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Transhemispheric functional reorganization of the motor cortex induced by the peripheral contralateral nerve transfer to the injured arm
Neuroscience 138 (2006) 1225–1231
TRANSHEMISPHERIC FUNCTIONAL REORGANIZATION OF THE MOTOR CORTEX INDUCED BY THE PERIPHERAL CONTRALATERAL NERVE TRANSFER TO THE INJURED ARM L. LOU,a T. SHOU,a,c* Z. LI,b W. LIb AND Y. GUb
with voluntary movements of the healthy hand. Subsequently, over a period of several years, some patients are able to move their injured hand without voluntary movement of the healthy hand (Gu et al., 1992, 1998; Gu and Shen, 1994). In rats with the same operation, we observed a similar process after transferring the C7 nerve. It is of interest to know the corresponding changes in the brain function after the operation and relative mechanisms. We hypothesize that there is extensive transhemispheric functional reorganization in the motor cortex that drives muscles in the injured limb after transferring the C7 nerves. Here, we tested this hypothesis in adult rats.
a
Vision Research Laboratory, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China b Department of Hand Surgery, Hua-shan Hospital, Fudan University, 12 Middle Wulumoqi Road, Shanghai 200040, China c
State Key Laboratory of Medical Neuroscience, Fudan University, 138 Yi Xue Road, Shanghai 200032, China
Abstract—Peripheral nerve injury in a limb usually causes functional reorganization of the contralateral motor cortex. However, a dynamic process of the novel transhemispheric functional reorganization in the motor cortex was found in adult rats after transferring the seventh cervical nerve root from the contralateral healthy side to the injured limb. Initially the ipsilateral motor cortex activated the injured forepaw for 5 months after the operation. Then, both hemispheres of the cortex activated the injured forepaw, and finally the contralateral cortex exclusively controlled the injured forepaw. It is concluded an extensive functional shift occurred between two hemispheres based on neural plasticity in the CNS. The experimental results of the later lesions of the ipsilateral cortex suggest that maintaining transhemispheric functional reorganization does not depend on the corpus callosum, but depends on mechanisms involving central axonal sprouting. Possible mechanisms underlying the alternative changes in cortical functions were discussed in rats and in patients having similar operations. © 2005 Published by Elsevier Ltd on behalf of IBRO.
EXPERIMENTAL PROCEDURES Experiments were conducted on 35 adult Sprague–Dawley (SD) rats. All investigations involving animals adhered to the Policy of Society for Neuroscience on the Use of Animals in Neuroscience Research. All animal use procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals described by the U.S. National Institutes of Health, and all experiments were designed to minimize the number of animals used and their suffering.
Animal preparation Group I. Rats with a unilateral nerve root avulsion of the left brachial plexus. Each rat was anesthetized by an i.p. injection of sodium pentobarbital (40 –50 mg/kg, Shanghai Reagent Company, Shanghai, China). While the rat was in a prostrate position, an incision was made from the occiput to the scapular angulus superior about 4 cm in length. The longissimus capitis cervicis muscle, semispinalis cervicis muscle, digastricus muscle, and complexus muscle were drawn to one side. The muscles on the processus spinosus and lamina arcus vertebrae were removed, and the nerves from C5 to T1 were exposed. The lamina arcus vertebrae from C5 to T1 on the left side were removed using pliers. The spinal cord was gently pulled to the left side by a hook. The left radix dorsalis and radix ventralis from C5 to T1 were exposed, and the roots from C5 to T1 were avulsed from the spinal cord. A segment about 2 mm in length in each ends from C5 to T1 nerves were cut off under a microscope.
Key words: motor cortex, hemisphere, organization, corpus callosum, sprouting.
Peripheral deafferentation produced by peripheral nerve lesions or forepaw amputations often leads to extensive functional reorganization of the contralateral motor cortex (Kolarik et al., 1994; Chen et al., 1998; Qi et al., 2000; Donoghue, 1995; Sanes and Donoghue, 1997). Arm paralysis induced by brachial plexus root avulsions has been widely treated by transferring a healthy cervical seventh (C7) nerve from the contralateral since 1986 (Gu et al., 1992, 1998; Gu and Shen, 1994). The C7 nerve forms the middle trunk of the brachial plexus. When it is transferred, the muscles previously innervated by it are still supplied by fibers in the upper and the lower trunk (Gu et al., 1992). Clinically, at the early stage after anastomosis, a patient is able to coordinately move his or her affected hand only
Group II. Rats with a nerve root avulsion of the brachial plexus and contralateral C7 nerve transfer. Anesthesia and root avulsion of the brachial plexus was identical to that done for Group I. Like the operation that have been done in patients (Gu et al., 1992, 1998; Gu and Shen, 1994), the nerve transfer operation in rats is shown schematically in Fig. 1. The proximal median nerve and ulnar nerve on the left side were sectioned under the axilla. They are coapted to each other, as shown by double arrows in Fig. 1. The distal ulnar nerve was dissected at the left wrist. The ulnar nerve with the collateralis ulnaris superior artery on the left side was used for transplantation. The root of the C7 nerve was cut off on the right side. The distal terminal of the left ulnar nerve with the collateralis ulnaris superior artery was moved to the contralateral
*Correspondence to: T. Shou, Vision Research Laboratory School of Life Science, Fudan University, 220 Handan Road, Shanghai 200433, China. Tel: ⫹86-21-65642355; fax: ⫹86-21-65643528. E-mail address: [email protected] (T. Shou). Abbreviations: C7, the seventh cervical nerve. 0306-4522/06$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.11.062
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Fig. 1. Diagram showing the operation of the contralateral (right) C7 nerve root transfer to connect to nerves of the injured hand in a rat like previously reported. Firstly, all the five brachial plexus roots of C5–C8 and T1 on the left side (the injured side) were totally pulled out and cut as shown on the left of the figure. Then, the normal C7 root was cut in the right side and anastomosed to the distal terminal of the ulnar nerve that was peeled off from the left injured side (the dashed lines denoting its original position of the ulnar nerve and the double arrows denoting the connecting position) and moved to the right side. Finally, the proximal terminal of the left ulnar nerve was coapted with the near terminal of the proximal median nerve injured (the double arrows indicating the connecting position). Here the ulnar nerve was employed as a graft of C7 to the median nerve to save limb function. All the operation was carried out in rats by careful microsurgery under a microscope.
body through the s.c. tissue of the front chest and connected with the terminal of the right C7 nerve root on the right side, as shown by another double arrow mark in Fig. 1. At the end of experiments of each rat the injured brachial plexus was checked. Data of any rat failed in complement of discontinuing C5–T1 nerve root in the left limb were not included in the study. The rats in different periods from 3 months to 16 months after the above operations were anesthetized with a single injection of sodium pentobarbital in an initial dose of 40 –50 mg/kg, i.p. and then maintained in a dose of 2.5– 4.0 mg/kg · h i.v. for continuous sedation. The animal’s body temperature was maintained at 37 °C using an animal body temperature controller (SS20-2, Dajiang Electronic Company, Bangbu, Anhui Province, China) adjusting the current of an electric heating pad according to rectal temperature. The rat was placed in a stereotaxic instrument (Jiangwan Type I-C, The Affiliated Instrument Factory of the Second Military Medical University, Shanghai, China) and then a portion of the left parietal and frontal bones of the skull was removed between 6 mm anterior to and 4 mm posterior to Bregma which corresponds to Horsley-Clarke coordinates A9.0, L0, and from 0 to 4.5 mm lateral to the midline. The dura was cut and retracted, and 37 °C mineral oil was poured over the cortex to prevent the cortex from dehydration.
motor representations in steps of 0.5 mm. The stimulus electrode was lowered perpendicularly to a depth of 1.8 mm below the cortical surface. In preliminary experiments, this depth was found to correspond to layer V of the frontal cortex. The penetration locations were examined by viewing the histology section. Mono-phasic cathodic pulse trains (train length 75 ms, pulse duration 250 s at temporal frequency 200 Hz) generated by a constant current stimulator (Model: U-ML180-OG-02A, MacLab, Powerlab, AD Instruments, Castle Hill, New South Wales, Australia) were used to evoke movement of the forepaw or the body. The threshold current for each tested point in motor cortex was defined as the minimal stimulating current that evoked a visible movement of a forepaw through adjusting the volume of current.
Intracortical microstimulation
Cortical lesion
Varnish-insulated tungsten microelectrodes with tip impedances of 0.2–1.0 M⍀ were used for cortical stimulation. The cortical surface covering the area between 5 mm anterior to and 1 mm posterior to Bregma and from 0.5– 4 mm lateral to the midline was tested to map
After the representation of the affected forepaw had been mapped in both motor cortices or exclusively in the contralateral cortex in two rats (one rat survived for 7 months after the operation, another rat for 10 months), the cortical area ipsilateral to the affected
Map construction Motor cortical maps were constructed by mapping the location of the penetrations in which electrical stimuli evoked a visible movement of the forepaw. Movements of other body parts such as the mouth or vibrissae were also elicited in cortical areas adjacent to the forelimb areas. These non-forepaw representations were not mapped in great detail, but served to delimit the boundaries of the forepaw representations.
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and regeneration. In contrast, all rats in Group I with total brachial plexus root avulsion failed to grasp with their injured forepaws throughout the experiments. The observers who measured grasping force were unaware of the previous treatments and behavioral observations. Dynamic changes in functional reorganization
Fig. 2. Behavioral measure of the grasping force of the rats’ injured forepaws at different periods after the operation. Error bars indicate standard deviation. To test the grasping force of a rat, the forepaw was allowed to hold a metal frame of 400 g that is fixed on an electronic balance. The maximum change in weight resulted from lifting the rat’s tail up indicated the grasping force of the forepaw tested once the forepaw released from the frame.
forepaw was ablated by aspirating the whole cortex up to the white matter with a glass pipette. A small piece of sterile absorbable gelatin sponge, soaked with normal saline, was then placed in the cavity to prevent bleeding. The representation of the affected forepaw in the contralateral motor cortex was measured again.
RESULTS Animal model and behavioral observation Transferring the contralateral C7 nerve root to the nerves of the injured hand was carried out in rats as shown in Fig. 1. Then, rats were reared to examine the function of the cortical area controlling the forepaw at different stages after the operation. Behavioral observations of the rats in Group I for reaching a goal were in an unstable manner, using the three healthy limbs and the injured left forelimb handing down all the time. They ate food with significant difficulty with the only right uninjured forelimb. These behavioral performances were maintained throughout the period of the experiment without exception. In contrast, the rats in Group II showed improvement in behavioral performance. They started to be able to reach a goal and manipulate food since the 5th month after the C7 nerve root transfer. They were able to run and could manipulate food by using the left injured forelimb combined with the other three. Until the 8th to 10th months, most rats performed the above tasks easily. The grasping force of the injured forepaw was measured using a method reported previously (Bertelli et al., 1995; Bertelli and Mira, 1995; Papalia et al., 2003). In eight rats of Group II, as shown in Fig. 2, grasping force initially appeared in the 5th month after the operation and reached about 17% of normal (about 300 g before the operation) another 3 months later. The grasping force recovered to about 2/3 (about 200 g) of normal in the 10th month after the operation. This progressive recovery of the injured forepaw indicates the success of establishing an animal model for the study of the contralateral C7 nerve transfer
In normal rats, the two forepaws were represented contralaterally in each motor cortex when electrical stimulation was used to evoke forepaw motion (Fig. 3A). In Group I, we failed to find any motion of the left forelimb when stimulating the motor cortex on either side after avulsing and cutting the left C5–T1 nerve roots. Similarly, in Group II, there was no representation of the injured forepaw in motor cortex on either side at a time less than 5 months after the operation. This indicates that the innervation of the transferred C7 nerve to the muscles of the left forepaw had not been completed in the injured limb while the left cortex controlled the right forepaw normally suggesting that cutting the right C7 did not affect control of the right limb significantly. Furthermore, in the right cortex, the area controlling neck and hindlimb extended significantly into the former forelimb area, like previous observations (Qi et al., 2000; Sanes et al., 1990; Donoghue et al., 1990; Kaas et al., 1990; Merzenich and Jenkins, 1993). From the 5th to the 7th months after the operation, the injured forepaw showed evoked movements only when stimulating the motor cortex on the ipsilateral side in five rats of Group II (Fig. 3B1, B2), but no motion was found in any rat of Group I when stimulating the cortex on either side. These facts indicate that regeneration of the transferred C7 axons had innervated some muscles in the left injured limb. Thus, of five rats in Group II, the injured forepaw was controlled by the ipsilateral motor cortex (L) but not by the contralateral side (R). The right healthy limb continued to be activated through the intact C5, C6, C8 and T1 nerves when the contralateral cortex was stimulated as shown in L= for comparison (see Fig. 3B, 3C, 3D). Thus, the left injured limb and the right limb whose C7 was transferred to the left side were both controlled by the left motor cortex simultaneously. In the remaining four rats of Group II, however motion of the injured forepaw was evoked by stimulating the motor cortex on either side before the 10th month after the operation (Fig. 3C1, C2). Accordingly, the injured forepaw was represented bilaterally in both the ipsilateral (L) and contralateral (R) primary motor cortex, in contrast to the maps in Fig. 3A. Note that the representation of the injured forepaw lay in the previously silent areas of the contralateral cortex (R) and extended into some positions of the healthy forelimb representation in the ipsilateral cortex (L=) simultaneously. Meanwhile, the movement of right limb was still controlled by left cortex through the intact C5, C6, C8 and T1 nerves. Obviously, in these nine rats mentioned above, the left motor cortex innervates the two limbs although how the contralateral cortex controls the injured limb in the latter four rats is unknown. This is similar to the clinical observations of patients with C7 transfer operations, in which they must initially move their intact limb in
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Fig. 3. Normal representation and six examples of the injured left forepaw representations in the primary motor cortex of the left (L) and the right (R) hemispheres. The healthy right forepaw representation in the left (L=) motor cortex are also shown as comparison. (A) Bilateral representations of two forepaws exclusively shown in each contralateral side of the primary motor cortex of a normal rat. These maps served as references of the representation of these peripheral parts of the body that was to be damaged. The size and shape of the forepaw representations varied somehow between hemispheres and animals. Positions of microelectrode stimuli that elicited the vibrissae, neck, mouth, hindlimb and forepaw movements were shown to delimit the boundary between them. () Cortical area responsible only for movement of forepaw, i.e. the wrist and digit flexion, or wrist intorsion. (●) Area for the forelimb movement in addition to the wrist and digit flexion, or wrist intorsion. Areas for movements of (□) the vibrissae, (Œ) the neck and (〫) the mouth, and (”) the hindlimb. (●) Area in which no detectable movement was observed when stimulated with electrical current. Hereinafter, the same meanings of the symbols are used for all figures in the paper. (B) Examples of two rats observed 5 months after the operation. Note that the injured forepaw representation lay only in the ipsilateral motor cortex (L) but not in the contralateral side (R). Besides, the representation of the injured limb entered forth inside positions of the healthy forelimb representations in the motor cortex in the ipsilateral side, as shown in L= as a reference. Thus, the two limbs shared the control of movement by ipsilateral hemisphere. (C) Functional maps of motor cortex of two rats observed 7–10 months after the operation. The injured forepaw representation appeared in both the ipsilateral (L) and contralateral (R) primary motor cortex, in contrast to those maps in A and B. Note that the representation of the injured forepaw lay in the previously silent areas of the contralateral cortex (R) of deafferentation and also covered some positions of the healthy forelimb representation in the ipsilateral cortex (L=). (D) Functional maps of the motor cortex of two rats obtained 10 months after the operation showing that the injured forepaw representation only lay in the contralateral motor cortex (R), but not in the ipsilateral side (L). Furthermore, the representation of the injured limb entered in the previously silent area caused by the deafferentation in the left limb (R). Meantime, the healthy forepaw representation was only shown in the left motor cortex (L=). Thus, the two forepaw representations of the rat appeared to be contralaterally distributed like those in normal rats, except the significant difference in threshold of electrical stimulating current used that elicit a minimum movement of a forepaw.
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Role of the corpus callosum Furthermore, the experiments of lesion in the left motor cortex in two rats (one showing bilateral cortical control of the injured forepaw and another rat showing sole contralateral control) were carried out in the period of 7–10 months after the operation. Ablation of the ipsilateral motor cortex did not prevent the contralateral cortex from evoking movements in the injured forepaw, without exception. Furthermore the cortical representation area and stimulus threshold current for evoking motion of the injured forepaw in the contralateral cortex were identical before and after the lesion. This suggests that the corpus callosum is not needed to maintain the function of the transhemispheric reorganization. Fig. 4. Threshold current ratios of the injured forepaw and the healthy forepaw for rats whose left ipsilateral motor cortex was able to be stimulated to activate both forepaws simultaneously after the contralateral C7 nerve transfer to the left injured limb. The column drawn by thick lines indicates the mean threshold current ratio for rats tested. Note that all ratios of nine rats were larger than 1 and the mean threshold current ratio denoted at left was 1.76, indicating that to initiate a minimal movement of the injured forepaw is always more difficult than to that of the healthy forepaw.
order to move their injured limb. However, the mean threshold current for evoking a minimum movement of the injured forepaw by stimulating the left cortex (38.2⫾5 A) was significantly higher than that of the intact forepaw (32⫾5 A) (t-test, P⫽0.045⬍0.005) (Fig. 4). This fact indicates that the transferred C7 axons from the right side have innervated some muscles in the injured limb, but the nerve regeneration is incomplete. Surprisingly, from the 8th to the 16th month after the operation, the injured forepaw of five rats in Group II could move exclusively by stimulating the contralateral motor cortex (Fig. 3D1, D2). Apparently, the injured forepaw representation had transferred to the contralateral motor cortex on the right side. Furthermore, the representation of the injured limb only reappeared in the previously silent area of the right cortex (R) that was caused by the deafferentation of the left limb. Meantime, the healthy forepaw representation was only found in the left motor cortex (L=). Thus, the representations of the rat’s two forepaws appeared to be contralaterally distributed like those in normal rats and the electrical current thresholds needed to elicit a minimum movement of the contralateral forepaw were similar for the right and the left hemispheres (34⫾24 A and 37⫾22 A respectively, P⫽0.83⬎0.05). This suggests that for these five rats there might be a functional shift from the left to right motor cortex in activating the C7 motoneurons in spinal cortex. Some descending nerves that were previously driven by the left ipsilateral hemisphere may withdraw from the right C7 motoneurons and a new innervation of descending nerves driven by the contralateral hemisphere of the injured limb may control the C7 motoneurons instead. This is in agreement with the behavior of some patients who can move their both limbs independently in several years after their C7 transfer treatment.
DISCUSSION In this study, we demonstrated a process of dynamical transhemispheric control of the injured limb caused by a peripheral contralateral nerve transfer. The process matched well with the animal behavioral and clinical observations after the operation, though there are some differences in the corticomotoneuronal pathway between rats and humans, suggesting possible differences in mechanisms underlying the cortical reorganization in the two species. The results also indicate that the corpus callosum does not play a role in maintaining the transhemispheric reorganization of the motor cortex, although it may play a possible role in establishing the functional shift initially. Peripheral lesion-induced change in cortical maps may result from reorganization at cortical and/or subcortical levels. In the somatosensory and visual systems, functional reorganization can occur at both cortical (Merzenich and Jenkins, 1993; Darian-Smith and Gilbert, 1994, 1995) and many subcortical sites, such as the thalamus, brain stem and spinal cord (Florence and Kaas, 1995; Davis et al., 1998). Sprouting of corticospinal axon terminals was found in redirected corticospinal axons after unilateral lesions of the sensorimotor cortex in neonatal rats (Rouiller et al., 1991). While most of these studies were done in postnatal or young animals, our study was concluded in adult rats, we found that the movement of the injured forepaw was driven first by the ipsilateral hemisphere, then by both hemispheres, and finally by the contralateral hemisphere only. It is easy to understand the initial ipsilateral control of the injured forepaw because the cross-transferred contralateral C7 was driven by the ipsilateral cortex. However, the mechanism underlying the transhemispheric cortical control in rats may not be the same as in patients. In humans, the number of direct corticomotoneural connections is very large, while in rats, nearly all the descending excitation in forelimb motoneurons are di- or polysynaptic via a corticoreticulospinal pathway, corticospinal fibers, and segmental interneurons indirectly (Alstermark et al., 2004). Possible mechanisms underlying the observation in the rats might be likely that shortly after the operation, movements in the left limb evoked by the left motor cortex produce sensory feedback signals, presumably via various levels of structures, including the corpus
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callosum, to the right hemisphere. This starts off a slow process whereby the right cortex increases the gain of already existing connections to the motoneurons of the C7 root on the right side that has been transferred to innervate the left limb. On the other hand, during the process, axonal sprouting and formation of functional synapses may occur in the indirect pathways. In fact, it was suggested, based on analysis of excitatory postsynaptic potentials in cat red nucleus, that sprouting and new synapses appeared on the proximal portion of the soma-dendritic membrane of the red nucleus neurons following cross-innervation of forelimb flexor and extensor nerves (Tsukahara et al., 1982; Fujita et al., 1982). As soon as the transhemispheric reorganization is established the contralateral control of the left limb may start to work independently from the corpus callosum. The direct corticospinal tract in human may play a more important role in functional reorganization than the indirect corticospinal pathways in the rat. Presumably the reorganization process may involve both an increase in the gain of the right hemisphere to the ipsilateral corticospinal control of existing connections to the transferred C7 root and the rewiring of the contralateral projection of major corticospinal tract to recross the middle line of the spinal cord to reach the motoneurons of the transferred C7 root, as shown in Fig. 5. If these do occur in humans, the enhanced right corticospinal fibers and the sprouting of the recrossed axons from the left may trigger the mechanism of withdrawing the left corticospinal fibers from the motoneurons of the transferred C7. They may play an essential role in establishing the new contralateral control of the injured limb in patients at later stage. There are several lines of evidence showing that the recrossing corticospinal tract axons occur either in the white or in the gray matter of the spinal cord following a unilateral lesion in the motor cortex in neonatal rats and hamsters (Kuang and Kalil, 1990; Rouiller et al., 1991; Joosten et al., 1992; Barth and Stanfield, 1990), or in patients (Carr et al., 1993). It is possible that the axons of the right corticospinal tract establish their synapses with the C7 motoneurons accompanying a gradual withdrawal of the left corticospinal axons from these motoneurons via competition though this remains to be confirmed further. Besides, it should be mentioned that indirect corticospinal pathways, such as the corticoreticulospinal and corticorubrospinal tracts, may also contribute to the functional reorganization in humans. Although the rat model might not be ideal for studying nerve transfer mechanisms in humans, the cortical plasticity that appeared in the adult rats is an interesting phenomenon for further study. Using a transhemispheric magnetic stimulator (TMS) and functional magnetic resonance imaging (fMRI) to probe corticospinal excitability in patients following a similar operation might provide a way to elucidate the mechanisms in humans. Rapidly induced changes in motor or sensory representations are reported through mechanisms involving modulation of GABAergic inhibition (Jacobs and Donoghue, 1991; Welker et al., 1989). Long-term changes may involve multiple mechanisms such as long-term potentia-
Fig. 5. Diagram showing the hypothetic sprouting appeared in somewhere of spinal cord in patients with an operation of the contralateral (right) C7 nerve root transfer to connect to nerves of the injured hand. The gain of the right hemisphere is increased unilaterally to the ipsilaterally corticospinal control of the already existing connections to the motoneurons of the transferred C7 root by axonal sprouting. The rewiring of the contralateral projection of the right major corticospinal tract recrosses the middle line of the spinal cord to reach the motoneurons of the transferred C7 root in the gray and the white matter, in addition to axonal sprouting and formation of functional synapses occurring in the indirect pathways (not shown here).
tion, axonal regeneration, and formation of new synapse (Chen et al., 2002). When somatosensory cortex is damaged, its basic sensory functions are taken over by the corresponding contralateral area. This transhemispheric cortical reorganization is reported to involve in cholinoreceptors and adrenoreceptors (Zarei and Stephenson, 2000; Zarei et al., 2001). Whether similar molecular mechanisms underlie the transhemispheric reorganization that we observed remains to be elucidated. Acknowledgments—We would like to thank Dr. Yoshikazu Shinoda at Tokyo Medical and Dental University in Japan for critical reviewing and helpful suggestions and to Dr. Kevin D. Alloway at Penn State University College of Medicine in the USA for his assistance in polishing English. This work was supported by grants of from the National Natural Science Foundation of China (No.90208013), the Laboratory of Visual Information Processing, Institute of Biophysics, Chinese Academy of Sciences, and the Ministry of Education of China (No.20010246071).
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(Accepted 29 November 2005) (Available online 19 January 2006)
TRANSHEMISPHERIC FUNCTIONAL REORGANIZATION OF THE MOTOR CORTEX INDUCED BY THE PERIPHERAL CONTRALATERAL NERVE TRANSFER TO THE INJURED ARM L. LOU,a T. SHOU,a,c* Z. LI,b W. LIb AND Y. GUb
with voluntary movements of the healthy hand. Subsequently, over a period of several years, some patients are able to move their injured hand without voluntary movement of the healthy hand (Gu et al., 1992, 1998; Gu and Shen, 1994). In rats with the same operation, we observed a similar process after transferring the C7 nerve. It is of interest to know the corresponding changes in the brain function after the operation and relative mechanisms. We hypothesize that there is extensive transhemispheric functional reorganization in the motor cortex that drives muscles in the injured limb after transferring the C7 nerves. Here, we tested this hypothesis in adult rats.
a
Vision Research Laboratory, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China b Department of Hand Surgery, Hua-shan Hospital, Fudan University, 12 Middle Wulumoqi Road, Shanghai 200040, China c
State Key Laboratory of Medical Neuroscience, Fudan University, 138 Yi Xue Road, Shanghai 200032, China
Abstract—Peripheral nerve injury in a limb usually causes functional reorganization of the contralateral motor cortex. However, a dynamic process of the novel transhemispheric functional reorganization in the motor cortex was found in adult rats after transferring the seventh cervical nerve root from the contralateral healthy side to the injured limb. Initially the ipsilateral motor cortex activated the injured forepaw for 5 months after the operation. Then, both hemispheres of the cortex activated the injured forepaw, and finally the contralateral cortex exclusively controlled the injured forepaw. It is concluded an extensive functional shift occurred between two hemispheres based on neural plasticity in the CNS. The experimental results of the later lesions of the ipsilateral cortex suggest that maintaining transhemispheric functional reorganization does not depend on the corpus callosum, but depends on mechanisms involving central axonal sprouting. Possible mechanisms underlying the alternative changes in cortical functions were discussed in rats and in patients having similar operations. © 2005 Published by Elsevier Ltd on behalf of IBRO.
EXPERIMENTAL PROCEDURES Experiments were conducted on 35 adult Sprague–Dawley (SD) rats. All investigations involving animals adhered to the Policy of Society for Neuroscience on the Use of Animals in Neuroscience Research. All animal use procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals described by the U.S. National Institutes of Health, and all experiments were designed to minimize the number of animals used and their suffering.
Animal preparation Group I. Rats with a unilateral nerve root avulsion of the left brachial plexus. Each rat was anesthetized by an i.p. injection of sodium pentobarbital (40 –50 mg/kg, Shanghai Reagent Company, Shanghai, China). While the rat was in a prostrate position, an incision was made from the occiput to the scapular angulus superior about 4 cm in length. The longissimus capitis cervicis muscle, semispinalis cervicis muscle, digastricus muscle, and complexus muscle were drawn to one side. The muscles on the processus spinosus and lamina arcus vertebrae were removed, and the nerves from C5 to T1 were exposed. The lamina arcus vertebrae from C5 to T1 on the left side were removed using pliers. The spinal cord was gently pulled to the left side by a hook. The left radix dorsalis and radix ventralis from C5 to T1 were exposed, and the roots from C5 to T1 were avulsed from the spinal cord. A segment about 2 mm in length in each ends from C5 to T1 nerves were cut off under a microscope.
Key words: motor cortex, hemisphere, organization, corpus callosum, sprouting.
Peripheral deafferentation produced by peripheral nerve lesions or forepaw amputations often leads to extensive functional reorganization of the contralateral motor cortex (Kolarik et al., 1994; Chen et al., 1998; Qi et al., 2000; Donoghue, 1995; Sanes and Donoghue, 1997). Arm paralysis induced by brachial plexus root avulsions has been widely treated by transferring a healthy cervical seventh (C7) nerve from the contralateral since 1986 (Gu et al., 1992, 1998; Gu and Shen, 1994). The C7 nerve forms the middle trunk of the brachial plexus. When it is transferred, the muscles previously innervated by it are still supplied by fibers in the upper and the lower trunk (Gu et al., 1992). Clinically, at the early stage after anastomosis, a patient is able to coordinately move his or her affected hand only
Group II. Rats with a nerve root avulsion of the brachial plexus and contralateral C7 nerve transfer. Anesthesia and root avulsion of the brachial plexus was identical to that done for Group I. Like the operation that have been done in patients (Gu et al., 1992, 1998; Gu and Shen, 1994), the nerve transfer operation in rats is shown schematically in Fig. 1. The proximal median nerve and ulnar nerve on the left side were sectioned under the axilla. They are coapted to each other, as shown by double arrows in Fig. 1. The distal ulnar nerve was dissected at the left wrist. The ulnar nerve with the collateralis ulnaris superior artery on the left side was used for transplantation. The root of the C7 nerve was cut off on the right side. The distal terminal of the left ulnar nerve with the collateralis ulnaris superior artery was moved to the contralateral
*Correspondence to: T. Shou, Vision Research Laboratory School of Life Science, Fudan University, 220 Handan Road, Shanghai 200433, China. Tel: ⫹86-21-65642355; fax: ⫹86-21-65643528. E-mail address: [email protected] (T. Shou). Abbreviations: C7, the seventh cervical nerve. 0306-4522/06$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.11.062
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Fig. 1. Diagram showing the operation of the contralateral (right) C7 nerve root transfer to connect to nerves of the injured hand in a rat like previously reported. Firstly, all the five brachial plexus roots of C5–C8 and T1 on the left side (the injured side) were totally pulled out and cut as shown on the left of the figure. Then, the normal C7 root was cut in the right side and anastomosed to the distal terminal of the ulnar nerve that was peeled off from the left injured side (the dashed lines denoting its original position of the ulnar nerve and the double arrows denoting the connecting position) and moved to the right side. Finally, the proximal terminal of the left ulnar nerve was coapted with the near terminal of the proximal median nerve injured (the double arrows indicating the connecting position). Here the ulnar nerve was employed as a graft of C7 to the median nerve to save limb function. All the operation was carried out in rats by careful microsurgery under a microscope.
body through the s.c. tissue of the front chest and connected with the terminal of the right C7 nerve root on the right side, as shown by another double arrow mark in Fig. 1. At the end of experiments of each rat the injured brachial plexus was checked. Data of any rat failed in complement of discontinuing C5–T1 nerve root in the left limb were not included in the study. The rats in different periods from 3 months to 16 months after the above operations were anesthetized with a single injection of sodium pentobarbital in an initial dose of 40 –50 mg/kg, i.p. and then maintained in a dose of 2.5– 4.0 mg/kg · h i.v. for continuous sedation. The animal’s body temperature was maintained at 37 °C using an animal body temperature controller (SS20-2, Dajiang Electronic Company, Bangbu, Anhui Province, China) adjusting the current of an electric heating pad according to rectal temperature. The rat was placed in a stereotaxic instrument (Jiangwan Type I-C, The Affiliated Instrument Factory of the Second Military Medical University, Shanghai, China) and then a portion of the left parietal and frontal bones of the skull was removed between 6 mm anterior to and 4 mm posterior to Bregma which corresponds to Horsley-Clarke coordinates A9.0, L0, and from 0 to 4.5 mm lateral to the midline. The dura was cut and retracted, and 37 °C mineral oil was poured over the cortex to prevent the cortex from dehydration.
motor representations in steps of 0.5 mm. The stimulus electrode was lowered perpendicularly to a depth of 1.8 mm below the cortical surface. In preliminary experiments, this depth was found to correspond to layer V of the frontal cortex. The penetration locations were examined by viewing the histology section. Mono-phasic cathodic pulse trains (train length 75 ms, pulse duration 250 s at temporal frequency 200 Hz) generated by a constant current stimulator (Model: U-ML180-OG-02A, MacLab, Powerlab, AD Instruments, Castle Hill, New South Wales, Australia) were used to evoke movement of the forepaw or the body. The threshold current for each tested point in motor cortex was defined as the minimal stimulating current that evoked a visible movement of a forepaw through adjusting the volume of current.
Intracortical microstimulation
Cortical lesion
Varnish-insulated tungsten microelectrodes with tip impedances of 0.2–1.0 M⍀ were used for cortical stimulation. The cortical surface covering the area between 5 mm anterior to and 1 mm posterior to Bregma and from 0.5– 4 mm lateral to the midline was tested to map
After the representation of the affected forepaw had been mapped in both motor cortices or exclusively in the contralateral cortex in two rats (one rat survived for 7 months after the operation, another rat for 10 months), the cortical area ipsilateral to the affected
Map construction Motor cortical maps were constructed by mapping the location of the penetrations in which electrical stimuli evoked a visible movement of the forepaw. Movements of other body parts such as the mouth or vibrissae were also elicited in cortical areas adjacent to the forelimb areas. These non-forepaw representations were not mapped in great detail, but served to delimit the boundaries of the forepaw representations.
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and regeneration. In contrast, all rats in Group I with total brachial plexus root avulsion failed to grasp with their injured forepaws throughout the experiments. The observers who measured grasping force were unaware of the previous treatments and behavioral observations. Dynamic changes in functional reorganization
Fig. 2. Behavioral measure of the grasping force of the rats’ injured forepaws at different periods after the operation. Error bars indicate standard deviation. To test the grasping force of a rat, the forepaw was allowed to hold a metal frame of 400 g that is fixed on an electronic balance. The maximum change in weight resulted from lifting the rat’s tail up indicated the grasping force of the forepaw tested once the forepaw released from the frame.
forepaw was ablated by aspirating the whole cortex up to the white matter with a glass pipette. A small piece of sterile absorbable gelatin sponge, soaked with normal saline, was then placed in the cavity to prevent bleeding. The representation of the affected forepaw in the contralateral motor cortex was measured again.
RESULTS Animal model and behavioral observation Transferring the contralateral C7 nerve root to the nerves of the injured hand was carried out in rats as shown in Fig. 1. Then, rats were reared to examine the function of the cortical area controlling the forepaw at different stages after the operation. Behavioral observations of the rats in Group I for reaching a goal were in an unstable manner, using the three healthy limbs and the injured left forelimb handing down all the time. They ate food with significant difficulty with the only right uninjured forelimb. These behavioral performances were maintained throughout the period of the experiment without exception. In contrast, the rats in Group II showed improvement in behavioral performance. They started to be able to reach a goal and manipulate food since the 5th month after the C7 nerve root transfer. They were able to run and could manipulate food by using the left injured forelimb combined with the other three. Until the 8th to 10th months, most rats performed the above tasks easily. The grasping force of the injured forepaw was measured using a method reported previously (Bertelli et al., 1995; Bertelli and Mira, 1995; Papalia et al., 2003). In eight rats of Group II, as shown in Fig. 2, grasping force initially appeared in the 5th month after the operation and reached about 17% of normal (about 300 g before the operation) another 3 months later. The grasping force recovered to about 2/3 (about 200 g) of normal in the 10th month after the operation. This progressive recovery of the injured forepaw indicates the success of establishing an animal model for the study of the contralateral C7 nerve transfer
In normal rats, the two forepaws were represented contralaterally in each motor cortex when electrical stimulation was used to evoke forepaw motion (Fig. 3A). In Group I, we failed to find any motion of the left forelimb when stimulating the motor cortex on either side after avulsing and cutting the left C5–T1 nerve roots. Similarly, in Group II, there was no representation of the injured forepaw in motor cortex on either side at a time less than 5 months after the operation. This indicates that the innervation of the transferred C7 nerve to the muscles of the left forepaw had not been completed in the injured limb while the left cortex controlled the right forepaw normally suggesting that cutting the right C7 did not affect control of the right limb significantly. Furthermore, in the right cortex, the area controlling neck and hindlimb extended significantly into the former forelimb area, like previous observations (Qi et al., 2000; Sanes et al., 1990; Donoghue et al., 1990; Kaas et al., 1990; Merzenich and Jenkins, 1993). From the 5th to the 7th months after the operation, the injured forepaw showed evoked movements only when stimulating the motor cortex on the ipsilateral side in five rats of Group II (Fig. 3B1, B2), but no motion was found in any rat of Group I when stimulating the cortex on either side. These facts indicate that regeneration of the transferred C7 axons had innervated some muscles in the left injured limb. Thus, of five rats in Group II, the injured forepaw was controlled by the ipsilateral motor cortex (L) but not by the contralateral side (R). The right healthy limb continued to be activated through the intact C5, C6, C8 and T1 nerves when the contralateral cortex was stimulated as shown in L= for comparison (see Fig. 3B, 3C, 3D). Thus, the left injured limb and the right limb whose C7 was transferred to the left side were both controlled by the left motor cortex simultaneously. In the remaining four rats of Group II, however motion of the injured forepaw was evoked by stimulating the motor cortex on either side before the 10th month after the operation (Fig. 3C1, C2). Accordingly, the injured forepaw was represented bilaterally in both the ipsilateral (L) and contralateral (R) primary motor cortex, in contrast to the maps in Fig. 3A. Note that the representation of the injured forepaw lay in the previously silent areas of the contralateral cortex (R) and extended into some positions of the healthy forelimb representation in the ipsilateral cortex (L=) simultaneously. Meanwhile, the movement of right limb was still controlled by left cortex through the intact C5, C6, C8 and T1 nerves. Obviously, in these nine rats mentioned above, the left motor cortex innervates the two limbs although how the contralateral cortex controls the injured limb in the latter four rats is unknown. This is similar to the clinical observations of patients with C7 transfer operations, in which they must initially move their intact limb in
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Fig. 3. Normal representation and six examples of the injured left forepaw representations in the primary motor cortex of the left (L) and the right (R) hemispheres. The healthy right forepaw representation in the left (L=) motor cortex are also shown as comparison. (A) Bilateral representations of two forepaws exclusively shown in each contralateral side of the primary motor cortex of a normal rat. These maps served as references of the representation of these peripheral parts of the body that was to be damaged. The size and shape of the forepaw representations varied somehow between hemispheres and animals. Positions of microelectrode stimuli that elicited the vibrissae, neck, mouth, hindlimb and forepaw movements were shown to delimit the boundary between them. () Cortical area responsible only for movement of forepaw, i.e. the wrist and digit flexion, or wrist intorsion. (●) Area for the forelimb movement in addition to the wrist and digit flexion, or wrist intorsion. Areas for movements of (□) the vibrissae, (Œ) the neck and (〫) the mouth, and (”) the hindlimb. (●) Area in which no detectable movement was observed when stimulated with electrical current. Hereinafter, the same meanings of the symbols are used for all figures in the paper. (B) Examples of two rats observed 5 months after the operation. Note that the injured forepaw representation lay only in the ipsilateral motor cortex (L) but not in the contralateral side (R). Besides, the representation of the injured limb entered forth inside positions of the healthy forelimb representations in the motor cortex in the ipsilateral side, as shown in L= as a reference. Thus, the two limbs shared the control of movement by ipsilateral hemisphere. (C) Functional maps of motor cortex of two rats observed 7–10 months after the operation. The injured forepaw representation appeared in both the ipsilateral (L) and contralateral (R) primary motor cortex, in contrast to those maps in A and B. Note that the representation of the injured forepaw lay in the previously silent areas of the contralateral cortex (R) of deafferentation and also covered some positions of the healthy forelimb representation in the ipsilateral cortex (L=). (D) Functional maps of the motor cortex of two rats obtained 10 months after the operation showing that the injured forepaw representation only lay in the contralateral motor cortex (R), but not in the ipsilateral side (L). Furthermore, the representation of the injured limb entered in the previously silent area caused by the deafferentation in the left limb (R). Meantime, the healthy forepaw representation was only shown in the left motor cortex (L=). Thus, the two forepaw representations of the rat appeared to be contralaterally distributed like those in normal rats, except the significant difference in threshold of electrical stimulating current used that elicit a minimum movement of a forepaw.
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Role of the corpus callosum Furthermore, the experiments of lesion in the left motor cortex in two rats (one showing bilateral cortical control of the injured forepaw and another rat showing sole contralateral control) were carried out in the period of 7–10 months after the operation. Ablation of the ipsilateral motor cortex did not prevent the contralateral cortex from evoking movements in the injured forepaw, without exception. Furthermore the cortical representation area and stimulus threshold current for evoking motion of the injured forepaw in the contralateral cortex were identical before and after the lesion. This suggests that the corpus callosum is not needed to maintain the function of the transhemispheric reorganization. Fig. 4. Threshold current ratios of the injured forepaw and the healthy forepaw for rats whose left ipsilateral motor cortex was able to be stimulated to activate both forepaws simultaneously after the contralateral C7 nerve transfer to the left injured limb. The column drawn by thick lines indicates the mean threshold current ratio for rats tested. Note that all ratios of nine rats were larger than 1 and the mean threshold current ratio denoted at left was 1.76, indicating that to initiate a minimal movement of the injured forepaw is always more difficult than to that of the healthy forepaw.
order to move their injured limb. However, the mean threshold current for evoking a minimum movement of the injured forepaw by stimulating the left cortex (38.2⫾5 A) was significantly higher than that of the intact forepaw (32⫾5 A) (t-test, P⫽0.045⬍0.005) (Fig. 4). This fact indicates that the transferred C7 axons from the right side have innervated some muscles in the injured limb, but the nerve regeneration is incomplete. Surprisingly, from the 8th to the 16th month after the operation, the injured forepaw of five rats in Group II could move exclusively by stimulating the contralateral motor cortex (Fig. 3D1, D2). Apparently, the injured forepaw representation had transferred to the contralateral motor cortex on the right side. Furthermore, the representation of the injured limb only reappeared in the previously silent area of the right cortex (R) that was caused by the deafferentation of the left limb. Meantime, the healthy forepaw representation was only found in the left motor cortex (L=). Thus, the representations of the rat’s two forepaws appeared to be contralaterally distributed like those in normal rats and the electrical current thresholds needed to elicit a minimum movement of the contralateral forepaw were similar for the right and the left hemispheres (34⫾24 A and 37⫾22 A respectively, P⫽0.83⬎0.05). This suggests that for these five rats there might be a functional shift from the left to right motor cortex in activating the C7 motoneurons in spinal cortex. Some descending nerves that were previously driven by the left ipsilateral hemisphere may withdraw from the right C7 motoneurons and a new innervation of descending nerves driven by the contralateral hemisphere of the injured limb may control the C7 motoneurons instead. This is in agreement with the behavior of some patients who can move their both limbs independently in several years after their C7 transfer treatment.
DISCUSSION In this study, we demonstrated a process of dynamical transhemispheric control of the injured limb caused by a peripheral contralateral nerve transfer. The process matched well with the animal behavioral and clinical observations after the operation, though there are some differences in the corticomotoneuronal pathway between rats and humans, suggesting possible differences in mechanisms underlying the cortical reorganization in the two species. The results also indicate that the corpus callosum does not play a role in maintaining the transhemispheric reorganization of the motor cortex, although it may play a possible role in establishing the functional shift initially. Peripheral lesion-induced change in cortical maps may result from reorganization at cortical and/or subcortical levels. In the somatosensory and visual systems, functional reorganization can occur at both cortical (Merzenich and Jenkins, 1993; Darian-Smith and Gilbert, 1994, 1995) and many subcortical sites, such as the thalamus, brain stem and spinal cord (Florence and Kaas, 1995; Davis et al., 1998). Sprouting of corticospinal axon terminals was found in redirected corticospinal axons after unilateral lesions of the sensorimotor cortex in neonatal rats (Rouiller et al., 1991). While most of these studies were done in postnatal or young animals, our study was concluded in adult rats, we found that the movement of the injured forepaw was driven first by the ipsilateral hemisphere, then by both hemispheres, and finally by the contralateral hemisphere only. It is easy to understand the initial ipsilateral control of the injured forepaw because the cross-transferred contralateral C7 was driven by the ipsilateral cortex. However, the mechanism underlying the transhemispheric cortical control in rats may not be the same as in patients. In humans, the number of direct corticomotoneural connections is very large, while in rats, nearly all the descending excitation in forelimb motoneurons are di- or polysynaptic via a corticoreticulospinal pathway, corticospinal fibers, and segmental interneurons indirectly (Alstermark et al., 2004). Possible mechanisms underlying the observation in the rats might be likely that shortly after the operation, movements in the left limb evoked by the left motor cortex produce sensory feedback signals, presumably via various levels of structures, including the corpus
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callosum, to the right hemisphere. This starts off a slow process whereby the right cortex increases the gain of already existing connections to the motoneurons of the C7 root on the right side that has been transferred to innervate the left limb. On the other hand, during the process, axonal sprouting and formation of functional synapses may occur in the indirect pathways. In fact, it was suggested, based on analysis of excitatory postsynaptic potentials in cat red nucleus, that sprouting and new synapses appeared on the proximal portion of the soma-dendritic membrane of the red nucleus neurons following cross-innervation of forelimb flexor and extensor nerves (Tsukahara et al., 1982; Fujita et al., 1982). As soon as the transhemispheric reorganization is established the contralateral control of the left limb may start to work independently from the corpus callosum. The direct corticospinal tract in human may play a more important role in functional reorganization than the indirect corticospinal pathways in the rat. Presumably the reorganization process may involve both an increase in the gain of the right hemisphere to the ipsilateral corticospinal control of existing connections to the transferred C7 root and the rewiring of the contralateral projection of major corticospinal tract to recross the middle line of the spinal cord to reach the motoneurons of the transferred C7 root, as shown in Fig. 5. If these do occur in humans, the enhanced right corticospinal fibers and the sprouting of the recrossed axons from the left may trigger the mechanism of withdrawing the left corticospinal fibers from the motoneurons of the transferred C7. They may play an essential role in establishing the new contralateral control of the injured limb in patients at later stage. There are several lines of evidence showing that the recrossing corticospinal tract axons occur either in the white or in the gray matter of the spinal cord following a unilateral lesion in the motor cortex in neonatal rats and hamsters (Kuang and Kalil, 1990; Rouiller et al., 1991; Joosten et al., 1992; Barth and Stanfield, 1990), or in patients (Carr et al., 1993). It is possible that the axons of the right corticospinal tract establish their synapses with the C7 motoneurons accompanying a gradual withdrawal of the left corticospinal axons from these motoneurons via competition though this remains to be confirmed further. Besides, it should be mentioned that indirect corticospinal pathways, such as the corticoreticulospinal and corticorubrospinal tracts, may also contribute to the functional reorganization in humans. Although the rat model might not be ideal for studying nerve transfer mechanisms in humans, the cortical plasticity that appeared in the adult rats is an interesting phenomenon for further study. Using a transhemispheric magnetic stimulator (TMS) and functional magnetic resonance imaging (fMRI) to probe corticospinal excitability in patients following a similar operation might provide a way to elucidate the mechanisms in humans. Rapidly induced changes in motor or sensory representations are reported through mechanisms involving modulation of GABAergic inhibition (Jacobs and Donoghue, 1991; Welker et al., 1989). Long-term changes may involve multiple mechanisms such as long-term potentia-
Fig. 5. Diagram showing the hypothetic sprouting appeared in somewhere of spinal cord in patients with an operation of the contralateral (right) C7 nerve root transfer to connect to nerves of the injured hand. The gain of the right hemisphere is increased unilaterally to the ipsilaterally corticospinal control of the already existing connections to the motoneurons of the transferred C7 root by axonal sprouting. The rewiring of the contralateral projection of the right major corticospinal tract recrosses the middle line of the spinal cord to reach the motoneurons of the transferred C7 root in the gray and the white matter, in addition to axonal sprouting and formation of functional synapses occurring in the indirect pathways (not shown here).
tion, axonal regeneration, and formation of new synapse (Chen et al., 2002). When somatosensory cortex is damaged, its basic sensory functions are taken over by the corresponding contralateral area. This transhemispheric cortical reorganization is reported to involve in cholinoreceptors and adrenoreceptors (Zarei and Stephenson, 2000; Zarei et al., 2001). Whether similar molecular mechanisms underlie the transhemispheric reorganization that we observed remains to be elucidated. Acknowledgments—We would like to thank Dr. Yoshikazu Shinoda at Tokyo Medical and Dental University in Japan for critical reviewing and helpful suggestions and to Dr. Kevin D. Alloway at Penn State University College of Medicine in the USA for his assistance in polishing English. This work was supported by grants of from the National Natural Science Foundation of China (No.90208013), the Laboratory of Visual Information Processing, Institute of Biophysics, Chinese Academy of Sciences, and the Ministry of Education of China (No.20010246071).
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(Accepted 29 November 2005) (Available online 19 January 2006)