Deep Brain Stimulation of the Midbrain Locomotor Region Improves Paretic Hindlimb Function After Spinal Cord Injury in Rats

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1 SPINAL CORD INJURY Deep Brain Stimulation of the Midbrain Locomotor Region Improve Paretic Hindlimb Function After Spinal Cord Injury in Rat Luka C. Bachmann, 1, Alina Mati, 1, Nicola T. Lindau, 1, Petra Felder, 1, Miriam Gullo, 1, Martin E. Schwab 1, In evere pinal cord injurie, the tract conveying motor command to the pinal cord are dirupted, reulting in paralyi, but many patient till have mall number of pared fiber. We have found that excitatory deep brain timulation (DBS) of the meencephalic locomotor region (MLR), an important control center for locomotion in the brain, markedly improved hindlimb function in rat with chronic, evere, but incomplete pinal cord injury. The medial medullary reticular formation wa eential for thi effect. Functional deficit of rat with to 3% pared reticulopinal fiber were comparable topatientabletowalkbutwithtrongdeficitin trength and peed [for example, individual with American Spinal Injury Aociation Impairment Scale (AIS) D core]. MLR DBS enabled cloe to normal locomotion in thee rat. In more extenively injured animal, with le than 1% pared reticulopinal fiber, hindlimb were almot fully paralyzed, comparable to wheelchair-bound patient (for example, AIS-A, B, and C). With MLR DBS, hindlimb function reappeared under gravity-releaed condition during wimming. We propoe that therapeutic MLR DBS uing the brain own motor command circuit may offer a potential new approach to treat peritent gait diturbance in patient uffering from chronic incomplete pinal cord injury. 1 Brain Reearch Intitute, Univerity of Zurich, 85 Zurich, Switzerland. Department of Health Science and Technology, ETH Zurich, Winterthurertrae 19, 85 Zurich, Switzerland. Correponding author. bachmann@hifo.uzh.ch INTRODUCTION After an incomplete pinal cord injury, both human and experimental animal often uffer from evere motor impairment including complete paralyi. Although in mot of the patient, ome tiue bridge and nerve fiber remain at the leion ite, about half of all pinal cord injured patient remain bound to a wheelchair for the ret of their live, and many of the le evere cae continue to uffer from locomotor impairment. Becaue of the preence of rhythm generator in the lumbar pinal cord, which are referred to a locomotor central pattern generator (CPG) (1, ), even fully iolated cat or rat pinal cord can be timulated to produce locomotor activity pattern (3 5). However, to function in a phyiological context, they are highly dependent on the decending control ytem coming from the brain, which include command ytem for pecific action a well a global excitatory input from the brain that regulate the baic level of activation of the pinal circuit (, ). Several experimental treatment have been introduced recently with the aim to recover motor control and locomotion after pinal cord injury in animal model. Promoting growth of interrupted fiber (8 1) orengraftment ofcell(11) to reconnect the uprapinal command center to ubleional neuron can induce ignificant recovery of function by retoring the miing control from the brain. Some of thee therapie are now in clinical trial in pinal cord injured patient (1 1). Thee trial are motly done in newly injured patient. However, induction of growth and new circuit formation in individual with chronic pinal cord injury who have been living with the injury for year i even more challenging (1). Reactivation of the inactive lumboacral network below a pinal cord injury by local pinal cord timulation with epidural or intrapinal electrode (15 1), application of neuromodulator (18 ), or combination thereof () have yielded promiing reult in rat and cat and are now being teted in the firt patient with chronic pinal cord injury (1). Whether uch global, nonpecific timulation of the pinal cord can lead to the retoration of phyiological movement pattern under voluntary control that help patient in their daily life activitie remain to be een. For mot of the pinal cord injured patient, rehabilitative training, which can include treadmill locomotor training for patient with ufficient paring of decending tract, remain the only option currently. Training can trigger change within the lumbar network that may allow for more autonomou function (3,,, 3), and decending ignal carried by pared fiber may be ued more effectively through mechanim of motor relearning (9, ). In rat, training can alo trigger anatomical retructuring of pared fiber termination (9,, 5). However, the effect of claical rehabilitation for impaired leg function are limited for the more everely injured patient a evidenced by the large number of wheelchair-bound patient who leave the rehab clinic. A trategy that wa o far not conidered i to reetablih locomotorfunctionbyincreaingthelocomotorcontrolcommandcarried by pared fiber after a large but anatomically incomplete pinal cord injury. Claical experiment howed that the midbrain tegmentum harbor a pecific locomotor command center that i conerved from fih to man (, ). In animal, a whole et of locomotor movement can be induced by timulating thi meencephalic locomotor region (MLR). Neverthele, even after complete removal of the forebrain in cat (), rodent (8), or alamander (9), thee tereotyped movement can be elicited. Decorticated nonhuman primate can move around (3), and anencephalic newborn how infant tepping (31). Hence, depite a modulatory role of the forebrain in locomotion in higher animal (3, 33), baic locomotion i initiated and maintained by ytem contained in the midbrain, the braintem, and pinal cord 3 October 13 Vol 5 Iue 8 8ra1 1

2 (3). Thee part compoe the evolutionarily ancient core of our brain. Studie with partial pinal cord tranection in animal howed that paring of mall part of the white matter including braintem reticulopinal fiber i aociated with coniderable recovery of locomotion (35, 3). The braintem reticular formation, when timulated optogenetically in vitro (3) or chemically in vivo (38), can induce rhythmic activity of the lumbar CPG network. Two of the key region of the MLR inducing and controlling locomotion upon electrical timulation in decerebrated animal are the cuneiform (CnF) and the pedunculopontine nucleu (PPN) (, 8), although their relative involvement in the oberved effect remain a matter of debate (, 39). The MLR receive input from the baal ganglia and the ubtantia nigra () and therefore may be one of the ite where locomotion-related ignal from the forebrain converge and are propagated to the braintem and pinal CPG network (1, ). Neuron of the MLR project and ignal to the medial medullary reticular formation and the raphe nuclei of the medulla oblongata (3 5). The locomotion-inducing effect of MLR timulation critically depend on the function of thee part of the reticular formation that give rie to a large part of the reticulopinal tract (, ). Here, we tudied the therapeutic manipulation of the MLR in pinal cord injury uing a deep brain timulation (DBS) approach. We how that increaing timulation of the adult rat MLR at phyiological frequencie induce a controllable, gradual activation of walking or wimming in intact animal. In rat with evere pinal cord injurie, MLR timulation immediately improved the reidual locomotor performance of the paretic hindlimb and reulted in the reappearance of fully or partially functional hindlimb movement. A C Ipilateral Contralateral B RESULTS The medial medullary braintem i the main ource of input to the lumbar pinal cord and receive input from the MLR The importance of the braintem in controlling the hindlimb i well illutrated by the number of decending fiber een by retrograde axonal tracing (uing Fat Blue tracer) from the lumbar pinal cord. Fat Blue i taken up by axon at the injection ite and label the oma of neuron by retrograde axonal tranport. In rat, more than % of all retrogradely labeled cell were found in the medulla oblongata, motly in the gigantocellular (NRGi) and magnocellular (NRGiA) reticular nuclei, and the raphe nuclei (8, 9), with only about 15% of the labeled lumbar-projecting neuron located in the neocortex (Fig. 1A). Thee projection are comparable to thoe of other mammal (9, ). Contralateral Ipilateral D Ipilateral Contralateral M1 S M1 S Vet HyT Me NRGi RN MLR MLR Pon Raphe NRGiA PAG mm Med 5 mm 5 mm Raphe : FatBlue injection unilateral, left pinal L : FatBlue injection unilateral, left NRGi / NRGiA L E SC F PAG L-EMG IC R-EMG CnF Me5 Stim 5 PPN L-EMG L-tim R-EMG mm Fig. 1. Braintem reticular nuclei dominate the input to the pinal cord and are driven by the MLR. (A) Recontruction of cell projecting to the left half of pinal egment L a hown by retrograde labeling (top view). M1, primary motor cortex; S, econdary enory cortex; HyT, hypothalamu; Me, meencephalic reticular formation; RN, red nucleu; Pon, pontine reticular formation; Med, medullary reticular formation and vetibular nuclei; M, agittal midline. Cell number are hown a hitogram on the left (bin,.1 mm; maximum, 13 cell). (B) Peak region in the rotral medulla oblongata (gray hading) i hown a a projection on a coronal ection plain. Vet, vetibular nuclei (horizontal bin,.1 mm; maximum, 1 cell; vertical bin,. mm; maximum, 391 cell). (C) Input to the reticular formation of the left ventral medulla oblongata a hown by retrograde labeling (top view). The highet number of neuron i found in the meencephalic tegmentum including the MLR nuclei (bin,.1 mm; maximum, cell). (D) Thee (gray-haded area) are hown a a projection on a coronal ection plain (horizontal bin,.1 mm; maximum, 393 cell; vertical bin,. mm; maximum, 1 cell). (E) Schematic view of the MLR. Orange quare, ite of timulation. IC, inferior colliculu; SC, uperior colliculu; Me5, meencephalic trigeminal nucleu; Stim, ite of MLR timulation. (F) Electromyographic (EMG) recording of hamtring mucle of an anethetized animal with left MLR timulation (L-tim) of. 3 October 13 Vol 5 Iue 8 8ra1

3 Two-third of the braintem-pinal neuron project ipilateral to the agittal midline, with mot neuron in cloe proximity to the midline (Fig. 1B). Similar to the moue(51), a very pare, ipilateral, direct projection from the MLR to the lumbar (L) region of the pinal cord wa een(fig.1a).to map the input to the reticular medullary nuclei NRGi andnrgia,weinjectedtheretrogradetracertereotacticallyintothee nuclei. In Fig. 1 (C and D), the tronget input come from the meencephalon, that i, from the periaqueductal gray (PAG) and the more lateral CnF and PPN nuclei and the colliculi (, 5). CnF and PPN comprie the claical MLR (, 53). The overall bilateral projection from the MLR to the medial medullary reticular formation how an ipilateral dominance (Fig. 1D) (). Hence, the indirect projection ytem repreent a prominent neuronal pathway tranmitting motor command ignal from the MLR to lumbar CPG network (, ). We therefore tudied thi ytem phyiologically in intact rat and in animal with incomplete pinal cord injury. A poible role of direct MLR-pinal projection remain unclear (51). DBS of the MLR modulate peed in different form of rat locomotion In patient with Parkinon dieae(pd), DBS, particularly of baal ganglia circuit, i a therapeutic approach ued to help alleviate motor dyfunction. It uually conit of high-frequency pule (for example, 1 ) that are believed to hut down neuronal activity (5). Here, we ued phyiological timulation parameter (- cathodal pule) in rat to drive neuronal activity of the MLR uing chronically implanted DBS electrode. Under anetheia, continuou unilateraltimulationofthemlrinnormalrat (Fig. 1E, orange quare) evoked rhythmic hindlimb tepping (Fig. 1F). Depite the predominantly ipilateral MLR to pinal cord pathway (Fig. 1, A to D), motly contralateral induction of tepping wa oberved. Ipilateral tep were weaker, le table, or abent. Forelimb remained in a emiflexed reting poition. A different ituation wa obtained in awake, freely moving animal. Unilateral MLR timulation induced regular, ymmetric tepping of both fore- and hindlimb (Fig. A). Compared to nontimulated animal, which occaionally get ditracted and top walking in the teting device, even lowet timulu intenity induced an uninterrupted walking acro the runway, demontrating the trong locomotor drive evoked by DBS in the MLR. Increaing intenity reulted in increaed walking peed A Walking Walking Walking Hindpaw footprint pattern (Fig. B; P < 1, repeated-meaure ANOVA, between column) paralleled by higher tep cycle frequency (Fig. C; P < 1). The overall range of hindlimb movement increaed only minimally (Fig. D; P < 1), wherea total tride length increaed coniderably (Fig. E; P < 1). Hence, peed i mainly influenced by the frequency and length of the tep. Linearly increaed timulation intenity led to a witch from alternating walking to gallop at about.5% of maximal timulation, reflected % 5% % % 1% B E H Walking peed % of maximal timulation % 5% % % 1% Stride length Swimming C F Fract. of tride length I Stepping frequency Speed at %.8 n = 13.9 Individual animal: D J Motion relative to hip 5.5 Stroke frequency 1 1 Spatial tep aynchrony G Temporal tep aynchrony Swimming peed Fract. of tep duration Fig.. MLR modulate the trength of locomotor output in intact, awake animal. (A)Repreentative cheme of intact, non-anethetized animal howing a gradual tranition from walking to gallop with increaing MLR timulation (indicated in % of maximal timulation). (B to E) Thi i reflected in an increaed walking peed (B), increaing frequency of tep (C), a minor increae in the range of hindlimb motion (D), and an increaed tride length (E). (F and G) The reduction of patial and temporal aynchrony of tep indicate the witch from walk to gallop around.5% of maximal timulation. (H) Repreentative hindpaw footprint pattern howing the tranition from walking to gallop. (I and J) Alo, the wimming peed (I) increaed with higher MLR timulation trength accompanied by gradually increaed wim troke frequency (J). (B to J) X axi indicate percentage of maximal timulation (158 ± 98 ma). Individual animal are color-coded according to their walking peed at % timulation from blue to dark red. P < 5, P < 1, P < 1, one-way analyi of variance (ANOVA) with Dunnett multiple comparion to % timulation (n = 13). 3 October 13 Vol 5 Iue 8 8ra1 3

4 in marked change in the patial and temporal ynchrony of the footprint pattern (Fig., F to H; P < 1 for both). During wimming, MLR DBS reulted in an increae in wimming peed at higher timulation trength and a gradual increae in hindlimb troke frequency (Fig., I and J; P < 1 for both). The typical wimming poture and movement were maintained at all timulation frequencie. MLR DBS in freely moving intact animal thu induce forward locomotion appropriate to contextual requirement, that i, walking or wimming, indicating the global role of the MLR in the production of locomotion. The unilateral MLR DBS at efficiently activate a uprapinally driven locomotor program. Spinal cord injurie induce peritent deficit in temporal execution and trength of tep All animal in the leion cohort decribed above howed almot complete initial paralyi of both hindlimb. The right hindlimb aumed a patically extended poture in the firt week after the injury. Over week, ome hindlimb function returned, but the gait remained highly abnormal and impaired (Fig. A). Thi deficit i comparable to that of patient who are unable to either execute weight-bearing tepping or regain a trongly impaired gait. Walking peed of the pinal cord injured rat, depite almot full initial hindlimb paraplegia and abdominal dragging, wa not ignificantly impaired becaue of the compenation by the forelimb (Fig. B). The hindlimb movement cycle (Fig., C to E) pontaneouly improved over time, but the impaired protraction reflected a remaining deficit Fiber of different decending tract in rat with large pinal cord leion are pared We next teted whether MLR DBS improve locomotion in 11 rat with large, but incomplete, pinal cord leion, with about 8% of the total white and gray matter cut at thoracic level T1. Spared tiue on one ide only wa located in the dorolateral (DLF) and ventrolateral (VLF) funiculi(fig.3,itos;leionofindividual rat). Taking the location of the individual decending fiber tract, a hown by anterograde tracing (Fig. 3, A to H), into account, the fraction of pared fiber for each tract could be etimated for each individual animal (Fig. 3T). On average, in thi cohort,.9 ±.% of the corticopinal tract (CST) wa remaining. The rubropinal tract (RST) wa variably pared becaue of variable dorolateral tiue paring (. ± 1.8%). Vetibulopinal (VST) and meencephalic reticulopinal (MESReST) tract were reduced to ±.% and 11. ±.% becaue of the leion of the ventromedial funiculi. Pontine (PReST) and medullary (MEDReST) reticulopinal tract retained 5. ± 5.9% and 5. ± 5.% of preleional projection. The erotonin-poitive raphepinal tract (5HT) wa reduced to 9.3 ± 9.%, and tyroine hydroxylae (TH) poitive dopamine (DA), noradrenaline (NA), and adrenaline (A) tract were reduced to 15.9 ± 8.% of intact value. Overall, 1 to 5% of the pinal cord cro ection wa pared, preerving only the left lateral funiculu. The medullary reticular formation retained about a fourth of initial projection to the ubleional pinal cord. White matter paring Decending pathway A B C D E F G H I J K L N O P T Q R S Fraction of intact M Etimated pared fiber Fig. 3. Poition of pinal cord decending pathway and extent of leion/white matter paring in rat with incomplete pinal cord injury. (A to H) Ditribution of the major pinally projecting fiber tract at thoracic level T1 hown a color-coded two-dimenional hitogram (left ide of pinal cord cro ection; pixel ize,. mm; color repreent percentage of all fiber within pixel) and anatomical location of fiber (right ide, compoite view of two hemicord). (I to S) Coronal projection of pared (white) and leioned (gray) part of the pinal cord at T1 in the 11 rat (labeled I to S) analyzed phyiologically (Fig. and 5). Percent indicate the fraction of the pared area of the pinal cord. Animal are color-coded according to their leion ize from blue (leat pared) to red (mot pared; ame animal are color-coded in Fig. and 5). (T) Etimation of the pared fraction of the major pinally projecting fiber tract. Color code repreent the animal from (I) to (S). 3 October 13 Vol 5 Iue 8 8ra1

5 Fig.. Impaired walking and wimming in rat with large, incomplete pinal cord injurie. Rat howed paring of 1 to 5% of the pinal cord cro ection, which correpond to evere AIS-D leion in human pinal cord injury patient. (A) Repreentative cheme of a walking animal at preleional baeline (), day pot-operation (dpo) with hindlimb dragging, dpo, and 8 dpo. The leion caued firt a flaccid then a patic overextenion of the hindlimb that reolved after about week when ome hindlimb walking movement reappeared. (B) Becaue of trong compenation by the forelimb, the walking peed wa little affected by the leion. (C to E) Motionofthe paw relative to the hip. The ability to bring the limb forward (D) and to retract it (E) remained trongly impaired. (F to H)Temporal execution of hindlimb tep wa impaired after injury, cauing a lower movement of the paw. (I) Height of the hip above the runway wa reduced and tayed deficient, particularly in thoe animal with the larget leion, illutrating a weakne of antigravitational trength. (J) Speed with which the paw wa brought forward (protractive paw peed) remained impaired, indicating a peritent deficit in dynamic trength. (K) Paw dragging wa complete after injury and recovered only minimally. (L) Repreentative cheme of a wimming animal at, dpo, dpo, and 8 dpo. animal generated propelling force with the hindlimb excluively; the forelimb remained immobile and flexed under the chin. After pinal cord injury, animal wam mainly with the forelimb. (M) Swimming peed depended on the forelimb andremainedlow.(n to P) The movement pattern of the hindlimb in water recovered relatively well, although (a in walking) the protraction of the foot remained lightly deficient. (Q to S) Temporal propertie of hindlimb troke were everely impaired after injury. (T and U) Propulive paw peed (T) and the peed (U) with which the paw i brought forward after full extenion (recovery paw peed) remained low. (A to U) The animal from Fig. 3 (I to S) are repreented according to the percentage of pared white matter tiue at T1 from blue (leat pared) to red (mot pared). P < 5, P < 1, P < 1, one-way ANOVA with Dunnett multiple comparion to. Swimming Walking A dpo D H... dpo 8 dpo Protraction to hip Swing duration Retraction to hip 8 L dpo O S dpo 8 dpo Protraction to hip Recovery duration E I Hip height P Retraction to hip T Propulive paw peed B F J Walking peed Day after injury Step frequency Protractive paw peed.8. Q M Swimming peed. U.. Stroke frequency Recovery paw peed Day after injury C 8 G... K.8... Fract. paw contact N R 8..1 Motion to hip % pared Stance duration Paw dragging % % 1% Motion to hip Propulion duration Individual animal: n = 11 (Fig. D). The temporal execution of the tep cycle remained impaired (Fig.,FtoH),wherebytancedurationrecovered(Fig.G),butthe time needed to protract wa and remained deficient (Fig. H). Hip height at midtance, reflecting the trength of the antigravity mucle, remained partially deficient (Fig. I). Initially, animal dragged the whole abdomen over the runway, and at week after the injury, they 3 October 13 Vol 5 Iue 8 8ra1 5

6 till dragged their paw extenively (Fig. K). Marked deficit remained in the peed with which the paw wa brought forward in the wing phae (Fig. J). Swimming allow for locomotion in a gravity-releaed environment, which i commonly ued in phyiotherapy a a rehabilitative tool that allow for movement otherwie overhadowed by the failure of antigravitational trength. In contrat to walking, coniderable hind- A % 5% % % 1% B % 5% % % 1% Retraction to hip 8 9 F Protraction to hip E Range of motion to hip 1 Walking D Walking peed C % of maximal timulation R Propulion duration S Recovery duration T Propulive paw peed U Recovery paw peed 1.15 Protraction October Stroke frequency O Q. Motion to hip 5 1 Retraction N Swim peed.8. 8 M Fraction of paw contact Paw dragging Walking P 5 Swimming L Hip height Protractive paw peed.3 K.3. J Swing duration.. I Stance duration 5 1 Walking H Stepping frequency G Swimming Fig. 5. DBS of the MLR in rat with evere leion of the pinal cord. (A) Repreentative cheme of a walking animal with increaing MLR timulation intenity at week after T1 pinal cord injury detroying to 88% of the white matter. (B) Repreentative cheme of the ame animal wimming with increaing MLR timulation. (C) Speed of overground locomotion gradually increaed with increaing MLR DBS. (D to F) Range of motion of the hindpaw relative to the hip joint increaed becaue of an increae in protraction to preleional value. (G to J) The temporal execution of tep, a function that remained deficient throughout recovery (Fig. ), improved with increaing MLR DBS along with a recovery of the peed with which the paw i brought forward (J). Thi ugget an MLR DBS induced recovery of dynamic trength cloe to preleional value in overground locomotion. (K) Deficient hip height recovered with MLR DBS even at low timulation intenitie, indicating an MLR DBS induced recovery of antigravitational trength. (L) There wa marked improvement in paw dragging with MLR DBS, the mot defective parameter of locomotion after pinal cord injury. (M to P) Swimming peed (M), a peritent deficit in thee animal, recovered with MLR DBS, a did (O) the protraction leading to increaed hindlimb flexibility (N to P). (Q to S) The temporal execution of troke cycle recovered to almot intact performance with MLR DBS. (T and U) Together with the propulive (T) and recovery (U) paw peed in relation to the hip, thi indicated a marked improvement in dynamic trength with MLR DBS. (C to U) Dahed horizontal line indicate the mean baeline performance in nontimulated, intact rat (Fig., ). The animal from Fig. 3 (I to S) are repreented according to the percentage of pared tiue at T1 from blue (leat pared) to red (mot pared). X axi, intenity of MLR DBS a a percent of maximal timulation ( ± 1.8 ma). P < 5, P < 1, P < 1, one-way ANOVA with Dunnett multiple comparion to % timulation. limb movement wa een in all but one pinal cord leioned animal at 1 week after injury (Fig. Q). Swimming performance improved with time, but imilar to walking, pecific impairment remained (Fig., M to U). rat wim with flexed, immobile forelimb, whereby propulive force i generated olely by the hindlimb. Forelimb are ometime ued for teering. In the pinal cord injured animal, the forelimb compenated for the weak or abent hindlimb propulion Vol 5 Iue 8 8ra1

7 (Fig. L). Initially, only weak hindlimb wim troke could be executed. At week, movement trajectorie reembled preleional performance(fig.,ntop),butthetemporalexecutionoftrokeremained deficient (Fig., Q to S). The deficient peed with which the paw wa moved againt water reitance, during troke and recovery phae, reflect a peritent deficit in trength (Fig., T and U). The deficient pattern in walking and wimming in thee everely pinal cord injured rat reemble clinical obervation that peed and trength of movement are the major deficit in human patient with incomplete pinal cord injury (55). MLR DBS almot fully retore hindlimb function during wimming When placed in a water-filled bain (Fig. 5B), wimming peed increaed with MLR DBS intenity. Crucial change were oberved in the propulive force generated by the hindlimb (Fig. 5M; P < 1), leg protraction (Fig. 5O; P = ) without a recovery of retraction (Fig. 5P; P = 3), and an overall normalization of the temporal execution of the troke induced by higher MLR timulation intenitie (Fig. 5, Q to U). Thee reult reflect a marked recovery of dynamic hindlimb trength during wimming week after the leion. MLR timulation retore walking in pinal cord injured rat cloe to preleional performance In the cohort of rat decribed in Fig. 3 and, DBS of the left MLR wa teted week after pinal cord injury. When the fully awake rat were placed in the teting device and timulation (, maintained until completion of the run) wa turned on, rat tarted to walk with a delay of 1 to. With increaing timulation trength (1% wa ± 1.8 ma), a marked increae in walking peed wa een (Fig. 5, A and C; P < 1, repeated-meaure ANOVA, between column). The range of hindlimb motion exceeded preleional level with MLR DBS, mainly due to recovery of limb protraction [Fig. 5, D (P < 1) and E (P = 11)]. However, a lipping of the foot at the end of tance phae wa preent (Fig. 5F). Hindlimb tepping frequency, a peritent deficit in thee animal, approached preleional value with MLR DBS (Fig. 5G; P < 1). Thi wa due to a reduced duration of tance phae and a gradual recovery of wing duration to baeline performance[fig.5,h(p < 1) and I (P < 1)]. The peed with which the foot wa moved forward during wing phae increaed with increaing timulation intenity back to the value of intact animal (Fig. 5J; P <1).Recoveryof hip height under MLR DBS reflect recovery of antigravitational trength (Fig. 5K; P < 1). Dragging of the hindpaw over the runway improved even at intermediate timulation intenitie, which indicate improvement in the quality of tep (Fig. 5L; P < 1). Overall, improvement in walking ability of the hindlimb in everely impaired rat could be demontrated at intermediate, that i, phyiological, timulation intenitie. Thi reult ugget that the 15 to 3% remaining braintem to pinal cord fiber in thee animal could be recruited by the timulated MLR to induce fully functional locomotor movement in the hindlimb pinal circuit. A Fraction of intact Functionally paralyzed animal regain baic movement with MLR timulation The American Spinal Injury Aociation Impairment Scale (AIS) rate the everity of deficit in pinal cord injured patient (A: no enory.5% B 3.% C 5.% D.% E 9.% F Etimated pared fiber G Walking peed H Stepping frequency (not functional).. 3 I Swimming peed J Stroke frequency K % L 1% (max. tim.) Area CST RST VST MESReST PReST MEDReST DA,NA,A 5HT 5 1 % of max. tim % of maximal timulation 5 1 Joint trajectorie (wimming) Iliac cret Hip Knee Ankle MTP Toe Fig.. MLR timulation improve wimming in animal with ubtotal pinal cord injurie (<1% remaining white matter). (A to E) Individual animal with evere, 91 to 9.5% leion of the T1 pinal cord are color-coded according to the amount of pared white matter from blue (leat pared) to red (mot pared). (F) Etimation of remaining fiber of the major decending tract from the brain in thee animal. (G) Walking peed without timulation wa fully compenated by the forelimb and increaed with increaing MLR DBS intenity. (H) Occaional, nonfunctional tep-like twitche were carce, illutrating the evere hindlimb deficit. Depite a light increae of tepping frequency, no functional tep with MLR DBS were een. (I) Under weight-releaed condition, wimming peed increaed with increaing timulation intenity. (J) Hindlimb troke frequency increaed lightly with timulation. (K and L) Repreentative joint trajectorie of the wimming animal hown in (D) without MLR DBS (K) and with 1% MLR DBS (L). Joint trajectorie are hown at -m interval. X axi, intenity of MLR DBS a a percent of maximal timulation (13 ±.1 ma). P < 5, P <1,P <1,one-wayANOVAwith Dunnett multiple comparion to % timulation. Dahed horizontal line indicate the mean intact baeline performance. 3 October 13 Vol 5 Iue 8 8ra1

8 A 11% B CST 1% C Walking peed D Stepping freq. E RST %. 5 VST 5% MESReST 8% PReST % MEDReST 1% DA,NA,A 5% 3 5HT 8% Joint trajectorie (walking) F % G 5% H % Iliac cret Hip Knee Ankle MTP Toe I % or motor function, B: enory but no motor function, C: limited motor function, D: coniderable motor function). All AIS-A and B and about half of AIS-C patient have no or very limited motor control of their lower body and remain wheelchair-bound depite the preence of ome tiue bridge acro the injury ite in mot of thee patient. We tudied a different, more everely injured, cohort of five rat with uch minimal tiue paring (.5 to 9%; average of 5.1 ±.% of the pinal cord cro ection) (Fig., A to E). CST and RST were fully interrupted (. ±.% and ± %). Remaining fiber of the MESReST were. ±.9%, of the VST 8.9 ±.5%, of the pontine reticular ytem 1.1 ± 3.%, and of the medullary reticular ytem.3 ± 3.%; DA, NA, and A tract retained.3 ± 3.%, and the 5HT fiber were reduced to.8 ±.8% (Fig. F). The hindlimb were trongly paretic in all animal; during walking, occaional, nonfunctional, tep-like twitche could be oberved. The abdomen wa dragged acro the runway when rat walked only with their forelimb. MLR DBS increaed walking and wimming peed [Fig., G(P < 1) and I (P = 11)], but thi effect wa largely forelimbdependent. In the hindlimb of ome of the MLR-timulated animal, an increae in troke frequency during wimming (almot abent without timulation) and in tepping during walking could be oberved [Fig., H (P = 1) and J (P = 13)]. Figure (K and L) how 5 1 % of max. tim. J 5 1 1% (maximal timulation) Hip height 5 1 Fig.. Bilateral white matter paring may increae timulation effectivene. (A) Animal with minimal bilateral ventral white matter paring (total of 11% pared). (B) Etimation of paring of individual tract. (C) Walking peed increaed with increaing MLR DBS. (D) Stepping frequency of the everely paretic hindlimb recovered with MLR DBS. (E) Increaing hip height with MLR DBS illutrate the capability for ome abdominal weight upport induced by the timulation (1%: 5 ma). (C to E) Dahed horizontal line indicate the mean intact baeline performance. (F to J) Repreentative joint trajectorie illutrate improved hindlimb motion during walking with MLR DBS. Joint trajectorie are hown at -m interval. the hindlimb joint trajectorie of a completely paralyzed animal, which would correpond clinically to an anatomically incomplete but paralyzed patient (for example, AIS-A). Thi animal wa able to produce functional wim troke cycle with or 1% MLR DBS intenity. The effect of MLR DBS may be even more pronounced in animal where there i bilateral paring of white matter. An individual rat received a large T-haped leion at T1. Only 11% of the pinal cord wa pared, with both halve of the pinal cord being equally affected (Fig. A).CSTandRSThowedcompleteinjury; MESReST and 5HT projection retained le than 1%; and VST, PReST, MEDReST, and DA, NA, and A tract retained about % of fiber (Fig. B). Again, MLR DBS wa able to induce walking and increaed locomotor peed (Fig. C). Already at 5% timulation intenity, the tepping frequency of the almot paralyzed hindlimb wa markedly increaed; it went above value for intact animalwithtrongermlrdbstimulation intenitie (Fig. D). Abdominal dragging diappeared and the hindlimb tepping became weight upporting (Fig. E) under MLR DBS treatment. Without timulation, only ome ankle movement were een (Fig. F), but MLR DBS progreively induced movement in the hip and knee and increaed flexibility in the ankle joint (Fig., G to J), reulting in much improved gait. Thee reult how that functional hindlimb movement can be induced in almot fully paralyzed rat by MLR DBS. Bilateral paring of white matter eem to be beneficial for the magnitude of the improvement obtained by MLR DBS. MLR effect are mediated by the medial medullary reticular formation The MLR of intact animal wa timulated under anetheia, and a rhythmic hamtring electromyogram reflecting hindlimb tepping wa recorded (Fig. 8, A and B). Cutaneou enory timulation of the plantar urface of the foot wa maintained throughout the time of recording to provide the phyiological enory feedback to elicit table tepping (ee Material and Method). Injection of the g-aminobutyric acid type A (GABA A ) receptor agonit mucimol into the primary motor cortex (Fig. 8C) or the red nucleu and the reticular formation of the meencephalon (Fig. 8D) did not interfere with the MLR-evoked tepping. Mucimol wa previouly etimated to hut down neuronal activity within a -mm radiu of the injection ite (5). When mucimol wa injected into the MLR directly, tepping wa completely abolihed (Fig. 8E). Becaue mucimol olely act on neuronal cell bodie and not on paing axon, thi reult indicate that cell bodie around the injection 3 October 13 Vol 5 Iue 8 8ra1 8

9 A C G Paramedian ventral medulla I Left paramedian NRGi and NRGiA K R-EMG Motor cortex Pot R-EMG E MLR Pot Pot Pot Left T1 hemiection Pot L-tim Mucimol 5x B 5x R-EMG FWR SLA D F Medial ventral medulla H Pot J Right paramedian NRGi and NRGiA L m at Red nucleu Pot Right paramedian ventral medulla Pot Right T1 hemiection Fig. 8. Hindlimb activation by MLR timulation i mediated by the reticulopinal nuclei. (A), anethetized animal were timulated in the left MLR (L-tim). EMG recording were done in the right hamtring mucle. (B) A - timulation of the MLR with -m cathodal pule at induced rhythmic burt of hamtring EMG, indicating hindlimb tepping. (C to L)EffectofinjectionoftheGABA A receptor agonit mucimol into different region of the motor ytem during MLR timulation. Trace are hown before () and after (Pot) the pecific intervention. (C) Bilateral injection of mucimol in the primary motor cortex did not interfere with the induction of tepping upon MLR DBS. (D) Bilateral injection of mucimol into the red nucleu had no effect on MLR timulation induced hindlimb EMG.(E)Mucimol injected into the MLR completely abolihed the timulation-induced tepping induction. (F) Inactivation of the medial ventral medulla oblongata along the agittal midline fully uppreed MLR tepping induction. (G) Inactivating the more lateral, paramedian apect of the medulla oblongata during MLR timulation reulted in a trong reduction of the force of the tep, but ome weak rhythmic activity remained (inet). (H) Thi effect wa preent only when inactivating the right paramedian ventral medulla. (I) Inactivation of NRGi and NRGiA of the medulla oblongata containing the larget number of pinal projection neuron (Fig. 1A) on the left ide, ipilateral to the MLR timulation, did not interfere with induced tepping. (J) Right-ided inactivation of NRGi and NRGiA by mucimol almot fully uppreed the hamtring EMG, howing the importance of thee nuclei in mediating MLR-induced hindlimb tepping. (K and L) Intead of mucimol injection, the pinal cord wa hemiected at thoracic level T1. Schematic, pinal cord; red triangle, hemiection. (K) Left ide hemiection fully uppreed left ide MLR-induced tepping. (L) Right-ided T1 hemiection uppreed rhythmic tepping induced by left ide MLR timulation, with the right hindlimb extended (inet). EMG trace are hown a full wave rectified (FWR) and 1-m liding window averaged (SLA) trace. Pot Pot 5x 5x 1 1 ite, that i, CnF and PPN neuron, are reponible for MLR-induced tepping, which i in line with previou obervation (5). When neuron at and around the agittal midline of the lower medullary braintem were targeted, tepping wa abolihed by mucimol, howing the key function of thee reticulopinal neuron in relaying the MLR ignal to the lumbar pinal cord (Fig. 8F). We then invetigated the medullary reticular complex in more detail. Bilateral inactivation of the more lateral apect of the medial medullary reticular formation reulted in almot complete abolihment of tepping (Fig. 8G), but pare weak tep could till be oberved, reflected in low-amplitude EMG ignal (Fig. 8G, inet). Some induction of rhythmicity wa therefore preerved, wherea the intenity of the neuronal activation (that i, the trength of the mucle contraction) wa greatly impaired. The ame injection unilaterally, contralateral to the ite of MLR timulation, again preerved ome rhythmicity of tep but with much reduced trength and peed (Fig. 8H, inet). Thi weak tepping wa even oberved when the ite of the highet denity of reticulopinal neuron (that i, NRGi and NRGiA) in the medulla oblongata (Fig. 1A) wa inactivated with mucimol (Fig. 8J). Unilateral inactivation of the ame area ipilateral to the timulation ite did not block rhythmicity or trength (Fig. 8I). Thi indicate that the reticulopinal neuron that project to the ipilateral lumbar pinal cord are critical for the maintenance of forceful hindlimb tep on thi ide. However, the eential ytem inducing the rhythmic hindlimb activity may be located cloe to the agittal midline of the medulla. After an acute unilateral T1 hemiection ipilateral to the MLR timulation, rhythmic activity wa completely abent (Fig. 8K). Hence, the MLR to pinal cord pathway doe not function without ipilateral decending tract depite an involvement of the contralateral reticular formation. When the pinal cord wa cut contralateral to the timulated MLR, tepping wa not elicited either. However, intead of reting immobile, the right hindlimb wa gradually extended (Fig. 8L, inet). Thi movement appeared to be an initial extenion that would normally be followed by a flexion and rhythmic tepping. The hamtring contracted continuouly, only 3 October 13 Vol 5 Iue 8 8ra1 9

10 interrupted by occaional interval of relaxation. Overall, thi how that MLR-induced tepping depend on the integrity of ipilaterally decending reticulopinal fiber. The uprapinal control of flexion and extenion eem to be differentially influenced by ipilateral or contralateral component of the medullary reticular formation. The efficacy of therapeutic MLR DBS may therefore be greater with bilateral white matter paring in the pinal cord. DISCUSSION Similar to human with evere but anatomically incomplete pinal cord injury, adult rat with to 8% detruction of the pinal cord white matter howed evere, permanent deficit in overground locomotion and wimming, in particular with regard to trength and temporal execution (55). The preent reult how that MLR DBS gradually improved locomotion and wimming to near baeline performance. In animal with ubtotal injurie (.5 to 11% white matter remaining), MLR timulation led to the reappearance of movement in the paralyzed leg under gravity-releaed condition. Pharmacological inactivation of relevant tructure howed that activation of the major uprapinal motor control pathway from the MLR to the medial braintem and from there via reticulopinal fiber to the lumbar pinal cord may underlie the oberved effect. DBS of the MLR may be a new approach for treating patient uffering from evere chronic pinal cord injury. The MLR i a control center for the trength of locomotor output Our reult are conitent with the concept that the MLR pace locomotion without activating a particular form of movement directly (, 9). Walking and wimming peed increaed with increaing timulation intenity, with a timulu-dependent tranition from walking to a gallop that occurred around.5% of maximal MLR timulation intenity (8). In bird, increaing timulu intenity in the medullary target of the MLR reulted in a witch from hopping to flapping of the wing (58). In alamander, weak timulation induced creeping, wherea wimming i induced by tronger timulation of the MLR (9). All of thee reult ugget that the role of the MLR i primarily to control peed of locomotion; the pecific pattern of limb activation to match a particular peed may depend on enory-motor integration in circuit located at braintem or pinal level. The iolated pinal cord can produce a variety of locomotor output in repone to different enory timuli (). MLR-induced tepping depend critically on the medial braintem Retrograde axonal tracing from the lumbar pinal cord howed that ventromedial braintem neuron repreent quantitatively the mot important uprapinal input with the larget contribution coming from NRGi and NRGiA. Thee nuclei are critically involved in the initiation and modulation of locomotion (3), and their activity i trongly coupled to hindlimb tepping (59). Indeed, inhibition of thi region by mucimol injection blocked MLR-induced tepping. The critical role of the MLR to braintem to pinal cord pathway for tepping wa alo hown by hypothermic inactivation of neuron along thi route (, ). Thi pathway i tronger ipilaterally, but it i bilaterally organized. The mechanim behind the preferential contralateral tepping of anethetized animal oberved in the preent tudy may be linked to an action of reticulopinal fiber on commiural interneuron of the pinal CPG (). Specific inactivation of the more lateral part of NRGi and NRGiA uggeted a ditinct regulation of rhythmicity and trength. Thu, the tepping engine, that i, the lumbar CPG and the force of the contraction, may be driven by ditinct, parallel pathway from the MLR via reticulopinal ubnuclei to the pinal cord. Our reult with acute pinal hemiection ugget that contralateral reticulopinal neuron, involved in extenor trength, may be part of the croed decending medullary reticulopinal tract. Overall, a full undertanding of the functional anatomy of thee and other reticulopinal ytem (for example, pontine reticular formation) require more detailed functional tudie (,, 1). Important projection into lower medullary reticulopinal nuclei alo come from neocortical area adjacent to the primary motor cortex (), that i, the upplementary motor area (3), which are critical for the complex killed forelimb movement requiring coordination of cortical and ubcortical activity (3, 33, ). Global excitatory input to the pinal cord may contribute to a bimodal action of therapeutic MLR DBS Retrograde tracing of the lumbar pinal cord alo howed labeled neuron in the raphe nuclei ituated at the midline of the medulla oblongata. Serotonergic, dopaminergic, and noradrenergic fiber are known to exert global excitatory action on the pinal neuron and circuit. Accordingly, interruption of thee decending tract reult in a hypoexcited tate of many pinal neuron including motor neuron (, 5). Application of erotonergic and/or noradrenergic agonit increae excitation and facilitate CPG-driven tepping movement (, 18, 5). The raphe nuclei were hown to receive a dene projection from the MLR (). Potentially, the timulation of the MLR alo excited the raphe nuclei, and ome pared erotonergic fiber globally excite lumbar CPG network. We propoe a bimodal mechanim of therapeutic MLR DBS. Nonpecific excitatory effect on the lumbar circuit are therefore to be expected, that i, the CPG i rendered receptive to the more pecific motor command ignal carried by the reticulopinal fiber. Nonpecific excitation may cofunction with pontaneou, leioninduced change in the pinal cord that modulate pinal excitability (3, ). Thu, MLR timulation can be expected to raie the general level of excitability of the pinal circuit in a global fahion with the merit of pecifically engaging locomotor circuit to generate movement and regulate their cycle peed and extenor trength via the reticulopinal pathway dicued above. MLR DBS ha potential for treating patient with gait diturbance after pinal cord injury The function of the MLR i exceptionally well conerved from fih and alamander to higher mammal (). In man, thee fundamental circuitintheancientcoreofthebrainarebelievedtobeveryimilar (3,, 8). The MLR wa previouly linked to neurological diorder in which walking i impaired. In rare cae of midbrain ataxia, the region of the CnF and PPN that comprie the MLR i damaged. Thee patient how a deficit in the initiation of walking (9, ). In patient with PD, where hypoactivity of uprapinal motor circuit i a hallmark, evere akineia and gait diturbance have been linked to the progreive lo of neuron in the MLR (1). Depite obviou difference in the pathology of thee condition to pinal cord injury, they all reult in a evere reduction of motor command ignal reaching the 3 October 13 Vol 5 Iue 8 8ra1 1

11 pinal cord. Excitatory timulation of the PPN, a part of the MLR, by DBS electrode implanted into the midbrain wa recently found to improve gait diturbance in patient with PD(, 3). Thi ugget the clinical feaibility of a therapeutic MLR DBS in human. Many pinal cord injured patient uffer from a evere compromie, but not a complete lo, of the ability to walk or to contract certain mucle (AIS-C and D patient). Small remaining white matter region, often in the periphery of the pinal cord, are frequently een by imaging, even in mot of the functionally complete pinal cord injured patient (AIS-A patient). In our experimental rat pinal cord injury cohort with 1 to 5% paring and evere locomotor impairment, MLR DBS greatly improved locomotion and wimming, almot to the level of intact animal. In the cohort with ubtotal leion (.5 to 11% pared cro ection; paralyzed hindlimb), MLR DBS wa till able to induce wimming movement in water, a condition that could probably be ued a a bai for further rehabilitative training (, ). We conclude that MLR DBS ha potential a an effective therapeutic approach to improve walking in human pinal cord injured individual. The ue of imaging technique to preciely ae the location and extent of the pinal tract leion will help to better predict the outcome and the poible gain by MLR DBS and thereby hould improve rehabilitative treatment. Patient with ome paring of medullary reticulopinal fiber within the ventral and ventrolateral funiculi have a good chance of benefitting from MLR DBS in a way that could improve their daily life activitie. MATERIALS AND METHODS Study deign The goal of the preent tudy wa to tet if electrical timulation of the MLR can improve locomotor performance in rat with evere, incomplete pinal cord injury. To tet thi, we implanted the rat with a DBS electrode in the MLR on one ide. Locomotor performance wa aeed during the firt week after having received an incomplete ectioning of the pinal cord. No timulation wa adminitered in the time of recovery. At week after injury, the locomotor performance wa aeed, wherea MLR DBS of different intenitie wa applied and compared with the performance without timulation. Animal in which locomotion could be elicited, indicating correct electrode placement, by timulation were included in the tudy independently of the outcome after pinal cord injury. All animal are preented in the tudy; no tatitical outlier were excluded. Data were acquired uing objective readout; blinding for timulation during data acquiition or analyi wa not poible becaue of the obviou effect of the MLR DBS on locomotion. General All experiment were performed with adult female Lewi rat ( to g) in accordance to the guideline of the veterinary authoritie. Animal were anethetized with Hypnorm ( mg/kg) and Dormicum 3. mg/kg) (HD), or ketamine ( mg/kg) and xylazine (5 mg/kg) (KX). Stereotaxic urgery Stereotaxic coordinate are indicated a [anterior-poterior (AP) reference point + ditance in millimeter/mediolateral (ML) reference point + ditance in millimeter/doroventral (DV) reference point + ditance in millimeter)/angle with orientation facing to the midline]. Reference point ued are a follow: bregma (B), lambda (L), dura (D), ventral bae of the cranial cavity (CCB), pinal doral midline (SDM), caudal end of vertebra T1 (T1), and lateral edge of pyramidal tract (LPT). Finally, all animal were perfued trancardially with ml of Ringer olution containing heparin followed by 3 ml of % phophatebuffered formalin. Brain and pinal cord were potfixed in formalin for 1 day and tranferred to 3% ucroe in phophate buffer for day. Retrograde tracing For unilateral pinal retrograde Fat Blue (EMS Chemie) tracing, animal (n = ) were anethetized with HD. Laminae of T1 13 were removed with the dura left intact. A total of nl of % Fat Blue () wa injected at two ite in pinal egment L with a tereotaxically guided 33-gauge needle penetrating through local dura opening at 1 nl/ (T1, /SDM+./D./ ). For unilateral retrograde tracing of the reticular formation, the ventral braintem urface wa expoed (n = ) a decribed previouly (). A ingle -nl injection wa performed with a tereotaxically guided 33-gauge needle at 1 nl/ (AP not defined/lpt+/d /5 ). Correct AP location wa confirmed potmortem. Seven day (pinal cord) or 3 day (braintem) after tracing, animal were perfued. Tiue wa embedded in a glutaraldehyde polymerized matrix of chicken egg albumin and gelatin. The traced cell in the brain were recontructed a decribed in fig. S1. Briefly, -mm horizontal vibratome ection were made and collected emifree floating in phophate buffer. All ection were imaged uing an epifluorecence microcope with an automated tage. Image were digitally tacked and aligned, and the poition of all Fat Blue containing cell wa manually recorded and put into reference to defined anatomical landmark. Poition of the cell in relation to the landmark were mathematically fitted into a tandard rat brain model. A repreentative animal per tracing i hown (Fig. 1). The method i ummarized in fig. S1. Anterograde tracing and mapping of fiber in T1 white matter For anterograde tracing, 3% biotinylated dextran amine in water wa injected at different tereotaxic location, ummarized in fig. S. Coronal -mm vibratome ection of the T1 pinal cord were collected in.1 M phophate buffer, and fiber were viualized with fluorecent treptavidin (1:, DyLight 59 treptavidin, Jackon ImmunoReearch). Staining protocol wa a follow: 5 5 min of.1 M phophate buffer; 1 1 min of.3% Triton X-1 in tri-buffered aline. Both hemicord of one animal were recontructed for all traced ytem (n = and 1 each). TH and 5HT were detected with primary antibodie againt TH (1:, moue, MAB318, Millipore) and 5HT (1:1,, rabbit, NeuroStar) a publihed previouly (). Two hemicord of two animal were recontructed per tranmitter ytem (n = each). Digitized ection were fitted to a T1 pinal cord template, and fiber in the white matter were mapped. Data are hown a compoite view of two mapped hemicord. Spinal cord injurie Animal were anethetized with HD. T9 lamina wa removed. With a 15 tip calpel blade and iridectomy cior, the pinal cord wa incompletely ectioned. Bladder were manually expreed after injury if required. Coronal -mm cryotat ection of the injury were manually recontructed in a tandard T1 pinal cord template on the bai of phae-contrat and green autofluorecence. Spared fiber by the pinal 3 October 13 Vol 5 Iue 8 8ra1 11