FIGURE LEGENDS

Figure 1 A focal unilateral traumatic brain injury using the controlled cortical impact (CCI) placed on the sensorimotor cortex (a-d) and analysis of hind limb-postural asymmetry (HL-PA) (e,f). (a) Schematic representation of the sensorimotor cortex of the rat brain (modified from (Tandon, Kambi & Jain, 2008). (b) An expanded region of cortex. Green circle denotes the intended lesion area although the actual lesion area slightly varied among the rats. Vertical black line indicates the bregma plane. Scales in the middle and on the right side indicate the distance in mm relative to the bregma rostrocaudally and to the midline mediolaterally, respectively. (c) Macro anatomical image shows the lesion site in the right hemisphere from a CCI-injured rat. (d) Five consecutive Toluidine blue-stained cortical sections with equal distance (250 µm) show the lesion site on the right side from another CCI rat brain. The dark blue areas indicate the damaged tissue. Scale bar = 2 mm. Caudal is to the left and rostral is to the right for (a – d). (e, f) Analysis of HL-PA in a sham-operated (e) and a CCI rat (f). The magnitude of postural asymmetry was measured in millimeter as the length of the projection of the line connecting symmetric hindlimb distal points (digits 2-4) on the longitudinal axis of the rat.
Figure 2 An experimental design. Rats were exposed to CCI or sham injury on Day 0 (Designs 1 and 2) followed by analysis of HL-PA on Day 1 (Design 1) or Day 3 (Design 2) that was performed immediately before and 30 and 60 min after complete spinal cord transection. Rats were treated with nor-BNI or β-FNA on Day 2 (Design 2 / Treatment 1), with naloxone, naltrindole or LY2444296 on Day 3 before the transection (Design 2 / Treatment 2), or with naloxone or naltrindole after it (Design 2 / Treatment 3). In Design 3, intact rats were treated with nor-BNI on Day 0 (Treatment 1) followed by administration of U50,488H or saline on Day 1 after spinal transection (Treatment 2). HL-PA was analyzed immediately before the injection and transection, and 30 and 60 min after them.
Figure 3 The right-side CCI induced formation of HL-PA that retained after complete spinal cord transection. HL-PA was analyzed before and 30 and 60 min after spinalization on the Day 1 (Design 1; D1) and Day 3 (Design 2; D2) after CCI or sham injury (SI). (a) Changes in the magnitude of HL-PA (MPA). MPA data is 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. Horizontal dashed line denotes the 2 mm threshold that was 94th MPA percentile in control group. (b) The mean probabilities (PA) and 95% CI for a rat to be asymmetric at MPA > 2 mm that corresponded to 94th MPA percentile in the SI group. (c) The mean probabilities (PC) and 95% CI for asymmetric rat to display contralesional flexion. No significant differences between the Design 1 and Design 2 sham injury groups (n = 5 / group) in the MPA and PA, were revealed and therefore these two groups we combined in the SS group (n = 10). No significant differences between the saline-treated CCI groups (Design 2, Treatment 2 and 3; n = 5 / group) in MPA and PA were found; they were pooled into the control CCI group (CCI-D2; n = 10). No significant differences among this control CCI group and the CCI rats not treated with saline were revealed and they were pooled into the combined CCI group (n = 20) for analysis of PC. The Design 1 CCI group (CCI-D1) consisted of 10 rats. * P < 0.05, significant differences among the rat groups in (a,b) or in comparison with the random (50/50) distribution (c). ANOVA with adjusted by Tukey method P-values in post hoc analysis was used in (a), and Fisher’s exact test with Bonferroni correction in (b) and (c)
Figure 4 Effects of the general opioid antagonist naloxone on formation of HL-PA induced by the right-side CCI and retention of the asymmetry after complete spinal cord transection. Rats exposed on Day 0 to the right-side CCI were treated on Day 3 with naloxone 50 min before spinal transection (Design 2 / Treatment 2, Tr2) or 40 min after it (Design 2 / Treatment 3, Tr3) (n = 10 / group). The control CCI group (n = 10) was treated with saline. For details, see legend to Figure 3
Figure 5 Effects of β-FNA and naltrindole, the selective µ- and δ-opioid antagonists, respectively, on formation of HL-PA induced by the right side CCI and retention of the asymmetry after complete spinal cord transection. Rats exposed on Day 0 to the right-side CCI were treated with β-FNA on Day 2 (Design 2; Treatment 1), or with naltrindole (NTI) on Day 3, 50 min before spinal transection (Design 2; Treatment 2, Tr2) or 40 min after it (Design 2; Treatment 3, Tr3) (n = 10 / group). For details, see legend to Figure 3
Figure 6 Effects of nor-BNI and LY2444296, the selective κ-opioid antagonists on formation of HL-PA induced by the right side CCI and retention of the asymmetry after complete spinal cord transection. Rats exposed on Day 0 to the right-side CCI were treated with nor-BNI on Day 2 (Design 2 / Treatment 1) (n = 10 in (a) and (b)), or with LY2444296 on Day 3, 90 min before spinal transection (Design 2; Treatment 2; n = 10). In (c), the nor-BNI group consisted of the exploratory (n = 10) and replication (n = 10) groups that were investigated to confirm the results; each of them was significantly different from the CCI group. For details, see legend to Figure 3
Figure 7 (a-c) Induction of HL-PA by U50,488H, the selective κ-opioid agonist in intact spinalized rats, and its prevention by nor-BNI. U50,488H or saline was administered intrathecally to caudal portion of transected spinal cord (Design 3, Treatment 2). nor-BNI was administered on Day 2 (Design 3 / Treatment 1). In (a) and (b), each the saline, U50,488H and nor-BNI groups consisted of 10 rats, and the U50,488H + nor-BNI group 9 animals. In (c), n = 18 for comparison with the random 50% left / 50% right distribution. (d) Effect of naltrindole on HL-PA with the right flexion induced by the right-side CCI in rats pretreated with nor-BNI (Design 2 / Treatment 2). The CCI rats were untreated or treated with saline (the CCI group; n = 20), NTI (n = 8), nor-BNI and saline (n = 20), and nor-BNI and NTI (n = 9). Naltrindole or saline were administered 50 min before asymmetry analysis (Design 2; Treatment 2) For details, see legend to Figures 3 and 6
Figure 8 Lateralization of opioid gene expression (a-c) and theOprk1 / Oprd1 mRNA ratio (d) in the lumbar spinal cord of intact rats (n = 10). Log-scaled data for the expression levels and the ratio are shown. The horizontal line in the box represents the median; the box hinges represent the first (Q1) and third quartiles (Q3). Upper and lower whiskers extend from the hinge to the highest/lowest value that lies within the 1.53 interquartile range (IQR) of the hinge. * P < 0.05; two tailed paired Student’s t test with Bonferroni correction
Figure 9 Hypothetical spinal mechanism of the opioid receptor mediated inhibition and side-to-side reversal of HL-PA induced by the right-side CCI. We hypothesize that there are two groups of endogenous substances (e.g. neurohormones, neuropeptides and growth factors) that regulate physiological processes either on the left or right side of the CNS (a), and which activity is balanced in bilaterally symmetric animals. These molecules may serve as postural asymmetry inducing factors (PAFs). A unilateral brain injury impairs the balance; an equilibrium in activity of the left-side and right-side PAFs may be shifted to favor the factors producing the contralesional hindlimb response (b). After the right-side CCI, activity of factors inducing the left side responses (the left-side PAFs) would dominate resulting in the left side flexion. Administration of µ- and κ-antagonists blocks the HL-PA (f) and reverses the side of the flexed limb (c), respectively, suggesting that the left-side PAFs act through the µ- and κ-receptors, respectively. Block µ-receptor may equalize the signaling stimulated by the left and right-side PAFs that reestablishes the balance and abolishes HL-PA formation (f). Factors targeting κ-receptor may prevail among the left-side PAFs (a,b), and therefore blocking their effects would lessen the left-side PAF signaling and change a balance to favor the signaling by the right-side PAFs (c). δ-Antagonist does not affect the CCI-induced HL-PA with left flexion (g) but inhibits HL-PA with right hindlimb flexed in rats pretreated with κ-antagonist (d). The left-side PAFs may consist of κ-agonists dynorphins and the endogenous ligand of µ-receptor Met-enkephalin with mixed µ-/δ-acticivity that could induce HL-PA with the left flexion in intact animals (h-j). Conversely, the right-side PAFs may contain Leu-enkephalin, an endogenous -agonist that induces flexion of the right hindlimb (k). HL-PA induced by the right-side CCI retains after complete spinal cord transection (e) suggesting the spinal underlying mechanism.