmacology.
Repeated measures of magnitude of hind limb postural asymmetry (MPA) and the side of the flexed leg for each rat for each measurement time(before, and 30 and 60 min after spinal transection) were analyzed by generalized linear mixed models using 2- and 3-way ANOVAs (Naomi & Krzywinski, 2015). Experimental factors were the day of CCI or sham injury, the day of spinalization, and treatment schedule (type of administered drug) (Figure 2). Analysis of interactions was included in the models. Inspection of data revealed deviations from normality for the residuals of MPA. Nonparametric ANOVA was computed in R 3.6 (Team, 2018) using package ARTool (Wobbrock, Findlater, Gergle & Higgins, 2011). Means, 95% confidence intervals (95% CI) and adjusted by Tukey method P-values (only in those tests where F achieved the necessary level of statistical significance, P < 0.05) were estimated from post hoc analysis using R packageemmeans 1.4 (Searle, Speed & Milliken, 2012). MPA data was presented as boxplots where the horizontal line in the box shows the median; the box covers 50% of all observations (the interquartile range, IQR) from the first (Q1) and third quartiles (Q3). The whisker extends from the bottom and top of the box by 1.5× IQR.
A rat was defined as asymmetric if MPA exceeded the 2 mm threshold that corresponded to 94th MPA percentile in the rats exposed to sham injury. The same effects were also significant at the 1 and 3 mm thresholds. The mean probabilities and 95% CI for a rat to be asymmetric (PA), to have contralesional flexion (PC) and to have left flexion (PL) were estimated by R package emmeans . Fisher’s exact test with Bonferroni correction for P-values was used to estimate differences between animal groups in odd ratios.
In analysis of gene expression, normality of data distribution of the log-scaled expression levels was tested using the Kolmogorov-Smirnov test and homogeneity of variances by Levene’s test. Differences between the groups were assessed two tailed paired Student’s t test (normally distributed data) or Kruskal-Wallis test of ranks (data with non-normal distribution). The Bonferroni procedure was used for multiple testing adjustments.
The size of the groups was decided by considering the accuracy and reproducibility of the detection method as well as the biological parameters involved. The number of animals in each group included for statistical tests is shown in the figure legend for analysis. Blinding was maintained as far as possible during data collection and evaluation which were performed by different investigators. Differences were considered significant when adjusted P < 0.05.

2.8 Materials

Naloxone, β-FNA, naltrindole, nor-BNI and U50,488H were purchased from Tocris (Minneapolis, MN). LY2444296 was synthesized at Lilly Research Laboratories (Indianapolis, IN). All test compounds were dissolved in saline for administration to animals.