Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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1 Title: Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats Authors: Gonzalez-Rothi EJ1*, Rombola AM2, Rousseau CA2, Mercier LM2, Fitzpatrick GM1, Reier PJ2, Fuller DD1, Lane MA2a, Addresses: 1 Department of Physical Therapy College of Public Health and Health Professions McKnight Brain Institute University of Florida P.O. Box 100154, 100 S. Newell Drive Gainesville, FL 32610, USA 2

Department of Neuroscience College of Medicine McKnight Brain Institute University of Florida P.O. Box 100154, 1149 S. Newell Drive Gainesville, FL 32610, USA a

Current address for Dr. Lane: Department of Neurobiology and Anatomy Drexel University 2900 W Queen Lane Philadelphia, PA 19129

*, corresponding author Email: [email protected]

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Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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2 Abstract: Cervical spinal cord injury (cSCI) disrupts bulbospinal projections to motoneurons controlling the upper limbs, resulting in significant functional impairments. Ongoing clinical and experimental research has revealed several lines of evidence for functional neuroplasticity and recovery of upper extremity function after SCI. The underlying neural substrates, however, have not been thoroughly characterized. The goals of the present study were to map the intraspinal motor circuitry associated with a defined upper extremity muscle and evaluate chronic changes in the distribution of this circuit following incomplete cSCI. Injured animals received a high cervical (C2) lateral hemisection (Hx), which compromises supraspinal input to ipsilateral spinal motoneurons controlling the upper extremities (forelimb) in adult rat. A battery of behavioral tests were used to characterize the time course and extent of forelimb motor recovery over a 16 week period post-injury. The retrograde transneuronal tracer – pseudorabies virus – was used to define the motor and premotor circuitry controlling the extensor carpi radialis longus (ECRL) muscle in spinal intact and injured animals. In the spinal intact rat, labeling was observed unilaterally within the ECRL motoneuron pool and within spinal interneurons bilaterally distributed within the dorsal horn and intermediate gray matter. No changes in labeling were observed 16 weeks post-injury, despite a moderate degree of recovery of forelimb motor function. These results suggest that recovery of the forelimb function assessed following C2 hemisection injury does not involve recruitment of new interneurons into the ipsilateral ECRL motor pathway. However the functional significance of these neurons to motor recovery requires further exploration.

Key Words: Spinal cord injury, upper extremity, functional plasticity, propriospinal interneurons

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Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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3 Introduction Although many examples of functional neuroplasticity and therapeutically-driven motor recovery have been reported in the spinal cord injury (SCI) literature, the underlying neural substrates mediating post-injury behavioral changes have not been extensively documented. A detailed understanding of the neuronal circuits underlying spontaneous recovery can significantly contribute to the design of cell-based and axonal growth-promoting approaches, as well as critical evaluation and prediction of their efficacy.1,2

Unfortunately,

comprehensive

neuroanatomical

demonstrations

of

functionally-defined intraspinal circuits are lacking in even the intact spinal cord.1 The more notable exceptions are studies of spinal bladder,3 autonomic,4 and respiratory1,5 networks which have employed the transneuronal tracer, pseudorabies virus (PRV) to identify pre-motor spinal interneurons. Treatments leading to restored upper extremity function are considered to be among the major therapeutic priorities of those who have endured cervical SCIs (cSCIs).6 Considerable attention has thus been directed at developing treatments to promote connectivity and functional improvements in rodent cSCI models. However, apart from demonstration of cervical motoneuron topography,7-9 the spinal neuronal substrate associated with forelimb function has not been fully investigated. Propriospinal neurons are of particular interest since they modulate lower motoneuron activity10 and have the potential to subserve partial recovery of motor function following incomplete SCI.11-15 Accordingly, the first goal of the present work was to characterize the anatomical distribution of the intraspinal pre-motor circuitry associated with a defined forelimb muscle, the extensor carpi radialis longus (ECRL), in the adult rat.

The ECRL is

significantly involved in facilitating wrist extension during a number of motor behaviors including locomotion, grooming, and feeding.

3

As in our previous definition of phrenic

Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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4 motor circuitry,1 PRV was used to map the distribution of ECRL motoneurons and the spinal interneurons that innervate them. Most studies investigating function of the upper limb after cSCI have utilized experimental lesions which directly damage forelimb motoneuron pools (i.e., mid- and low-cervical contusions).16-20 Those studies clearly established that injuries damaging forelimb motoneuron pools, as well as propriospinal networks in surrounding gray matter, lead to persistent forelimb motor control deficits. On the other hand, injuries rostral to forelimb motor pools, which could be considered as primarily descending tract (i.e., white matter) lesions, have received very little formal evaluation.21 The second goal of the present study was to determine the effect of C2Hx on ECRL pre-motor interneuron connectivity relative to the temporal progression of forelimb behavioral improvements involving the ECRL. These results provide the first detailed documentation of spinal motor- and interneurons associated with forelimb muscle in the intact and injured adult rat spinal cord.

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Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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5 Materials and Methods

Animals. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Florida. A total of 60 adult female Sprague-Dawley rats (200-250 g; ~16 weeks of age) were obtained from Harlan Laboratories Inc. (Indianapolis, IN, USA). Animals were housed within the McKnight Brain Institute at the University of Florida and maintained on a 12/12 hour light-dark cycle. In addition, they had free access to water and food pellets. Animals were assigned to one of two groups: spinal intact (n=22) or C2 hemisection groups (n=38). Animals within the spinal intact group were injected with a neuroanatomical tracer and assigned to either a 24 hour (n=2), a 48 hour (n=4), a 72 hour (n=6), a 96 hour (n=6), or a 120 hour (n=4) postinjection incubation period. Within the chronic hemisection group (n=38), all animals underwent behavioral assessments prior to- and at bi-weekly intervals following C2Hx injury. A subset of 14 animals from the C2Hx group were allocated for neuroanatomical assessments at a post-PRV incubation interval of either 72 (n=6) or 96 hours (n=8). All animals assigned to the C2Hx group were assessed for lesion completeness prior to inclusion in the final analyses.

General Surgical Methods. Anesthesia and injury methods have been previously described.22-24 Briefly, rats were anesthetized by injection of xylazine (10mg/kg, s.q.) and ketamine (140 mg/kg, i.p., Fort Dodge Animal Health, IA, USA). Following completion of the surgical procedure, anesthesia was reversed via injection of yohimbine (1.2 mg/kg s.q.). Upon recovery, animals were given injections of buprenorphine (0.03 mg/kg s.q., Hospira, IL, USA) for analgesia and sterile lactated Ringers solution (5 ml s.q.) to prevent dehydration. Post-

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Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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6 surgical care included administration of buprenorphine (0.03 mg/kg, s.q.) for the initial 48 hours post-injury and delivery of lactate Ringers solution (5ml/day, s.q.) and oral Nutrical supplements (1-3 ml, Webster Veterinary, MA, USA) until adequate volitional drinking and eating resumed. Spinal Cord Hemisection Injury. As previously described,1,23,25 a 1-inch midline dorsal incision was made from the base of the skull extending caudally to approximately the fourth cervical segment (C4). A laminectomy was performed at the second cervical segment (C2) to expose the spinal cord. A small incision was then made in the dura and a lateral hemisection (Hx) performed on the left side of the spinal cord using a microscalpel, followed by gentle aspiration. Using this approach, the completeness of the lesion was readily visible and the extent of the lesion was reproducible. The dura was then closed with interrupted 9-0 sutures and durafilm was placed over the dura. The overlying muscle was then sutured in layers and the skin was closed with stainless steel surgical wound clips.

Anatomical Tracing Protocols. Recombinants of the Bartha strain of pseudorabies virus (PRV; in minimal essential

medium with 10% fetal bovine serum) were used as anatomical tracers to examine the neural circuitry associated with the ECRL muscle in cohorts of spinal-intact and C2Hx rats. PRV is a transynaptically transported tracer which will label the entire motor circuitry over time. Propagation and culture methods for PRV have been extensively detailed.1 Two recombinants of PRV were used in the present study: either PRV152 or PRV614 (each ~2.0 x 108 pfu) was used to trace the circuitry associated with the ECRL muscle.

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Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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7 Application of PRV was made via a small incision in the skin of the distal forelimb above the ECRL muscle in spinal-intact and C2Hx rats. The skin and fascia overlying the ECRL were dissected to expose the ECRL muscle belly. PRV (10l) was injected into the ECRL via Hamilton syringe (Hamilton Company, Reno, NV). Animals were left to survive 24 - 120 hours following tracer injection. A careful time-course study was conducted in uninjured rats to determine the appropriate post-injection time point for first-order ECRL motoneuron labeling and subsequent, second-order transneuronal labeling of pre-motor ECRL spinal interneurons was observed. Thus these data guided the selection of 72 and 96 hour PRV incubation times in the chronic C2Hx group, as there was evidence of both first- and second-order labeling at these time points. In a subset of animals (n=2 from the 72 hour control group, n=2 from the 96 hour control group, and n=2 from the 96 hour C2Hx group), the left radial nerve was severed prior to injection of PRV to dennervate the ECRL muscle. The muscle was then injected with PRV. These control experiments were used to determine whether any of the observed PRV labeled cells in the cervical spinal cord arose from labeling of presympathetic neurons. Any neuronal PRV labeling in these rats would be the result of uptake and transport of PRV via sympathetic innervation of blood vessels, not via retrograde infection from the ECRL.

Behavioral Testing of Forelimb Function. Prior to initiation of experimental testing, rats were handled for 3-5 minutes daily for 710 days prior to testing by laboratory personnel to familiarize the animals with test administrators. All testing was conducted at the same time of day for each behavioral test. All procedures were videotaped and subsequently quantified by a blinded observer. Rats were tested prior to injury, at 1- and 2-weeks post-injury, then at bi-weekly intervals until termination of the study at 16 weeks post-injury.

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Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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8 Limb-use Asymmetry (Cylinder) Test The cylinder test was conducted on awake, unrestrained animals. Testing consisted of a single trial in which rats were placed in a clear Plexiglas cylinder (20 cm in diameter, 20 cm high) for 5 minutes. Forelimb use was measured during vertical exploration as the number of individual and simultaneous forepaw contacts with the cylinder wall.26-28 Ipsilateral forelimb use was calculated as the proportion of ipsilateral forepaw contacts to the total number of forepaw contacts. Scores were calculated with and without dorsal paw contacts included in the quantification. Forelimb Locomotor Scale (FLS) Rats were placed in an open-field plastic enclosure measuring 2.5 ft. x 3 ft. and were observed for a period lasting no longer than 5 minutes. Trials were scored in real-time by an examiner, and were also videotaped for later viewing and scoring. Scoring was conducted according to the Forelimb Locomotor Scale (FLS), a modified version of the Basso-Beattie-Bresnahan locomotor rating scale29,30 as previously described.19,31,32 The FLS is 17-point scale that defines deficits based on range of motion (Table 1), level of weight support, and whether the paw is parallel to the body. Vermicelli Pasta Handling Test Rats were given 7cm lengths of uncooked vermicelli pasta, marked at 1.75cm intervals to facilitate visualization of paw and pasta movement. For 10 days prior to initiation of behavioral testing, all rats were given the vermicelli pieces in their home cages to familiarize them with the pasta handling. Testing consisted of 3-5 trials in which the rats were given a single piece of pasta per trial while still in the clear Plexiglas cylinder. Test trials were video-taped with a Sony Digital Camcorder for frame-by-frame analysis. Quantification of this test involved counting the number of adjustments made by each forepaw, (left vs right), the time to eat the entire length of the pasta, and documentation of any atypical behaviors (Table 2), including atypical paw positions,

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Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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9 failure to contact the pasta, drops, abnormal pasta grasping or compensatory head and neck positions.33,34 Adjustments were defined as any visually confirmed release and regrasp of the length of pasta. The time to eat was defined by when the pasta was first placed in the mouth, ending when the piece was released from both hands and completely consumed.19,33,34 Trials were ignored if the rat did not consume the entire length of pasta or turned away from the camera in such a way that the pasta and the digits were obscured from view. Spinal Cord Histology At the pre-determined termination point, rats were euthanized by systemic perfusion with saline followed by 4% paraformaldehyde (Sigma, St. Louis, MO, USA). For lesion verification, the spinal cord was dissected and removed, embedded in paraffin, and 5m transverse sections were prepared. Spinal cord sections were mounted on glass slides (Fisher Scientific, Pittsburgh, PA, USA), stained with Cresyl violet, and evaluated using light microscopy. C2Hx lesions were considered to be anatomically complete only if there was a complete absence of apparently healthy white or gray matter in the ipsilateral spinal cord at the lesion epicenter as previously described.22-25,35,36 For anatomical tracing studies, the cervical spinal cord was removed, and 40m vibratome sections were made in the longitudinal plane. A subset of tissue was sectioned transversely (40m vibratome sections) for assessing the cross-sectional distribution of labeled neurons. For immunocytochemical analyses of PRV labeling, longitudinal vibratome sections were washed in PBS (0.1M, pH 7.4, 3 x 5 minutes), blocked against endogenous peroxidase activity (30% methanol, 0.6% hydrogen peroxide in 0.1 M PBS, incubated for 1-2 hours), rewashed in PBS, and blocked against nonspecific protein labeling (10% normal goat serum in 0.1 M PBS with 0.03% Triton-X). Sections were then incubated at 4o C overnight with primary antibodies against PRV

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Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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10 (rabbit anti-PRV; Rb133/134, raised against whole purified PRV particles that were acetone inactivated; 1:10,000; provided by Dr. Lynn Enquist). The following day, tissue was washed in PBS (0.1 M, 3 x 5 minutes), incubated for 2 hours at room temperature in a biotinylated secondary antibody (goat anti-rabbit; Jackson Immunocytochemicals, West Grove, PA; 1:200) and rewashed in PBS (3 x 5 minutes). Sections were further incubated for 2 hours in an avidin-biotin complex (ABC Elite Vectastain Kit; Vector Laboratories, Burlingame, CA), and given a third series of washes in PBS, and processed for antigen visualization with diaminobenzidine (DAB; Sigma, St. Louis, MO).1 A subset of tissue sections were counterstained with Cresyl violet for visualization of neuronal cell bodies.

Microscopy and Quantitative Analyses Spinal cord sections were examined using brightfield microscopy and photographs were taken using a Zeiss AxioPhot microscope with and AxioCam digital camera linked to a PC. PRV-positive cells (ECRL motoneurons and pre-motor interneurons) were quantified in serial longitudinal 40m sections separated by 120m (every 4th section) by a blind observer. Cell counts were made bilaterally in the dorsal horn, intermediate gray matter and the ventral horn and separated rostro-caudally into three cervical regions: C1-C3, C4-C6, and C7-T1. The average number of cells within each region ( + standard error of the mean) was sampled 72 and 96 hours following application of PRV. Motoneurons (primary PRV labeling) were distinguished from spinal interneurons (secondary labeling) based on their location and size. All other cells within the cervical spinal cord were considered spinal interneurons that innervate the ECRL motoneurons. In addition to reporting ‘raw’ cell numbers, the number of PRV-positive interneurons was then expressed as a function of the number of labeled ECRL motoneurons in order to

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Journal of Neurotrauma Spinal Interneurons and Forelimb Plasticity Following Incomplete Cervical Spinal Cord Injury in Adult Rats (doi: 10.1089/neu.2014.3718) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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11 control for variations in primary labeling between animals (secondary labeling was normalized against primary labeling).1,23

Data analyses All statistical analyses were performed using SigmaPlot 12.5 statistical software (SigmaPlot, SPSS, Chicago, IL). Research hypotheses were tested at an alpha level of 0.05 using repeated measures (RM), one-, two- and three-way analyses of variance (ANOVAs) to determine differences between groups. Post hoc tests were performed using the Student-Newman-Keuls method to correct for multiple pair-wise comparisons. One-way RM ANOVAs were used to assess changes in body weight, cylinder test scores, time to eat and atypical behavior scores on the vermicelli pasta handling test. 2way repeated measures ANOVA and the Student-Newman-Keuls post-hoc tests were conducted for FLS test scores and the number of adjustments in the vermicelli pasta test. For this ANOVA, factor 1 was “treatment” (i.e. pre-injury or the various post-C2Hx time points) and factor 2 was “side” (i.e. relative to the side of injury-contralateral vs. ipsilateral). All data are presented as the mean + standard error of the mean. A P-value of

Spinal interneurons and forelimb plasticity after incomplete cervical spinal cord injury in adult rats.

Cervical spinal cord injury (cSCI) disrupts bulbospinal projections to motoneurons controlling the upper limbs, resulting in significant functional im...
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