Neuroscience Research 78 (2014) 3–8

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Review article

Plasticity for recovery after partial spinal cord injury – Hierarchical organization Tadashi Isa a,b,∗ , Yukio Nishimura a,b,c a b c

Department of Developmental Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan Department of Life Sciences, Graduate University for Advanced Studies (SOKENDAI), Hayama, Japan Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan

a r t i c l e

i n f o

Article history: Received 6 October 2013 Received in revised form 8 October 2013 Accepted 10 October 2013 Available online 25 October 2013 Keywords: Spinal cord injury Plasticity Recovery Hand dexterity Monkey

a b s t r a c t To cure the impaired physiological functions after the spinal cord injury, not only development of molecular therapies for axonal regeneration, but also that of therapeutic strategies to induce appropriate rewiring of neural circuits should be necessary. For this purpose, understanding the plastic changes in the central nervous system during spontaneous recovery following the injury would be helpful. In this article, a series of studies conducted in the authors’ laboratory on the reorganization of neural networks in the partial spinal cord injury model using macaque monkeys are reviewed. In this model, after selective lesion of the lateral corticospinal tract at the fifth cervical segment, dexterous digit movements are once impaired, but recover through rehabilitative training in a few weeks to a few months. During the recovery, synaptic transmission and organization of the neural circuits exhibit drastic changes depending on the time after the injury, not only in the spinal cord, but also in hierarchically higher order structures such as motorrelated cortical areas and even in limbic structures. It is suggested that on top of the molecular therapies, neurorehabilitative and neuromodulatory therapies targeting such higher order structures should be helpful in inducing appropriate rewiring of the neural circuits. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of neural mechanism of recovery in partial spinal cord injury model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Spinal cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cerebral cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. And beyond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and future perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Over the last several decades, a great deal of effort has been devoted to the researches to cure the spinal cord injury (SCI), such as stem cell graft and drug administration, in human patients and animal models. These researches were initially focused on stimulating axonal regeneration, in which providing the tissue with a

∗ Corresponding author at: Department of Developmental Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan. Tel.: +81 564 55 7761; fax: +81 564 55 7868. E-mail addresses: [email protected], [email protected] (T. Isa).

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growth-stimulatory environment was expected to enable the severed axons to regenerate across the injury and re-connect with their target neurons (David and Aguayo, 1981). This line of studies was further facilitated by finding of myelin-derived inhibitors of axonal growth and trials to neutralize such inhibitory myelin proteins (Caroni and Schwab, 1988; Schnell and Schwab, 1990; Akbik et al., 2012). Moreover, understanding the reactive changes that occur after injury and impede the recovery processes, such as inflammation, glial scar formation, cavitation and demyelination, led to development of therapeutic approaches to target these processes (Bunge et al., 1993; Fitch et al., 1999; Schwab and Bartholdi, 1996). In these studies, significant axonal regeneration has been shown, but the extent was relatively modest. On the other

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hand, a recent study by Mark Tsuzynski and colleagues showed remarkable extension of axons and synapse formation from the human neuronal stem cells grafted into the site of complete thoracic spinal cord injury in rats, in which a variety of treatments, such as fixing the implanted cells in the tissue with fibrin, administration of the cocktail of immune-depressant drugs and more than 20 agents to facilitate the axonal growth were combined (Lu et al., 2012). For further facilitation of functional recovery, now importance of induction of appropriate plasticity and removal of mal-plasticity of the surviving neurons or grafted cells, by combination with neurorehabilitative or neuromodulatory therapies is becoming more and more seriously recognized (Brown and Weaver, 2011; García-Alías and Fawcett, 2011). Previous studies investigating the effects of stem cell graft or drug administration on the functional recovery were mainly performed in rodent models of the spinal cord injury. In many of them, apparent recovery of locomotor functions could be observed (e.g. in BBB score), and the effectiveness of the treatment was claimed. However, the justification of such paradigm has been challenged, because the recovery of locomotor functions on the flat floor does not always indicate the recovery of descending tract across the injury level; the spinal cord by itself can generate highly coordinated locomotor behavior without the descending input from the brain (Courtine et al., 2009). This is because apparent locomotor ability could be explained by up-regulation of the central pattern generator circuits intrinsic to the lumbar spinal cord (GrahamBrown, 1911; Grillner and Zangger, 1984; Barbeau and Rossignol, 1987; De Leon et al., 1998). Thus, to demonstrate the regeneration of the injured spinal cord, researchers are now required to show that the voluntary control has been recovered, and that the descending tract, either from the cerebral cortex or brainstem, was surely re-connected to the neurons caudal to the lesion either directly or indirectly via the intercalated neurons, anatomically and electrophysiologically (van den Brand et al., 2012). To demonstrate the voluntary control, movement repertories which require more volitional control, such as forelimb reach and grasp movements, or ladder walk with varying step intervals or bipedal locomotion (over the obstacle) should be tested. Collectively, for further development of the treatment against the spinal cord injury, understanding the recovery process at the systems level is necessary (Baker, 2011). In this review article, we will introduce our studies during the last decade on the reorganization of large-scaled neuronal network which occurs during functional recovery after the partial cervical spinal cord injury in the non-human primate model. It will be shown that remarkable reorganization occurs not only at the spinal cord level (Sasaki et al., 2004; Nishimura et al., 2009), but also in higher hierarchical levels in the central nervous system (Nishimura et al., 2007a,b, 2011; Nishimura and Isa, 2012).

2. Analysis of neural mechanism of recovery in partial spinal cord injury model It is well known that the corticospinal tract plays a major role in the control of dexterous hand movements (Tower, 1940; Liu and Chambers, 1964; Lawrence and Kuypers, 1968). Especially, the direct connection from the motor cortex to spinal motoneurons, direct cortico-motoneuronal (CM) connection, developed only in higher primates and paralleled the evolution of fractionated digit movements (Bernhard and Bohm, 1954; Heffner and Masterton, 1975, 1983; Lemon, 2008). Damage to the corticospinal tract, either by stroke or by spinal cord injury, etc., causes severe deficit in hand movements, which is one of the most serious complaints of the patients with such neuronal damage and should be one of the major targets of therapeutic strategies against the injury

(Anderson, 2004). On the other hand, spinal motoneurons are not the sole target of the corticospinal tract; actually majority of the corticofugal fibers are terminated in the brainstem level, and in the spinal cord, many of the corticospinal fibers are targeted not only on the motoneurons but also interneurons in the intermediate zone and dorsal horn (Kuypers, 1981; Bortoff and Strick, 1993; Dum and Strick, 1991, 1996; Yoshino-Saito et al., 2010). In cats, which lack direct CM connection, the shortest pathway from the motor cortex to forelimb motoneurons is disynaptic, and significant portion of the cortical disynaptic excitation of motoneurons is mediated by propriospinal neuorns (PNs) in the midcervical segments (Alstermark and Lundberg, 1992; Alstermark and Isa, 2012). Recently, it has been shown that in addition to the CM connection, there is an indirect route through the PNs in the intermediate zone of the midcervical segments, which mediate cortical command to motoneurons of hand and arm muscles also in macaque monkeys (Alstermark et al., 1999; Isa et al., 2006). Recently, it has been demonstrated that these PNs are involved in the control of dexterous digit movements in normal monkeys by applying viral vector-mediated pathway-selective and reversible transmission blocking technique (Kinoshita et al., 2012). To study the role of the indirect route in the functional recovery after lesion of the direct CM connection, specific lesion was made to the lateral corticospinal tract (l-CST) at the C5 segment of the spinal cord by transecting the dorsolateral funiculus, where the cluster of the corticospinal fibers is located. This lesion transected the direct corticospinal input to hand and arm motoneurons located in the ventral horn of the C6Th1 segments and to segmental interneurons in the same segments as these motoneurons, while the indirect input to motoneurons via the PNs were mostly left intact, because the descending axons of the PNs are located in the ventral part of the lateral funiculus and lateral part of the ventral funiculus. Interestingly, after the lesion, the precision grip of the monkeys was impaired, however through rehabilitative training, it showed remarkable recovery in a few weeks to a few months after the lesion. Success ratio of the precision grip recovered to higher than 90% during this period (Sasaki et al., 2004; Nishimura et al., 2007a, 2009). Postmortem studies of these monkeys which experienced the recovery, by both electrophysiological and neuroanatomical analyses, indicated no regeneration of the corticospinal fibers over the injured tissue of the spinal cord. When the corticospinal tract was severed at the brainstem level (Lawrence and Kuypers, 1968), the precision grip was permanently impaired. Comparing these two different types of lesions to the corticofugal fibers, it is suggested that the PNs located rostral to the C5 segment play a major role in the recovery of hand dexterity. When the recovery of animals which initiated training immediately after the lesion was compared with those which were not trained at all during the first one month after the lesion, the ability of fractionated digit movement was by far better in the former group than in the latter group of animals, suggesting that early rehabilitative training is important (Sugiyama et al., 2013; Higo, 2014). We have been using this recovery model to study how the change in the large scaled network of the monkey brain could be the basis for functional recovery after the partial spinal cord injury. The results of the analysis at different level of the neural systems such as the spinal cord, cerebral cortex and further beyond, will be described. 2.1. Spinal cord Electrophysiological analysis was conducted to investigate the transmission from the brainstem pyramid to spinal motoneurons in the animals after recovery. The monkeys which received the C5 l-CST lesion and showed recovery of precision grip was anesthetized and the acute electrophysiological experiments were performed. Stimulating electrodes were placed on both sides of the brainstem pyramid and intracellular recordings were made

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from forelimb motoneurons in the C6–C8 segments, including deep radial motoneurons, and the effects of the pyramidal stimulation were investigated. Interestingly, disynaptic or oligosynaptic excitatory postsynaptic potentials (EPSPs) were found in about half of the motoneurons on the affected side (Sasaki et al., 2004). In normal monkeys, it was necessary to administer strychnine intravenously to reduce the glycinergic inhibition to unmask such di- or oligosynaptic pyramidal EPSPs in motoneurons (Alstermark et al., 1999). Such di- or oligosynaptic EPSPs were mostly abolished by additional lesion to the l-CST at the C2 segment, suggesting that these EPSPs were mediated by intercalated neurons caudal to the C2 segment, namely the PNs. These results suggested that the efficacy of signal transmission from the motor cortex to motoneurons through the PNs were enhanced, either by strengthening the excitatory connection from the motor cortex to the PNs, or PNs to motoneurons, or reduction in inhibition onto the PNs or motoneurons. Physiological and neuroanatomical evidence for such plastic change is currently pursued by our group. On the other hand, the functional coupling between the primary motor cortex (M1) and spinal motoneurons was investigated along the course of lesion and recovery by recording the local field potential in the hand and arm area of the M1 and electromyography of hand and arm muscles (Nishimura et al., 2009). While the monkeys were performing the force-controlled precision grip task with the index finger and thumb, the cortico-muscular coherence at the ␤band frequency (peak at 17 Hz) could be recorded as previously reported in non-human primates and human subjects (Baker et al., 1997; Mima and Hallett, 1999; Mima et al., 2001). The corticomuscular coherence disappeared completely after the C5 l-CST lesion and did not recover at all even three months after the lesion, when behavioral performance in the precision grip task was recovered to nearly 100% level. On the other hand, musculo-muscular coherence at the ␥-band (peak at 33 Hz), which was not observed before the lesion, appeared and increased in parallel to the recovery of performance in the precision grip task. The coherent activity was observed over a large number of forelimb muscles, from proximal to distal. Causality of such coherent activity to the functional recovery was not demonstrated, however, the time course of growth of the coherence was closely related to that of the functional recovery, which suggested that the mechanism might be linked to the recovery process. The oscillatory activity at this frequency range could not be recorded in the electrodes implanted in the M1 and surrounding cortical areas. These results suggested that the ␥-band musculo-muscular coherence should be derived through reorganization of the subcortical structures such as the spinal cord and brainstem. Neurophysiological basis of such reorganization will be an interesting target in future studies. 2.2. Cerebral cortex During the course of experiments, we had to make treatment of the animals which recovered from the spinal cord injury. Before the anesthesia, the monkey appeared to have completely recovered, however when anesthesia was introduced to these animals with ketamine, in some monkeys, as they became drowsy, the paralysis appeared again in the affected forelimb, suggesting the recovered side of the brain required higher level of activation to maintain the recovered condition of the forelimb. Driven by such observation, we initiated the experiments to study the brain activation related to the reach and grasp movements in our recovery model from the partial spinal cord injury. Brain imaging with positron emission tomography (PET) with H2 15 O was performed in three animals during performance of the reach and precision grip task in three macaque monkeys. Before injury, higher activation was observed in the sensorimotor cortex and fronto-parietal stream on the contralateral side to the

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moving arm (Nishimura et al., 2007b). After the lesion, during the early stage of recovery (1 month after the lesion), when the performance of precision grip recovered above 80% of the success ratio, activation was increased compared to the intact state in the hand representation areas of the bilateral sensorimotor cortex. In contrast, at the late stage of recovery (3–4 months after the lesion), when the recovery was more stabilized, the amplitude of activation was enhanced and activated area was expanded in the contralesional sensorimotor cortex. In addition to the sensorimotor cortex, higher activation was found in the bilateral ventral premotor cortex (PMv) (see Fig. 1). Interestingly, the increased activation in the ipsilesional M1 (ipM1), which was observed at the early recovery stage, disappeared. Thus, the activation area was shifted depending on the recovery stage. However, just with such observations, it was not clear whether the activated areas really contributed to the recovery. To demonstrate the causal relationship between the higher activation in each area detected by the PET scan and the functional recovery, we made reversible inactivation of these cortical areas (bilateral M1 and bilateral PMv) with microinjection of muscimol, a GABAA receptor agonist, during preoperative, early recovery and late recovery stages. Inactivation of contralesional M1 (coM1) caused severe deficit in reach and precision grip behavior all through the three stages. Especially, the deficit was most severe when the coM1 was inactivated during the early recovery stage, which indicated that the recovery mechanism at this stage was significantly dependent on the activity of the coM1. Interestingly, the effect of inactivation of the coM1 was moderate or weaker at the late recovery stage, suggesting that the other areas besides the center of digit area of the coM1, such as PMv or M1 area surrounding the digit area might have participated in the recovery. Inactivation of the ipM1 caused no deficit at the preoperative stage, however, interestingly, the same manipulation caused deficit in the precision grip at the early recovery stage. But at the late recovery stage, the inactivation of ipM1 caused no effect. These results suggested that the ipM1, which does not play a significant role in the control of hand movements in the normal condition (see also a recent work by Soteropoulos et al., 2011), is critically involved in the control of dexterous digit movements only during the early recovery stage, which well fitted with the results of the PET imaging. A recent study by Jankowska and colleagues showed that in cats, stimulation of ipsilateral brainstem pyramid induce oligosynaptic excitation of hindlimb motoneurons, presumably via contralateral reticulospinal tract and commissural spinal interneurons when excitability of the pathway was facilitated by intravenous administration of 4-aminopyridine (Jankowska et al., 2006). These results suggest that there are latent indirect pathways from the ipsilateral motor cortex to limb motoneurons which are usually masked by inhibition. In the monkeys with C5 l-CST lesion, we could often observe the mirror movements, which might also suggest the activation of the ipM1 (unpublished result). Thus, some sort of disinhibitory mechanism operates during the early stage after lesion and enables the latent pathways to help the recovery of the whole system. Inactivation of both contralesional PMv (coPMv) and ipsilesional PMv (ipPMv) caused no consistent deficit in two monkeys in both preoperative and early recovery stages. However, inactivation of ipPMv caused deficit consistently in two monkeys at the late recovery stage. Although it is known that the PMv projects to cervical segments in monkeys (Bortoff and Strick, 1993; Dum and Strick, 1991; Borra et al., 2010), whether the PMv controls the hand movements, either via the direct spinal projection, or via the M1 (Schmidlin et al., 2008) is still elusive. These results clarified that coM1, ipM1 and ipPMv plays significant contribution to the different aspects of recovery. Investigation of plasticity-related gene expression such as GAP-43 by in situ hybridization showed enhanced expression of GAP-43 in the laminae II/III of the M1 and PMv and large cells (presumably the corticofugal neurons)

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Fig. 1. Schematic diagram of the change in the large scaled network during the recovery from the spinal cord injury (C5 l-CST lesion). For simplicity, the supraspinal structures on the contralateral side (“contra”) are placed on the same side as the spinal motoneurons (MNs) in this figure. (A) Before lesion (intact, left panel), corticospinal axons, primarily originating from the M1, control the motoneurons (MNs) innervating the hand muscles via the direct CM connection and through the pathway via the propriospinal neurons (PNs). Contribution of PMv to direct control of muscle activity would not be significant. The limbic structures such as the nucleus accumbens (NAc), anterior cingulate cortex (ACC) and the orbitofrontal cortex (OFC) do not cooperate with the M1 and/or PMv. The descending pathway from the ipsilateral M1 to the MNs might be mediated either by the reticulospinal neurons (RSNs) or more directly spinal projection, but the pathway is supposed to be inhibited. (B) During the early recovery stage, bilateral M1 is activated. Inhibition of the descending effects from the ipM1 should have been unmasked. ipM1 would presumably control the MNs via the reticulospinal neurons (RSNs). The NAc, ACC and OFC cooperate with the M1 and/or PMv. (C) During the late recovery stage, larger number of M1 neurons become involved in the control of hand muscles, presumably via indirect route through PNs. Then, the pathway from the ipM1 should be inhibited again. In addition, the activity of bilateral PMv is enhanced, but the pathway which mediates the activity to motoneurons is not clear yet (either via cortico-cortical connection to M1, or more direct, descending pathways to subcortical centers). The NAc, ACC and OFC cooperate much with the M1 and/or PMv. Thus, during the recovery process, the activity of coM1 becomes closely linked to the contralateral NAc and other limbic centers. Dotted lines in this figure indicate that existence of the pathways has been suggested but not empirically demonstrated. Updated from the Fig. 4 in Nishimura and Isa (2009).

in M1 on both sides in the animals with C5 l-CST lesion (Higo et al., 2009). These results suggest that the change in activation of bilateral M1 and PMv accompanies the morphological change with neurite extension in the cortico-cortical association circuits and descending corticofugal pathways originating from these areas. In addition, secreted phosphoprotein 1 (SPP1), also known as osteopontin, which is specifically expressed in, which is specifically expressed in the corticospinal neurons (especially high expression in M1) of higher primates (not expressed in rats, domestic pigs and common marmosets), increased their expression in the PMv during the recovery from the C5 l-CST lesion (Yamamoto et al., 2013). These results also suggested the involvement of PMv in the direct control of hand movements during recovery. Detailed mechanism should be a future target of the research.

2.3. And beyond In the brain imaging study of recovery process by using PET, in addition to the cortical areas as described above, we found increased activation in the contralesional ventral striatum including the nucleus accumbens (NAc) during recovery from the C5 l-CST lesion (Nishimura et al., 2007a). Higher activation was observed during the late recovery stage than during the early stage. The ventral striatum comprises the neural circuits for the control of motivation together with other limbic structures such as the anterior cingulate cortex (ACC) and the orbitofrontal cortex (OFC). As the first step to explore the contribution of these limbic structures in the recovery mechanisms, we evaluated the connectivity among the coM1, NAc, ACC and OFC during the preoperative stage and during recovery by analyzing the correlation between the regional cerebral blood flow during each task sessions between individual areas (Nishimura et al., 2011). It was turned out that virtually no significant correlation could be found preoperatively in all

the three monkeys tested, except for a weak correlation (p < 0.05) between the coM1 and ACC in one monkey. However, during the recovery, significantly strong connectivity could be detected in a variety of combinations among these areas. Thus, these limbic structures became closely correlated with the motor cortex (coM1) not under the normal condition, but only during the recovery from the spinal cord injury (Fig. 1). It is well known that the ventral striatum including NAc and M1 is indirectly connected in both directions (Ohnishi et al., 2004; Miyachi et al., 2005). In the present case, it was not clear which direction of the signal flow played a critical role in the cooperative activation of both areas. To show the causal involvement of these limbic structures in the recovery process, manipulation of activities in the ventral striatum at preoperative stage and different stages during the recovery, and clarifying the neural pathways underlying such interaction between the motor-related structures and limbic structures are essential in the future studies.

3. Conclusion and future perspective As summarized in this review (see Fig. 1), reorganization of neuron circuit and change in activation occurs at various hierarchical levels in the central nervous system during the spontaneous functional recovery after the spinal cord injury. Cortical reorganization after the spinal cord injury has also been reported in other recent reports (Endo et al., 2007; Ghosh et al., 2009). After the C5 l-CST lesion, the precision grip recovered to almost normal level through the time course of a few months. However, even in this type of recovery model, which showed near-complete recovery, the structure and function of the brain is no longer the same as the preoperative state; the pattern of oscillatory activity of muscles changed (Nishimura et al., 2009), relationship between the M1 and limbic system changed (Nishimura et al., 2011), etc. All

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these observations described in this review would suggest that for treatment of the spinal cord injury, rewiring the spinal pathways across the injury is not enough, but rewiring the whole networks in an appropriate manner should be necessary. In this regard, more critical evaluation of the recovery process, both physiological and neuroanatomical, is required for the researches to develop the therapeutic strategies in the animal models. Furthermore, therapeutic strategies to facilitate the network reorganization, such as neurorehabilitation and neuromodulation using the neuroprosthetic devices, should be combined with the cellular and molecular therapies, such as the stem cell graft and drug administrations (Freund et al., 2006; Brown and Weaver, 2011; García-Alías and Fawcett, 2011). For the future research, appropriate evaluation of the recovery should be critically made. The effort to standardize the process of evaluating the results of therapeutic researches would be the short-cut for the development of evidence-based therapies against the spinal cord injury.

Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Synapse Neurocircuit Pathology” (No. 25110735 to T.I.) by the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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