Quantification of Myelinated Nerve Fraction and Degeneration in Spinal Cord Neuropil by SHIFT MRI.

BACKGROUND: Neurodegeneration is a complex cellular process linked to prompt changes in myelin integrity and gradual neuron loss. Current imaging techniques offer estimations of myelin volumes in lesions/remyelinated areas but are limited to detect subtle injury. PURPOSE: To investigate whether measurements detected by a signal hierarchically isolated as a function of time-to-echo (SHIFT) MRI technique can determine changes in myelin integrity and fiber axolemma. STUDY TYPE: Prospective animal model. ANIMAL MODEL: Surgically demyelinated spinal cord (SC) injury model in rodents (n = 6). FIELD STRENGTH/SEQUENCE: Gradient-echo spin-echo at 3T. ASSESSMENT: Multicompartment T2 relaxations were computed by SHIFT MRI in 75-microns-resolution images of the SC injury penumbra region 2 weeks post-trauma. G-ratio and axolemma delamination were assessed by transmission electron microscopy (TEM) in intact and injured samples. SC myelinated nerve fraction was computed by SHIFT MRI prospectively and assessed histologically. STATISTICAL TESTS: Relations between SHIFT-isolated T2 -components and TEM measurements were studied using linear regression and t-tests. Pearson's correlation and significance were computed to determine the SHIFT's sensitivity to detect myelinated fibers ratio in gray matter. Regularized least-squares-based ranking analysis was employed to determine SHIFT MRI's ability to discern intact and injured myelinated nerves. RESULTS: Biexponential signals isolated by SHIFT MRI for intact vs. lesion penumbra exhibited changes in T2 , shifting from intermediate components (25 ± 2 msec) to long (43 ± 11 msec) in white matter, and similarly in gray matter regions-of-interest (31 ± 2 to 46 ± 16 msec). These changes correlated highly with TEM g-ratio and axon delamination measurements (P < 0.05). Changes in short T2 components were observed but not statistically significant (8.5 ± 0.5 to 7 ± 3 msec, P = 0.445, and 4.0 ± 0.9 to 7 ± 3 msec, P = 0.075, respectively). SHIFT MRI's ability to detect myelinated fibers within gray matter was confirmed (P < 0.001). DATA CONCLUSION: Changes detected by SHIFT MRI are associated with abnormal intermembrane spaces formed upon mild injury, directly correlated with early neuro integrity loss. Level of Evidence 1 Technical Efficacy Stage 2.

Formulation of thrombin-inhibiting hydrogels via self-assembly of ionic peptides with peptide-modified polymers.

Cell therapy for spinal cord injuries offers the possibility of replacing lost cells after trauma to the central nervous system (CNS). In preclinical studies, synthetic hydrogels are often co-delivered to the injury site to support survival and integration of the transplanted cells. These hydrogels ideally mimic the mechanical and biochemical features of a healthy CNS extracellular matrix while also providing the possibility of localized drug delivery to promote healing. In this work, we synthesize peptide-functionalized polymers that contain both a peptide sequence for incorporation into self-assembled peptide hydrogels along with bioactive peptides that inhibit scar formation. We demonstrate that peptide hydrogels formulated with the peptide-functionalized polymers possess similar mechanical properties (soft and shear-thinning) as peptide-only hydrogels. Small angle neutron scattering analysis reveals that polymer-containing hydrogels possess larger inhomogeneous domains but small-scale features such as mesh size remain the same as peptide-only hydrogels. We further confirm that the integrated hydrogels containing bioactive peptides exhibit thrombin inhibition activity, which has previously shown to reduce scar formation in vivo. Finally, while the survival of encapsulated cells was poor, cells cultured on the hydrogels exhibited good viability. Overall, the described composite hydrogels formed from self-assembling peptides and peptide-modified polymers are promising, user-friendly materials for CNS applications in regeneration.

Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration.

A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.

Fabrication of multipoint side-firing optical fiber by laser micro-ablation.

A multipoint, side-firing design enables an optical fiber to output light at multiple desired locations along the fiber body. This provides advantages over traditional end-to-end fibers, especially in applications requiring fiber bundles such as brain stimulation or remote sensing. This Letter demonstrates that continuous wave (CW) laser micro-ablation can controllably create conical-shaped cavities, or side windows, for outputting light. The dimensions of these cavities determine the amount of firing light and their firing angle. Experimental data show that a single side window on a 730 µm fiber can deliver more than 8% of the input light. This can be increased to more than 19% on a 65 µm fiber with side windows created using femtosecond laser ablation and chemical etching. Fine control of light distribution along an optical fiber is critical for various biomedical applications such as light-activated drug-release and optogenetics studies.

Tunable, Injectable Hydrogels Based on Peptide-Cross-Linked, Cyclized Polymer Nanoparticles for Neural Progenitor Cell Delivery.

A PEG-based cyclized vinyl polymer was synthesized via one-step RAFT polymerization and used as a precursor of injectable hydrogels. Dithiol linkers including laminin-derived peptides containing IKVAV and YIGSR sequences and DTT were used for gelation. Fast and adjustable gelation rate was achieved through nucleophile-initiated thiol-Michael reaction under physiological conditions. Low swelling ratio and moderate degradation rate of the formed hydrogels were observed. 3D encapsulation of neural progenitor cells in the synthetic hydrogel showed good cell viability over 8 days. The long-term cell survival and proliferation were promoted by the introduction of laminin-derived peptides. This hydrogel platform based on peptide-cross-linked, cyclized vinyl polymers can be used as a universal hydrogel template for 3D cell encapsulation.

Nano-Sized Sunflower Polycations As Effective Gene Transfer Vehicles.

The architecture of polycations plays an important role in both gene transfection efficiency and cytotoxicity. In this work, a new polymer, sunflower poly(2-dimethyl amino)ethyl methacrylate) (pDMAEMA), is prepared by atom transfer radical polymerization and employed as nucleic acid carriers compared to linear pDMAEMA homopolymer and comb pDMAEMA. The sunflower pDMAEMAs show higher IC50 , greater buffering capacity, and stronger binding capacity toward plasmid DNA than their linear and comb counterparts. In vitro transfection studies demonstrate that sunflower pDMAEMAs exhibit high transfection efficiency as well as relatively low cytotoxicity in complete growth medium. In vivo gene delivery by intraventricular injection to the brain shows that sunflower polymer delivers plasmid DNA more effectively than comb polymer. This study provides a new insight into the relationship between polymeric architecture and gene delivery capability, and as well as a useful means to design potent vectors for successful gene delivery.

Neural Stem Cells in the Adult Subventricular Zone Oxidize Fatty Acids to Produce Energy and Support Neurogenic Activity.

Neural activity is tightly coupled to energy consumption, particularly sugars such as glucose. However, we find that, unlike mature neurons and astrocytes, neural stem/progenitor cells (NSPCs) do not require glucose to sustain aerobic respiration. NSPCs within the adult subventricular zone (SVZ) express enzymes required for fatty acid oxidation and show sustained increases in oxygen consumption upon treatment with a polyunsaturated fatty acid. NSPCs also demonstrate sustained decreases in oxygen consumption upon treatment with etomoxir, an inhibitor of fatty acid oxidation. In addition, etomoxir decreases the proliferation of SVZ NSPCs without affecting cellular survival. Finally, higher levels of neurogenesis can be achieved in aged mice by ectopically expressing proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), a factor that increases cellular aerobic capacity by promoting mitochondrial biogenesis and metabolic gene transcription. Regulation of metabolic fuel availability could prove a powerful tool in promoting or limiting cellular proliferation in the central nervous system. Stem Cells 2015;33:2306-2319.

Early astrocyte redistribution in the optic nerve precedes axonopathy in the DBA/2J mouse model of glaucoma.

Glaucoma challenges the survival of retinal ganglion cell axons in the optic nerve through processes dependent on both aging and ocular pressure. Relevant stressors likely include complex interplay between axons and astrocytes, both in the retina and optic nerve. In the DBA/2J mouse model of pigmentary glaucoma, early progression involves axonopathy characterized by loss of functional transport prior to outright degeneration. Here we describe novel features of early pathogenesis in the DBA/2J nerve. With age the cross-sectional area of the nerve increases; this is associated generally with diminished axon packing density and survival and increased glial coverage of the nerve. However, for nerves with the highest axon density, as the nerve expands mean cross-sectional axon area enlarges as well. This early expansion was marked by disorganized axoplasm and accumulation of hyperphosphorylated neurofilamants indicative of axonopathy. Axon expansion occurs without loss up to a critical threshold for size (about 0.45-0.50 µm(2)), above which additional expansion tightly correlates with frank loss of axons. As well, early axon expansion prior to degeneration is concurrent with decreased astrocyte ramification with redistribution of processes towards the nerve edge. As axons expand beyond the critical threshold for loss, glial area resumes an even distribution from the center to edge of the nerve. We also found that early axon expansion is accompanied by reduced numbers of mitochondria per unit area in the nerve. Finally, our data indicate that both IOP and nerve expansion are associated with axon enlargement and reduced axon density for aged nerves. Collectively, our data support the hypothesis that diminished bioenergetic resources in conjunction with early nerve and glial remodeling could be a primary inducer of progression of axon pathology in glaucoma.

Remyelination reporter reveals prolonged refinement of spontaneously regenerated myelin.

Neurological diseases and trauma often cause demyelination, resulting in the disruption of axonal function and integrity. Endogenous remyelination promotes recovery, but the process is not well understood because no method exists to definitively distinguish regenerated from preexisting myelin. To date, remyelinated segments have been defined as anything abnormally short and thin, without empirical data to corroborate these morphological assumptions. To definitively identify regenerated myelin, we used a transgenic mouse with an inducible membrane-bound reporter and targeted Cre recombinase expression to a subset of glial progenitor cells after spinal cord injury, yielding remarkably clear visualization of spontaneously regenerated myelin in vivo. Early after injury, the mean length of sheaths regenerated by Schwann cells and oligodendrocytes (OLs) was significantly shorter than control, uninjured myelin, confirming past assumptions. However, OL-regenerated sheaths elongated progressively over 6 mo to approach control values. Moreover, OL-regenerated myelin thickness was not significantly different from control myelin at most time points after injury. Thus, many newly formed OL sheaths were neither thinner nor shorter than control myelin, vitiating accepted dogmas of what constitutes regenerated myelin. We conclude that remyelination, once thought to be static, is dynamic and elongates independently of axonal growth, in contrast to stretch-based mechanisms proposed in development. Further, without clear identification, past assessments have underestimated the extent and quality of regenerated myelin.

Differential progression of structural and functional alterations in distinct retinal ganglion cell types in a mouse model of glaucoma.

Intraocular pressure (IOP) elevation is a principal risk factor for glaucoma. Using a microbead injection technique to chronically raise IOP for 15 or 30 d in mice, we identified the early changes in visual response properties of different types of retinal ganglion cells (RGCs) and correlated these changes with neuronal morphology before cell death. Microbead-injected eyes showed reduced optokinetic tracking as well as cell death. In such eyes, multielectrode array recordings revealed that four RGC types show diverse alterations in their light responses upon IOP elevation. OFF-transient RGCs exhibited a more rapid decline in both structural and functional organizations compared with other RGCs. In contrast, although the light-evoked responses of OFF-sustained RGCs were perturbed, the dendritic arbor of this cell type remained intact. ON-transient and ON-sustained RGCs had normal functional receptive field sizes but their spontaneous and light-evoked firing rates were reduced. ON- and OFF-sustained RGCs lost excitatory synapses across an otherwise structurally normal dendritic arbor. Together, our observations indicate that there are changes in spontaneous activity and light-evoked responses in RGCs before detectable dendritic loss. However, when dendrites retract, we found corresponding changes in receptive field center size. Importantly, the effects of IOP elevation are not uniformly manifested in the structure and function of diverse RGC populations, nor are distinct RGC types perturbed within the same time-frame by such a challenge.

iDISCO Tissue Clearing Whole-Brain and Light Sheet Microscopy for High-Throughput Imaging in a Mouse Model of Traumatic Brain Injury.

Immunolabeling-enabled imaging of solvent-cleared organs (iDISCO) (Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier-Lavigne M, Cell 159:896-910, 2014) aims to match the refractive index (RI) of tissue to the surrounding medium, thereby facilitating three-dimensional (3D) imaging and quantification of cellular points and tissue structures. Once cleared, transparent tissue samples allow for rapid imaging with no mechanical sectioning. This imaging technology enables us to visualize brain tissue in situ and quantify the morphology and extent of glial cell branches or neuronal processes extending from the epicenter of a traumatic brain injury (TBI). In this way, we can more accurately assess and quantify the damaging consequences of TBI not only in the impact region but also in the extended pericontusional regions.

Novel TCF4:TCF12 heterodimer inhibits glioblastoma growth.

TWIST1 (TW) is a pro-oncogenic basic helix-loop-helix (bHLH) transcription factor and promotes the hallmark features of malignancy (e.g., cell invasion, cancer cell stemness, and treatment resistance), which contribute to poor prognoses of glioblastoma (GBM). We previously reported that specific TW dimerization motifs regulate unique cellular phenotypes in GBM. For example, the TW:E12 heterodimer increases periostin (POSTN) expression and promotes cell invasion. TW dimer-specific transcriptional regulation requires binding to the regulatory E-box consensus sequences, but alternative bHLH dimers that balance TW dimer activity in regulating pro-oncogenic TW target genes are unknown. We leveraged the ENCODE DNase I hypersensitivity data to identify E-box sites and tethered TW:E12 and TW:TW proteins to validate dimer binding to E-boxes in vitro. Subsequently, TW knockdown revealed a novel TCF4:TCF12 bHLH dimer occupying the same TW E-box site that, when expressed as a tethered TCF4:TCF12 dimer, markedly repressed POSTN expression and extended animal survival. These observations support TCF4:TCF12 as a novel dimer with tumor-suppressor activity in GBM that functions in part through displacement of and/or competitive inhibition of pro-oncogenic TW dimers at E-box sites.

A novel minimally invasive and versatile kyphoplasty balloon-based model of porcine spinal cord injury.

Introduction: Spinal cord injury (SCI) animal models often utilize an open surgical laminectomy, which results in animal morbidity and also leads to changes in spinal canal diameter, spinal cord perfusion, cerebrospinal fluid flow dynamics, and spinal stability which may confound SCI research. Moreover, the use of open surgical laminectomy for injury creation lacks realism when considering human SCI scenarios. Methods: We developed a novel, image-guided, minimally invasive, large animal model of SCI which utilizes a kyphoplasty balloon inserted into the epidural space via an interlaminar approach without the need for open surgery. Results: The model was validated in 5 Yucatán pigs with imaging, neurofunctional, histologic, and electrophysiologic findings consistent with a mild compression injury. Discussion: Few large animal models exist that have the potential to reproduce the mechanisms of spinal cord injury (SCI) commonly seen in humans, which in turn limits the relevance and applicability of SCI translational research. SCI research relies heavily on animal models, which typically involve an open surgical, dorsal laminectomy which is inherently invasive and may have untoward consequences on animal morbidity and spinal physiology that limit translational impact. We developed a minimally invasive, large animal model of spinal cord injury which utilizes a kyphoplasty balloon inserted percutaneously into the spinal epidural space. Balloon inflation results in a targeted, compressive spinal cord injury with histological and electrophysiological features directly relevant to human spinal cord injury cases without the need for invasive surgery. Balloon inflation pressure, length of time that balloon remains inflated, and speed of inflation may be modified to achieve variations in injury severity and subtype.

Electrokinetic convection-enhanced delivery for infusion into the brain from a hydrogel reservoir.

Electrokinetic convection-enhanced delivery (ECED) utilizes an external electric field to drive the delivery of molecules and bioactive substances to local regions of the brain through electroosmosis and electrophoresis, without the need for an applied pressure. We characterize the implementation of ECED to direct a neutrally charged fluorophore (3 kDa) from a doped biocompatible acrylic acid/acrylamide hydrogel placed on the cortical surface. We compare fluorophore infusion profiles using ECED (time = 30 min, current = 50 µA) and diffusion-only control trials, for ex vivo (N = 18) and in vivo (N = 12) experiments. The linear intensity profile of infusion to the brain is significantly higher in ECED compared to control trials, both for in vivo and ex vivo. The linear distance of infusion, area of infusion, and the displacement of peak fluorescence intensity along the direction of infusion in ECED trials compared to control trials are significantly larger for in vivo trials, but not for ex vivo trials. These results demonstrate the effectiveness of ECED to direct a solute from a surface hydrogel towards inside the brain parenchyma based predominantly on the electroosmotic vector.

Hemin-Induced Transient Senescence Via DNA Damage Response: A Neuroprotective Mechanism Against Ferroptosis in Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) poses acute fatality and long-term neurological risks due to hemin and iron accumulation from hemoglobin breakdown. Our observation that hemin induces DNA double-strand breaks (DSBs), prompting a senescence-like phenotype in neurons, necessitating deeper exploration of cellular responses. Using experimental ICH models and human ICH patient tissue, we elucidate hemin-mediated DNA damage response (DDR) inducing transient senescence and delayed expression of heme oxygenase (HO-1). HO-1 co-localizes with senescence-associated ß-Galactosidase (SA-ß-Gal) in ICH patient tissues, emphasizing clinical relevance of inducible HO-1 expression in senescent cells. We reveal a reversible senescence state protective against acute cell death by hemin, while repeat exposure leads to long-lasting senescence. Inhibiting early senescence expression increases cell death, supporting the protective role of senescence against hemin toxicity. Hemin-induced senescence is attenuated by a pleiotropic carbon nanoparticle that is a catalytic mimic of superoxide dismutase, but this treatment increased lipid peroxidation, consistent with ferroptosis from hemin breakdown released iron. When coupled with iron chelator deferoxamine (DEF), the nanoparticle reduces hemin-induced senescence and upregulates factors protecting against ferroptosis. Our study suggests transient senescence induced by DDR as an early potential neuroprotective mechanism in ICH, but the risk or iron-related toxicity supports a multi-pronged therapeutic approach.

Axonal thinning and extensive remyelination without chronic demyelination in spinal injured rats.

Remyelination following spinal cord injury (SCI) is thought to be incomplete; demyelination is reported to persist chronically and is proposed as a compelling therapeutic target. Yet most reports do not distinguish between the myelin status of intact axons and injury-severed axons whose proximal stumps persist but provide no meaningful function. We previously found full remyelination of spared, intact rubrospinal axons caudal to the lesion in chronic mouse SCI. However, the clinical concept of chronically demyelinated spared axons remains controversial. Since mouse models may have limitations in clinical translation, we asked whether the capacity for full remyelination is conserved in clinically relevant chronic rat SCI. We determined myelin status by examining paranodal protein distribution on anterogradely labeled, intact corticospinal and rubrospinal axons throughout the extent of the lesion. Demyelination was evident on proximal stumps of severed axons, but not on intact axons. For the first time, we demonstrate that a majority of intact axons exhibit remyelination (at least one abnormally short internode, <100 µm). Remarkably, shortened internodes were significantly concentrated at the lesion epicenter and individual axons were thinned by 23% compared with their rostral and caudal zones. Mathematical modeling predicted a 25% decrease in conduction velocity at the lesion epicenter due to short internodes and axonal thinning. In conclusion, we do not find a large chronically demyelinated population to target with remyelination therapies. Interventions may be better focused on correcting structural or molecular abnormalities of regenerated myelin.

Distal axonopathy with structural persistence in glaucomatous neurodegeneration.

An early hallmark of neuronal degeneration is distal transport loss and axon pathology. Glaucoma involves the degeneration of retinal ganglion cell (RGC) neurons and their axons in the optic nerve. Here we show that, like other neurodegenerations, distal axon injury appears early in mouse glaucoma. Where RGC axons terminate in the superior colliculus, reduction of active transport follows a retinotopic pattern resembling glaucomatous vision loss. Like glaucoma, susceptibility to transport deficits increases with age and is not necessarily associated with elevated ocular pressure. Transport deficits progress distal-to-proximal, appearing in the colliculus first followed by more proximal secondary targets and then the optic tract. Transport persists through the optic nerve head before finally failing in the retina. Although axon degeneration also progresses distal-to-proximal, myelinated RGC axons and their presynaptic terminals persist in the colliculus well after transport fails. Thus, distal transport loss is predegenerative and may represent a therapeutic target.

Rigor and reproducibility in analysis of rodent behavior utilizing the forelimb reaching task following a cervical spinal cord injury.

Spinal cord injury (SCI) research with animals aims to understand the neurophysiological responses resultant of injury and to identify effective interventions that can translate into clinical treatments in the future. Consistent and reliable assessments to properly measure outcomes are essential to achieve this aim and avoid issues with reproducibility. The objective of this study was to establish a baseline for implementing the forelimb reaching task (FRT) assessment and analysis that increased reproducibility of our studies. For this study, we implemented a weekly FRT training program for six weeks. During this time the language of the scoring rubric for movement elements that comprise a reaching task was simplified and expanded. We calculated intra- and inter-rater variability among participants of the study both before and after training to determine the effect changes made had on rigor and reproducibility of this behavioral assessment in a cervical SCI rodent model. All animals (n = 19) utilized for FRT behavioral assessments received moderate contusion injuries using the Ohio State University device and were tested for a period of 5 weeks post-SCI. Videos used for scoring were edited and shared with all participants of this study to test FRT score variability and the effect simplification of the scoring rubric had on overall inter-rater reliability. From our results we determined training for a minimum of three weeks in FRT analysis is necessary for rigor and reproducibility of our behavioral studies, as well as the need for two raters to be assigned per animal to ensure accuracy of results.

Assessment of spinal cord injury using ultrasound elastography in a rabbit model in vivo.

The effect of the mechanical micro-environment on spinal cord injury (SCI) and treatment effectiveness remains unclear. Currently, there are limited imaging methods that can directly assess the localized mechanical behavior of spinal cords in vivo. In this study, we apply new ultrasound elastography (USE) techniques to assess SCI in vivo at the site of the injury and at the time of one week post injury, in a rabbit animal model. Eleven rabbits underwent laminectomy procedures. Among them, spinal cords of five rabbits were injured during the procedure. The other six rabbits were used as control. Two neurological statuses were achieved: non-paralysis and paralysis. Ultrasound data were collected one week post-surgery and processed to compute strain ratios. Histologic analysis, mechanical testing, magnetic resonance imaging (MRI), computerized tomography and MRI diffusion tensor imaging (DTI) were performed to validate USE results. Strain ratios computed via USE were found to be significantly different in paralyzed versus non-paralyzed rabbits. The myelomalacia histologic score and spinal cord Young's modulus evaluated in selected animals were in good qualitative agreement with USE assessment. It is feasible to use USE to assess changes in the spinal cord of the presented animal model. In the future, with more experimental data available, USE may provide new quantitative tools for improving SCI diagnosis and prognosis.

Optogenetic spinal stimulation promotes new axonal growth and skilled forelimb recovery in rats with sub-chronic cervical spinal cord injury.

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.