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.