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Objective. Spinal cord injury (SCI) leads to debilitating sensorimotor deficits that greatly limit quality of life. This work aims to develop a mechanistic understanding of how to best promote functional recovery following SCI. Electrical spinal stimulation is one promising approach that is effective in both animal models and humans with SCI. Optogenetic stimulation is an alternative method of stimulating the spinal cord that allows for cell-type-specific stimulation. The present work investigates the effects of preferentially stimulating neurons within the spinal cord and not glial cells, termed 'neuron-specific' optogenetic spinal stimulation. We examined forelimb recovery, axonal growth, and vasculature after optogenetic or sham stimulation in rats with cervical SCI. Approach. Adult female rats received a moderate cervical hemicontusion followed by the injection of a neuron-specific optogenetic viral vector ipsilateral and caudal to the lesion site. Animals then began rehabilitation on the skilled forelimb reaching task. At four weeks post-injury, rats received a micro-light emitting diode (µLED) implant to optogenetically stimulate the caudal spinal cord. Stimulation began at six weeks post-injury and occurred in conjunction with activities to promote use of the forelimbs. Following six weeks of stimulation, rats were perfused, and tissue stained for GAP-43, laminin, Nissl bodies and myelin. Location of viral transduction and transduced cell types were also assessed. Main Results. Our results demonstrate that neuron-specific optogenetic spinal stimulation significantly enhances recovery of skilled forelimb reaching. We also found significantly more GAP-43 and laminin labeling in the optogenetically stimulated groups indicating stimulation promotes axonal growth and angiogenesis. Significance. These findings indicate that optogenetic stimulation is a robust neuromodulator that could enable future therapies and investigations into the role of specific cell types, pathways, and neuronal populations in supporting recovery after SCI., (Creative Commons Attribution license.)

To date, relatively few studies have used optogenetic stimulation to address basic science and therapeutic questions within the spinal cord. Even less have reported optogenetic stimulation in the rat spinal cord. This is likely due to a lack of accessible optogenetic implants. The development of a device that can be fabricated and operated by most laboratories, requiring no special equipment, would allow investigators to begin dissecting the functions of specific neuronal cell-types and circuitry within the spinal cord, as well as investigate therapies for spinal ailments like spinal cord injury. Here, we describe a long-term implantable μLED device designed for optogenetic stimulation of the spinal cord in awake, freely moving rats that is simple enough to be fabricated, implanted and operated by most laboratories. This device, which sits above the dorsal cord, can induce robust movements for at least 6 weeks without causing physical or thermal damage to the underlying spinal cord. In this regard, the presented μLED device could help tease apart the complexities of the spinal cord and uncover potential future therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Electrical intraspinal microstimulation (ISMS) at various sites along the cervical spinal cord permits forelimb muscle activation, elicits complex limb movements and may enhance functional recovery after spinal cord injury. Here, we explore optogenetic spinal stimulation (OSS) as a less invasive and cell type-specific alternative to ISMS. To map forelimb muscle activation by OSS in rats, adeno-associated viruses (AAV) carrying the blue-light sensitive ion channels channelrhodopsin-2 (ChR2) and Chronos were injected into the cervical spinal cord at different depths and volumes. Following an AAV incubation period of several weeks, OSS-induced forelimb muscle activation and movements were assessed at 16 sites along the dorsal surface of the cervical spinal cord. Three distinct movement types were observed. We find that AAV injection volume and depth can be titrated to achieve OSS-based activation of several movements. Optical stimulation of the spinal cord is thus a promising method for dissecting the function of spinal circuitry and targeting therapies following injury. NEW & NOTEWORTHY Optogenetics in the spinal cord can be used both for therapeutic treatments and to uncover basic mechanisms of spinal cord physiology. For the first time, we describe the methodology and outcomes of optogenetic surface stimulation of the rat spinal cord. Specifically, we describe the evoked responses of forelimbs and address the effects of different adeno-associated virus injection paradigms. Additionally, we are the first to report on the limitations of light penetration through the rat spinal cord.

Traumatic spinal cord injury (SCI) often leads to permanent neurological impairment. Currently, the only clinically effective intervention for patients with acute SCI is surgical decompression by removal of impinging bone fragments within 24 h after injury. Recent clinical studies suggest that elevated intraparenchymal spinal pressure (ISP) limits functional recovery following SCI. Here, we report on the temporal and spatial patterns of elevated ISP following a moderate rodent contusion SCI. Compared with physiological ISP in the intact cord (2.7 ± 0.5 mm Hg), pressures increase threefold 30 min following injury (8.9 ± 1.1 mm Hg, p < 0.001) and remain elevated for up to 7 days (4.3 ± 0.8 mm Hg). Measurements of rostrocaudal ISP distribution reveal peak pressures in the injury center and in segments rostral to the injury during the acute phase(≤ 24 h). During the subacute phase(≥ 72 h), peak ISP decreases while a 7.5 mm long segment of moderately elevated ISP remains, centered on the initial contusion site. Interestingly, the contribution of the dural and pial compartments toward increased ISP changes with time after injury: Dural and pial linings contribute almost equally to increased ISP during the acute phase, whereas the dural lining is primarily responsible for elevated ISP during the subacute phase (78.9%). Our findings suggest that a rat contusion SCI model in combination with novel micro-catheters allows for direct measurement of ISP after SCI. Similarly to traumatic brain injury, raised tissue pressure is likely to have detrimental effects on spontaneous recovery following SCI.

Cervical spinal cord contusion is the most common human spinal cord injury, yet few rodent models replicate the pathophysiological and functional sequela of this injury. Here, we modified an electromechanical injury device and characterized the behavioral and histological changes occurring in response to a lateralized C4 contusion injury in rats. A key feature of the model includes a non-injurious touch phase where the spinal cord surface is dimpled with a consistent starting force. Animals were either left intact as a control, received a non-injury-producing touch on the surface of the cord ("sham"), or received a 0.6 mm or a 0.8 mm displacement injury. Rats were then tested on the forelimb asymmetry use test, CatWalk, and the Irvine, Beatties, and Bresnahan (IBB) cereal manipulation task to assess proximal and distal upper limb function for 12 weeks. Injuries of moderate (0.6 mm) and large (0.8 mm) displacement showed consistent differences in forelimb asymmetry, metrics of the CatWalk, and sub-scores of the IBB. Overall findings indicated long lasting proximal and distal upper limb deficits following 0.8 mm injury but transient proximal with prolonged distal limb deficits following 0.6 mm injury. Significant differences in loss of ipsilateral unmyelinated and myelinated white matter was detected between injury severities. Demyelination was primarily localized to the dorsolateral region of the hemicord and extended further rostral following 0.8 mm injury. These findings establish the C4 hemi-contusion injury as a consistent, graded model for testing novel treatments targeting forelimb functional recovery.

MMP9-responsive bivalirudin-HPMA copolymers were synthesized for direct, local administration in rat spinal cord contusion injury models. Polymer-conjugated bivalirudin peptides maintained activity while demonstrating enzyme-mediated release upon MMP9 exposure and prolonged release from hyaluronic acid/methylcellulose (HAMC) hydrogels compared to free bivalirudin peptide. Localized administration of bivalirudin copolymers in vivo at the site of rat spinal cord injury decreased cellular proliferation and astrogliosis, suggesting the bivalirudin copolymer and HAMC hydrogel system are a promising therapeutic intervention for reducing immediate inflammatory responses and long term scarring.