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).