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.