3.1 The CCI-induced HL-PA: contralesional flexion and spinal fixation
The HL-PA was analyzed before, and 30 and 60 min after complete spinal cord transection at the thoracic level under pentobarbital anesthesia. The CCI rats developed HL-PA that was evident on both Days 1 and 3 after the operation, although HP-PA was not observed in sham-injured rats (Figure 3). Analysis of the MPA by ANOVA showed significant main effect of the CCI. Post hoc analysis revealed that MPA was significantly higher both on Day 1 and Day 3 in the CCI groups compared to the control group. At every measurement time, the MPA was significantly higher, approximately 3-fold in the CCI rats compared to sham-injured animals (Figure 3a). In the CCI groups the MPA was significantly greater on Day 3 compared to Day 1. The odds of a CCI rat to develop HL-PA were significantly greater than those of a rat exposed to sham injury (Figure 3b). Most control rats did not develop asymmetry, and therefore the odds of asymmetric rats to display contralesional flexion was analyzed for the CCI groups only. The odds of the asymmetric CCI rats to have contralateral flexion were significantly greater than the random 50 / 50 distribution before the transection; and 30 and 60 min after the transection (Figure 3c).

3.2 Effects of the general opioid antagonist naloxone

To examine whether opioid receptors mediate effects of the CCI on formation of HL-PA the general opioid antagonist naloxone was administered to the CCI rats on Day 3 after the brain injury either 50 min before (Design 2 / Treatment 2) or 40 min after (Design 2 / Treatment 3) complete spinal transection. Naloxone injection at both treatment designs resulted in substantial decrease in the MPA and the probability to develop HL-PA (Figure 4). The MPA was significantly reduced before spinal transection (Treatment 2), and 60 min after it (Treatments 2 and 3). The odds for the naloxone treated rats to be asymmetric were significantly decreased at the 30 and / or 60 min time points. To identify subtypes of the opioid receptors involved we analyzed effects of selective µ-, δ- and κ-antagonists.

3.3 Effect of β-FNA

After a single injection of β-FNA, various effects last for weeks and become selective for the µ-receptor 24 h after administration (Petrillo et al., 2003). Therefore, the antagonist was administered to CCI rats 24 h before HL-PA analysis (Design 2 / Treatment 1) (Figure 5). Administration of β-FNA resulted in substantial decrease in the MPA before, and 30 and 60 min after spinal transection (Figure 5a). The odds for the β-FNA treated rats to be asymmetric were significantly reduced before and after the spinal transection (Figure 5b).

3.4 Effect of naltrindole

No significant effects on formation and maintenance of HL-PA, and the side of the flexed hindlimb were revealed after naltrindole administration to the CCI animals before and after spinalization (Design 2 / Treatments 2 and 3) (Figure 5a,b). The left hind limb was still flexed after the treatment (Figure 5c).

3.5 Effects of κ-antagonists

nor-BNI is long acting κ-antagonist which selectively blocks -receptor 24 h after a single injection (Horan, Taylor, Yamamura & Porreca, 1992; Patkar et al., 2013; Rutten, Schroder, Christoph, Koch & Tzschentke, 2018) , and therefore was administered to the CCI rats 24 h before HL-PA analysis (Design 2 / Treatment 1). Administration of nor-BNI did not produce significant changes in the MPA and the odds to develop asymmetry (Figure 6a,b). Unexpectedly, the CCI rats treated with nor-BNI displayed flexion of the ipsilesional (right) hindlimb instead of the contralesional (left) hindlimb (Figure 6c). The odds of the nor-BNI treated CCI rats to produce ipsilesional (right side) flexion were significantly higher than those of the control CCI group (the CCI rats received saline) at each of three time points.
The nor-BNI effects were replicated with LY2444296, a κ-antagonist characterized by shorter onset and shorter duration of action (Melief et al., 2011). No significant main effect of LY2444296 administered to the CCI rats 90 min before spinal transection on both the MPA and the odds to develop HL-PA were revealed. Similarly with nor-BNI, administration of LY2444296 resulted in the left-to-right flexion side transition (Figure 6c); the odds of the LY2444296 treated rats to develop flexion on the ipsilesional (right) side were significantly higher than those of the control CCI group before and after the transection. These results suggest that the development of the contralesional (left) hindlimb flexion as the primary effect of the right side CCI is mediated through activation of the spinal κ-opioid receptor by endogenous κ-ligands.

3.6 Effects of U50,488H

To test this hypothesis, we assessed if U50,488H, a selective κ-agonist could induce HL-PA in intact animals, and whether the left or right-hind limb would be flexed. U50,488H was administered intrathecally to intact rats after transection of their spinal cord, and the formation of HL- PA was examined 30 and 60 min after the injection (Figure 7a-c). U50,488H significantly elevated MPA compared to both saline injection and co-treatment with U50,488H and nor-BNI at both the 30 and 60 min time points. The odds of the U50,488H treated rats to be asymmetric were significantly higher than those of the saline treated rats. The odds of the rats receiving both U50,488H and nor-BNI to be asymmetric did not differ from those of the saline treated rats.
Most asymmetric U50,488H treated animals developed the left flexion; the odds for it were significantly different from the random (50% left/50% right) distribution.

3.7 Naltrindole effect on HL-PA in the CCI rats treated with nor-BNI

Previous studies demonstrated that δ-agonist Leu-enkephalin may induce HL-PA with right hindlimb flexion (Bakalkin & Kobylyansky, 1989; Chazov, Bakalkin, Yarigin, Trushina, Titov & Smirnov, 1981). We examined whether formation of HL-PA with right flexion in the CCI rats treated with nor-BNI is mediated through -receptor (Figure 7d). Naltrindole or saline was administered on Day 3 (Design 2 / Treatment 2) to the CCI rats pretreated with nor-BNI (Treatment 1), and HL-PA was analyzed 50 min after injection of the δ-antagonist. The MPA was significantly decreased in the nor-BNI pretreated CCI rats receiving naltrindole compared to the CCI rats that were not treated with an antagonist or treated either with not-BNI or naltrindole alone (Figure 7d). Thus -opioid receptor may mediate formation HL-PA with the flexion of the right but not left hindlimb.

3.8 Expression of opioid receptor genes in the lumbar spinal cord

The side-specific effects of opioid agonists or antagonists (Bakalkin & Kobylyansky, 1989; Chazov, Bakalkin, Yarigin, Trushina, Titov & Smirnov, 1981, and present study) may be mediated through opioid receptors if they are lateralized in the lumbar spinal cord. We previously demonstrated that the expression of opioid receptors was lateralized to the left, while the proportion of κ- and δ-receptors analyzed as the Oprk1 / Oprd1 mRNA ratio to the right in the cervical spinal cord in the rats (Kononenko et al., 2017). We addressed the hypothesis by analysis of the κ- (Oprk1 ), µ- (Oprm1 ) and δ- (Oprd1 ) opioid receptor mRNA in the left and right halves of the lumbar spinal cord of intact rats (Figure 8). Significantly higher, 1.28-fold expression of Oprd1 was revealed in the left compared to the right-half. Furthermore, the proportion of opioid receptor mRNA was significantly different between the left and right sides; the Oprk1 / Oprd1 mRNA ratio was higher, 1.16-fold in the right side compared to the left side part. Essentially the same results were obtained in the replication study of sham-injured rats (n = 11; 1.23-fold significantly higher Oprd1 expression on the left side, and 1.32-fold significantly higher Oprk1 / Oprd1ratio on the right side).