4 DISCUSSION
The first principal finding of this study is that the unilateral focal
CCI of the sensorimotor cortex, a rat model of focal TBI induced
formation of the HL-PA, an inherent feature of brain injury-induced
motor deficits. The CCI-induced HL-PA was retained after complete spinal
cord transection suggesting that neuroplastic changes in the spinal cord
or “pathological spinal memory” is the mechanism underling the
asymmetry.
In the previous and present studies no nociceptive stimulation was
applied and tactile stimulation was negligible when the HL-PA was
analyzed. As established, the stretch and postural limb reflexes are
abolished immediately and for days after complete spinal cord
transection (Frigon, Johnson & Heckman,
2011; Miller, Paul, Lee, Rymer &
Heckman, 1996; Musienko, Zelenin,
Orlovsky & Deliagina, 2010) and substantially decreased under
anesthesia (Fuchigami et al., 2011;
Zhou, Jin, Qin & Turndorf, 1998).
Therefore, the nociceptive withdrawal reflexes and stretch reflex could
not contribute to HL-PA formation in preparations of the spinalized CCI
rats under anesthesia. The HL-PA may be mediated by the group II muscle
afferents that remain active after spinalization in acute experiments
(Jankowska, 1992;
Lavrov, Gerasimenko, Burdick, Zhong, Roy
& Edgerton, 2015; Valero-Cabre, Fores &
Navarro, 2004) . On the other hand, the asymmetry induced by the
right-side localized brain injury was not eliminated by bilateral lumbar
dorsal rhizotomy suggesting that it did not depend on the somatosensory
afferent input (Bakalkin et al., 2018;
Zhang, Watanabe, Sarkisyan, Thelin,
Schouenborg & Bakalkin, 2018). Instead, it may develop due to
sustained muscle contractions that are evoked by the efferent drive.
Thus, the HL-PA is a complex phenomenon that is developed either due to
a persistent asymmetric activity of lumbar motoneurons not stimulated by
afferent input, or discharge of proprioceptive neurons activated perhaps
by group II muscle afferents, which are tonically active and maintain
muscle tone.
The second principal finding is that the formation and maintenance of
the CCI-induced HL-PA is mediated by the spinal opioid system. Both
naloxone, the general opioid antagonist and β-FNA, a selective
µ-antagonist blocked the asymmetry formation. nor-BNI and LY2444296,
selective κ-antagonists did not produce significant changes in the MPA
but reversed the side of flexed limb; instead of the contralesional
(left) hindlimb, the right (ipsilesional) hindlimb was flexed in rats
after the right-side CCI. Naltrindole, a selective δ-antagonist produced
no effect on the HL-PA with the left flexion, but eliminated the
asymmetry if the CCI rats were pretreated with nor-BNI and displayed the
right limb flexion.
The findings with the antagonists are complemented by observations that
opioid peptides and synthetic opioids induce HL-PA in intact rats after
their spinalization. U50,488H, bremazocine and dynorphin, selective
κ-agonists along with the endogenous µ- and δ-agonist Met-enkephalin,
induced HL-PA with flexion of the left hindlimb (the present study and
(Bakalkin & Kobylyansky, 1989;
Chazov, Bakalkin, Yarigin, Trushina, Titov
& Smirnov, 1981). In contrast, Leu-enkephalin that acts through
δ-receptor, caused the right limb to flex. Relative affinity of
Met-enkephalin for binding to µ- vs . δ-receptor is much higher
than that of Leu-enkephalin (Gacel,
Fournie-Zaluski & Roques, 1980;
Jankowska, 1992;
Mansour, Hoversten, Taylor, Watson &
Akil, 1995). The asymmetric motor responses were induced by intrathecal
agonist administration suggesting that they are medicated through spinal
opioid receptors. In the spinal cord, the μ-, δ- and κ-opioid receptors
are expressed both in the dorsal and ventral horns
(Kononenko et al., 2017;
Wang et al., 2018). δ-Opioid receptor is
expressed in multiple classes of neurons that regulate spinal motor
control while δ- and µ-receptors are co-expressed in V1 ventral horn
interneurons (Wang et al., 2018). Opioid
agonists exert their action on ventral root reflexes via presynaptic
inhibition of afferent signaling, the postsynaptic inhibition of the
dorsal horn interneurons and actions on ventral horn interneurons
regulating motoneurons activity (Wang et
al., 2018). This may result in suppression of the ipsilateral reflexes
(Faber, Chambers, Brugger & Evans, 1997)
while targeting of opioid receptors in neurons surrounding the central
canal (Mansour et al., 1994;
Wang et al., 2018) may inhibit the spinal
commissural pathways (Light & Perl,
1979; Petko, Veress, Vereb,
Storm-Mathisen & Antal, 2004) and contralateral reflexes
(Duarte et al., 2019). The endogenous
opioid peptides suppressed reflexes evoked by electrical stimulation of
the skin (Clarke, Galloway, Harris, Taylor
& Ford, 1992; Steffens & Schomburg,
2011) that may attenuate pain and to promote healing
(Steffens & Schomburg, 2011). The opioid
system is also engaged in a motor control operated under conditions of
pain and stress.
The side-specific opioid effects suggest that spinal neural circuits
regulating the left and right hindlimb muscles differ in sensitivity
towards the opioid agonists (Bakalkin &
Kobylyansky, 1989; Chazov, Bakalkin,
Yarigin, Trushina, Titov & Smirnov, 1981). An asymmetric expression of
opioid receptor and peptide genes was identified in the cervical spinal
cord (Kononenko et al., 2017). All three
opioid receptors were lateralized to the left but in different
proportions. Expression was coordinated between the dorsal and ventral
domains but with different patterns on the left and right spinal sides.
The present study identified generally the same lateralization patterns
in the lumbar spinal cord. Expression of δ-receptor (Oprd1 ) was
lateralized to the left whereas a proportion of κ- and δ-receptors (theOprk1 / Oprd1 expression ratio) was higher on the right
side. Neural circuits controlling motor functions of the left and right
hindlimbs are mirror symmetric but may be differentially regulated
through opioid receptor subtypes; the unilateral CCI-induced flexion of
the left and right hindlimb may be controlled by κ- and δ-receptors,
respectively.
We and others previously described multiple peptide factors in the brain
and spinal cord that may induce HL-PA (the postural asymmetry inducing
factors, PAFs) (Bakalkin, Pivovarov,
Kobylyansky, Yarygin & Akparov, 1989;
Kryzhanovskii, Lutsenko, Karganov &
Beliaev, 1984; Vartanian, Shatik, Tokarev
& Klement’ev, 1989). The PAFs of the left hemisphere induced flexion
of the left hindlimb, while the right hindlimb was flexed after
administration of the right hemisphere PAFs. The PAF fraction prepared
from the whole brain however did not produce the asymmetry suggesting
that activity of the left and right-side factors is equalized in the
CNS. Effects of PAFs were partially blocked by naloxone whereas
biochemical analysis demonstrated that PAFs were multiple short
peptides. Similar factors were identified in the left and right
hemisphere of the turtle; they inhibited the evoked potentials
preferentially of the ipsilateral side in the visual cortex
(Bakalkin, Pivovarov, Kobylyansky,
Nesterenko & Yarygin, 1989; Bakalkin,
Pivovarov, Kobylyansky, Yarygin & Akparov, 1989) acting through the
lateralized opioid receptors as demonstrated in electrophysiological and
receptor binding experiments. The left visual cortex was enriched in κ-
and µ-opioid receptors while the right-side cortex in δ-receptors
(Bakalkin, Pivovarov, Kobylyansky, Yarygin
& Akparov, 1989). Thus, differential lateral distribution of opioid
receptors has been demonstrated for other types of somatosensory input
as well.
The side-specific effects of κ- and µ-antagonists may be interpreted in
the frame of the PAF balance hypothesis (Figure 9). A balance in
activity of PAFs producing the left- or right-side response may be
impaired after a unilateral brain injury; an equilibrium may be shifted
to favor the factors that elicit the contralesional hindlimb response.
After the right-side CCI, activity of factors that induce the left
hindlimb flexion including dynorphins and Met-enkephalin may be
increased and become dominant over those producing the right side
response. Injection of either of these peptides to spinalized rats
resulted in formation of the HL-PA with left-side flexion. Selective
blockade by µ-receptor selective antagonist may equalize the potency of
the left and right-side PAFs and abolish HL-PA formation in the
right-side CCI rats. The PAFs targeting κ-receptor may dominate among
the left-side factors, and κ-receptor-selective blockade would change
the balance to favor the signaling produced by the right-side PAFs,
leading to formation of right-side flexion (Figure 9). Effects of the
right-side PAFs may be mediated through δ-opioid receptor because i)
naltrindole, a δ-antagonist blocked HL-PA with right hindlimb flexion in
the CCI rats pretreated with nor-BNI; and because ii) Leu-enkephalin, a
δ-agonist produced HL-PA with right hindlimb flexion in intact rats.
In conclusion, our study revealed a role of the endogenous opioid system
in the brain injury-induced neuroplastic adaptations in the spinal cord
that may underlie pathological changes in motor reflexes. The general
and µ-receptor selective opioid antagonists abolished pathological
changes by re-establishing hindlimb postural symmetry whereas κ- and
δ-antagonists interfered with processes that determine the side (leftvs . right) of motor deficits. Effects of the antagonists
demonstrate that spinal neural circuits are not irreversibly impaired
after the brain injury but may be rescued by pharmacological means.
These findings corroborate earlier observations demonstrating that
naloxone can reverse asymmetric neurological deficits secondary to focal
unilateral cerebral ischemia in gerbils, baboons and humans
(Baskin & Hosobuchi, 1981;
Baskin, Kieck & Hosobuchi, 1984;
Hosobuchi, Baskin & Woo, 1982). It is
important to identify clinical features of asymmetric motor deficits
e.g. hemiparesis and hemiplegia, which are encoded by the opioid
system-mediated spinal neuroplasticity, and to establish whether
targeting of these features by selective antagonists may promote
recovery and / or compensation of motor functions impaired in TBI
patients.