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.