Locomotor Recovery Research Articles

Intracerebral Hemorrhage-Induced Cognitive Impairment in Rats Is Associated With Brain Atrophy, Hypometabolism, and Network Dysconnectivity.

The mechanisms underlying intracerebral hemorrhage (ICH)-related cognitive impairment (CI) remain unclear. Long-term structural and functional changes were investigated in the brains of healthy male and female Wistar rats after experimental ICH. Following double injection of autologous blood, rats underwent short-term (onset, 3 and 7 days) and long-term (3 and 6 months) radiological assessment and behavioral tests exploring spontaneous locomotion, anxiety-like behavior and working memory, spatial recognition memory and visual recognition memory. Volumetric and metabolic changes in brain areas were examined by 7Tesla-MRI and [18F] FDG-PET, respectively. Brain connectomic disorders and maladaptive processes were seeked through brain metabolic connectivity analysis and atrophy-related network analysis. From an initial hematoma mean volume of 23.35 ± 9.50 mm3, we found early spontaneous locomotor recovery and significant spontaneous blood resorption (≈ 40% of the initial lesion) from days 0 to 7. After 3 and 6 months, ICH rats exhibited CI in several domains as compared to the sham group (working memory: 58.1 ± 1.2 vs. 70.7 ± 1.2%, p < 0.001; spatial recognition memory: 48.7 ± 1.9 vs. 64 ± 1.8%, p < 0.001 and visual recognition memory: 0.14 ± 0.05 vs. 0.33 ± 0.04, p = 0.013, in female only). Rats that experienced ICH had remote and concomitant cerebral atrophy and hypometabolism of ipsilateral striatum, thalamus, limbic system and cortical areas (temporal and parietal lobes). Interestingly, both structural and metabolic deterioration was found in the limbic system connected to the affected site, but remotely from the initial insult. On the other hand, increased activity and functional connectivity occurred in the contralateral hemisphere. These connectomics results showed that both maladaptative and compensation processes coexist in the rat brain following ICH, even at young age and in a disease-free setting. These radiological findings deepen our understanding of ICH-related CI and may serve as biomarkers in the view of future therapeutic intervention.

Novel Noninvasive Spinal Neuromodulation Strategy Facilitates Recovery of Stepping after Motor Complete Paraplegia.

It has been suggested that neuroplasticity-promoting neuromodulation can restore sensory-motor pathways after spinal cord injury (SCI), reactivating the dormant locomotor neuronal circuitry. We introduce a neuro-rehabilitative approach that leverages locomotor training with multi-segmental spinal cord transcutaneous electrical stimulation (scTS). We hypothesized that scTS neuromodulates spinal networks, complementing the neuroplastic effects of locomotor training, result in a functional progression toward recovery of locomotion. We conducted a case-study to test this approach on a 27-year-old male classified as AIS A with chronic SCI. The training regimen included task-driven non-weight-bearing training (1 month) followed by weight-bearing training (2 months). Training was paired with multi-level continuous and phase-dependent scTS targeting function-specific motor pools. Results suggest a convergence of cross-lesional networks, improving kinematics during voluntary non-weight-bearing locomotor-like stepping. After weight-bearing training, coordination during stepping improved, suggesting an important role of afferent feedback in further improvement of voluntary control and reorganization of the sensory-motor brain-spinal connectome.

Abstracts from The 39th Annual Symposium of the National Neurotrauma Society, including the AANS/CNS Joint Section on Neurotrauma and Critical Care

Abstracts from The 39^th Annual Symposium of the National Neurotrauma Society, including the AANS/CNS Joint Section on Neurotrauma and Critical Care

Collagen scaffold loaded allogeneic neural stem cells promoted locomotion recovery of spinal cord injury mainly through secreting neurotrophic factors

The combination of biological scaffold and neural stem cells (NSCs) is a promising treatment strategy for spinal cord injury (SCI). But adverse niche at injures spinal cord severe hamper the transplanted NSCs to preferentially differentiate neurons. In this study, we compared the therapeutic outcomes in rat completely transected SCI model after transplantations of collagen scaffold loaded with NSCs in different forms including unicellular NSCs, neurospheres, differentiated unicellular NSCs, and differentiated neurospheres. The results showed collagen scaffold combined each form of NSCs could markedly restore the motor function of the hindlimbs by Basso-Beattie-Bresnahan(BBB) scoring to similar levels. The transcriptome analysis showed there were similar expressions of neurotrophic factors among neurospheres, differentiated unicellular NSCs, and differentiated neurospere. Next, we enriched neurotrophic factors secreted from culture medium of these three forms of NSCs respectively and combined with collagen scaffold were transplanted into SCI to further assay therapeutic effects of secreting neurotrophic factors on SCI. BBB score analysis showed that the secreted neurotrophic factors from above three forms of NSCs had similar functional recovery of SCI. Therefore, we proposed that allogeneic NSCs transplant promotes functional recovery of SCI predominantly by secreting neurotrophic factors, not by direct neuronal replacement of differentiated neurons from transplanted cells.

Neurotransmitter phenotype switching by spinal excitatory interneurons regulates locomotor recovery after spinal cord injury.

Severe spinal cord injury in adults leads to irreversible paralysis below the lesion. However, adult rodents that received a complete thoracic lesion just after birth demonstrate proficient hindlimb locomotion without input from the brain. How the spinal cord achieves such striking plasticity remains unknown. In this study, we found that adult spinal cord injury prompts neurotransmitter switching of spatially defined excitatory interneurons to an inhibitory phenotype, promoting inhibition at synapses contacting motor neurons. In contrast, neonatal spinal cord injury maintains the excitatory phenotype of glutamatergic interneurons and causes synaptic sprouting to facilitate excitation. Furthermore, genetic manipulation to mimic the inhibitory phenotype observed in excitatory interneurons after adult spinal cord injury abrogates autonomous locomotor functionality in neonatally injured mice. In comparison, attenuating this inhibitory phenotype improves locomotor capacity after adult injury. Together, these data demonstrate that neurotransmitter phenotype of defined excitatory interneurons steers locomotor recovery after spinal cord injury.

Treadmill Training for Common Marmoset to Strengthen Corticospinal Connections After Thoracic Contusion Spinal Cord Injury.

Spinal cord injury (SCI) leads to locomotor dysfunction. Locomotor rehabilitation promotes the recovery of stepping ability in lower mammals, but it has limited efficacy in humans with a severe SCI. To explain this discrepancy between different species, a nonhuman primate rehabilitation model with a severe SCI would be useful. In this study, we developed a rehabilitation model of paraplegia caused by a severe traumatic SCI in a nonhuman primate, common marmoset (Callithrix jacchus). The locomotor rating scale for marmosets was developed to accurately assess the recovery of locomotor functions in marmosets. All animals showed flaccid paralysis of the hindlimb after a thoracic contusive SCI, but the trained group showed significant locomotor recovery. Kinematic analysis revealed significantly improved hindlimb stepping patterns in trained marmosets. Furthermore, intracortical microstimulation (ICMS) of the motor cortex evoked the hindlimb muscles in the trained group, suggesting the reconnection between supraspinal input and the lumbosacral network. Because rehabilitation may be combined with regenerative interventions such as medicine or cell therapy, this primate model can be used as a preclinical test of therapies that can be used in human clinical trials.

Dual-functional hydrogel system for spinal cord regeneration with sustained release of arylsulfatase B alleviates fibrotic microenvironment and promotes axonal regeneration

Traumatic damage to the spinal cord does not spontaneously heal, often leading to permanent tissue defects. We have shown that injection of imidazole-poly(organophosphazene) hydrogel (I-5) bridges cystic cavities with the newly assembled fibronectin-rich extracellular matrix (ECM). The hydrogel-created ECM contains chondroitin sulfate proteoglycans (CSPGs), collagenous fibrils together with perivascular fibroblasts, and various fibrotic proteins, all of which could hinder axonal growth in the matrix. In an in vitro fibrotic scar model, fibroblasts exhibited enhanced sensitivity to TGF-β1 when grown on CSPGs. To alleviate the fibrotic microenvironment, the I-5 hydrogel was equipped with an additional function by making a complex with ARSB, a human enzyme degrading CSPGs, via hydrophobic interaction. Delivery of the I-5/ARSB complex significantly diminished the fibrotic ECM components. The complex promoted serotonergic axonal growth into the hydrogel-induced matrix and enhanced serotonergic innervation of the lumbar motor neurons. Regeneration of the propriospinal axons deep into the matrix and to the lumbar spinal cord was robustly increased accompanied by improved locomotor recovery. Therefore, our dual-functional system upgraded the functionality of the hydrogel for spinal cord regeneration by creating ECM to bridge tissue defects and concurrently facilitating axonal connections through the newly assembled ECM.

Alginate hydrogel cross-linked by Ca2+ to promote spinal cord neural stem/progenitor cell differentiation and functional recovery after a spinal cord injuryhh.

Alginate capillary hydrogels seeded with differentiated cells can fill the lesion cavity and promote axonal regeneration after grafting into the injured spinal cord. Neural stem/progenitor cells (NSPCs) can potentially repair the spinal cord; however, effects of alginate hydrogels (AHs) on NSPCs remain unknown. In this study, we fabricated AHs cross-linked by Ca2+ and seeded hydrogels with rat embryonic day 14 NSPCs. Immunocytochemistry and electron microscopy show that NSPCs survive, proliferate and differentiate into neurons in vitro within the capillaries. After transplantation into an acute T8 complete spinal cord transection site in adult rats, approximately one-third (38.3%) of grafted cells survive and differentiate into neurons (40.7%), astrocytes (26.6%) and oligodendrocytes (28.4%) at 8 weeks post-grafting. NSPCs promote the growth of host axons within the capillaries in a time-dependent manner. Host axons make synapse-like contacts with NSPC-derived neurons within the hydrogel channels, and graft-derived axons extend into the host white and gray matter making putative synapses. This is paralleled by improved electrophysiological conductivity across the lesion and partial hindlimb locomotor recovery.

Knockdown of a Cyclic Nucleotide-Gated Ion Channel Impairs Locomotor Activity and Recovery From Hypoxia in Adult Drosophila melanogaster.

Cyclic guanosine monophosphate (cGMP) modulates the speed of recovery from anoxia in adult Drosophila and mediates hypoxia-related behaviors in larvae. Cyclic nucleotide-gated channels (CNG) and cGMP-activated protein kinase (PKG) are two cGMP downstream targets. PKG is involved in behavioral tolerance to hypoxia and anoxia in adults, however little is known about a role for CNG channels. We used a CNGL (CNG-like) mutant with reduced CNGL transcripts to investigate the contribution of CNGL to the hypoxia response. CNGL mutants had reduced locomotor activity under normoxia. A shorter distance travelled in a standard locomotor assay was due to a slower walking speed and more frequent stops. In control flies, hypoxia immediately reduced path length per minute. Flies took 30–40 min in normoxia for >90% recovery of path length per minute from 15 min hypoxia. CNGL mutants had impaired recovery from hypoxia; 40 min for ∼10% recovery of walking speed. The effects of CNGL mutation on locomotor activity and recovery from hypoxia were recapitulated by pan-neuronal CNGL knockdown. Genetic manipulation to increase cGMP in the CNGL mutants increased locomotor activity under normoxia and eliminated the impairment of recovery from hypoxia. We conclude that CNGL channels and cGMP signaling are involved in the control of locomotor activity and the hypoxic response of adult Drosophila.

The Effect of Galvanic Vestibular Stimulation on Visuospatial Cognition in an Incomplete Bilateral Vestibular Deafferentation Mouse Model.

ObjectivesTo evaluate the efficacy of galvanic vestibular stimulation (GVS) for recovering from the locomotor and spatial memory deficits of a murine bilateral vestibular deafferentation (BVD) model.MethodsMale C57BL/6 mice (n = 36) were assigned to three groups: bilateral labyrinthectomy with (BVD_GVS group) and without (BVD_non-GVS group) the GVS intervention, and a control group with the sham operation. We used the open field and Y maze, and Morris water maze (MWM) tests to assess locomotor and visuospatial cognitive performance before (baseline) and 3, 7, and 14 days after surgical bilateral labyrinthectomy. For the GVS group, a sinusoidal current at the frequency at 1 Hz and amplitude 0.1 mA was delivered for 30 min daily from the postoperative day (POD) 0 to 4 via electrodes inserted subcutaneously close to both the bony labyrinths.ResultsShort-term spatial memory was significantly impaired in bilaterally labyrinthectomized mice (BVD_non-GVS group), as reflected by decreased spontaneous alternation performance in the place recognition test and time spent in the novel arm and increased same arm return in the Y-maze test, compared with the control. Long-term spatial memory was also impaired, as indicated by a longer escape latency in the hidden platform trial and a lower percentage of time spent in the target quadrant in the probe trial of the MWM. GVS application significantly accelerated the recovery of locomotion and short-term and long-term spatial memory deficits in the BVD mice.ConclusionsOur data demonstrate that locomotion, short-term, and long-term (at least 2 weeks) spatial memory were impaired in BVD mice. The early administration of sinusoidal GVS accelerated the recovery of those locomotion and spatial memory deficiencies. GVS could be applied to patients with BVD to improve their locomotion and vestibular cognitive functioning.

Fatty Acid-Binding Protein 4 Inhibition Promotes Locomotor and Autonomic Recovery in Rats following Spinal Cord Injury.

The inflammatory response associated with traumatic spinal cord injury (SCI) contributes to locomotor and sensory impairments. Pro-inflammatory (M1) macrophages/microglia (MϕMG) are the major cellular players in this response as they promote chronic inflammation resulting in injury expansion and tissue damage. Fatty acid-binding protein 4 (FABP4) promotes M1 MϕMG differentiation; however, it is unknown if FABP4 also plays a role in the etiology of SCI. The present study investigates whether FABP4's gene expression influences functional recovery following SCI. Analysis of quantitative polymerase chain reaction data shows a robust induction of FABP4 messenger RNA (mRNA; >100 fold) in rats subjected to a T9-T10 contusion injury compared with control. Western blot experiments reveal significant upregulation of FABP4 protein at the injury epicenter, and immunofluorescence analysis identifies that this upregulation occurs in CD11b+ MϕMG. Further, upregulation of FABP4 gene expression correlates with peroxisome proliferator-activated receptor γ (PPARγ) downregulation, inactivation of Iκβα, and the activation of the NF-κB pathway. Analysis of locomotor recovery using the Basso-Beattie-Bresnahan's locomotor scale and the CatWalk gait analysis system shows that injured rats treated with FABP4 inhibitor BMS309403 have significant improvements in locomotion compared with vehicle controls. Additionally, inhibitor-treated rats exhibit enhanced autonomic bladder reflex recovery. Immunofluorescence experiments also show the administration of the FABP4 inhibitor increases the number of CD163+ and liver arginase+ M2 MϕMG within the epicenter and penumbra of the injured spinal cord 28 days post-injury. These findings show that FABP4 may significantly exacerbate locomotor and sensory impairments during SCI by modulating macrophage/microglial activity.

Mechanics-electro-adaptive multifunctional bioactive nanocomposites hydrogel for inducing spinal cord regeneration

Traumatic spinal cord injury (SCI) is one of the most serious injuries, which will cause severe sensory and motor obstacles. The current effective treatment methods remain a worldwide challenge for both clinic and research. The lack of electrical signal conduction and cell growth microenvironment in SCI is one of the main obstacles that inhibit spinal cord regeneration. Herein, we report a multifunctional polycitrate-based nanocomposite hydrogel (PMEAC) scaffold with spinal cord-biomimetic mechanical and electrical properties for promoting spinal cord repair and regeneration. PMEAC scaffolds were prepared using poly(citrate-maleic)-ε-polylysine (PME) and multi-walled carbon nanotubes (MWCNT) by the facile self-crosslinking route. PMEAC scaffolds possessed multifunctional properties including injectability, self-healing ability, tissue-adhesiveness, broad-spectrum antibacterial property and UV-shielding performance, especially the biomimetic mechanical modulus (G’ = 291.69 Pa) and electroconductivity (0.15 S/m) with spinal cord tissue. PMEAC scaffolds also exhibited good cytocompatibility, hemocompatibility and biodegradability. In vivo results from a spinal cord defect model showed that PMEAC scaffolds could significantly improve locomotion recovery (BBB score 6–7), reduce inflammation, promote remyelination and axon regeneration after SCI. This study showed that multifunctional bioactive biomaterials with mechanics-electro-biomimetic properties should be the effective strategy to promote SCI recovery and regeneration.

Suppressing CSPG/LAR/PTPσ Axis Facilitates Neuronal Replacement and Synaptogenesis by Human Neural Precursor Grafts and Improves Recovery after Spinal Cord Injury.

Traumatic spinal cord injury (SCI) is a leading cause of permanent neurologic disabilities in young adults. Functional impairments after SCI are substantially attributed to the progressive neurodegeneration. However, regeneration of spinal-specific neurons and circuit re-assembly remain challenging in the dysregulated milieu of SCI because of impaired neurogenesis and neuronal maturation by neural precursor cells (NPCs) spontaneously or in cell-based strategies. The extrinsic mechanisms that regulate neuronal differentiation and synaptogenesis in SCI are poorly understood. Here, we perform extensive in vitro and in vivo studies to unravel that SCI-induced upregulation of matrix chondroitin sulfate proteoglycans (CSPGs) impedes neurogenesis of NPCs through co-activation of two receptor protein tyrosine phosphatases, LAR and PTPσ. In adult female rats with SCI, systemic co-inhibition of LAR and PTPσ promotes regeneration of motoneurons and spinal interneurons by engrafted human directly reprogramed caudalized NPCs (drNPC-O2) and fosters their morphologic maturity and synaptic connectivity within the host neural network that culminate in improved recovery of locomotion and sensorimotor integration. Our transcriptomic analysis of engrafted human NPCs in the injured spinal cord confirmed that inhibition of CSPG receptors activates a comprehensive program of gene expression in NPCs that can support neuronal differentiation, maturation, morphologic complexity, signal transmission, synaptic plasticity, and behavioral improvement after SCI. We uncovered that CSPG/LAR/PTPσ axis suppresses neuronal differentiation in part by blocking Wnt/β-Catenin pathway. Taken together, we provide the first evidence that CSPGs/LAR/PTPσ axis restricts neurogenesis and synaptic integration of new neurons in NPC cellular therapies for SCI. We propose targeting LAR and PTPσ receptors offers a promising clinically-feasible adjunct treatment to optimize the efficacy and neurologic benefits of ongoing NPC-based clinical trials for SCI.SIGNIFICANCE STATEMENT Transplantation of neural precursor cells (NPCs) is a promising approach for replacing damaged neurons after spinal cord injury (SCI). However, survival, neuronal differentiation, and synaptic connectivity of transplanted NPCs within remain challenging in SCI. Here, we unravel that activation of chondroitin sulfate proteoglycan (CSPG)/LAR/PTPσ axis after SCI impedes the capacity of transplanted human NPCs for replacing functionally integrated neurons. Co-blockade of LAR and PTPσ is sufficient to promote re-generation of motoneurons and spinal V1 and V3 interneurons by engrafted human caudalized directly reprogramed NPCs (drNPC-O2) and facilitate their synaptic integration within the injured spinal cord. CSPG/LAR/PTPσ axis appears to suppress neuronal differentiation of NPCs by inhibiting Wnt/β-Catenin pathway. These findings identify targeting CSPG/LAR/PTPσ axis as a promising strategy for optimizing neuronal replacement, synaptic re-connectivity, and neurologic recovery in NPC-based strategies.

484 The Role of Plasma Derived Purified Exosome Product (PEP) for Improved Locomotor Recovery in Spinal Cord Injured Rats

INTRODUCTION: Preclinical studies indicate that mesenchymal stem cell-derived secretomes exert immunomodulatory and neuroprotective effects in various spinal cord injury models. Exosomes as one of the secretomes, have been shown to successfully aid wound healing for the skin and following myocardial infarction. However, the effects of human plasma-derived purified exosomal products (PEP) on spinal cord injury (SCI) have not been explored to this date. METHODS: Twenty-four female rats were randomly divided into five groups. These included 2 control groups for the I.V or I.T injected PEP groups and a sham group. Groups were subject to a T9 laminectomy, followed by a moderate contusion SCI. After 24 hours, lactated ringer solution or PEP was injected through I.V or IT routes. Rats were assessed weekly for a total period of 10 weeks by an open field Basso, Beattie, and Bresnahan (BBB) motor score and a postmortem quantification of external scaring, neuronal regeneration, cavity volume, and glial scarring was carried out at the end of the follow-up period. RESULTS: Animals treated with I.V PEP or IT PEP had significant improvement within the first-week post-injury (1.05±1.2 vs. 2.16±3.15) and (1.08±3.5 vs. 4.5±0.083) respectively, with sustained improvement in BBB score at 10 weeks (11±1.16 vs. 13.8±1.90), (11.3±1.4 vs. 14±1.16). CONCLUSION: Our preliminary results suggest that I.V or IT PEP injection without immunosuppression might provide beneficial effects initially following traumatic SCI.

483 Improvement in Behavioral, Cellular and Molecular Outcomes with Connexin Inhibitors Administration in a Porcine Spinal Cord Injury Model

INTRODUCTION: Connexin inhibition has demonstrated promising results in rodents spinal cord injury models (SCI) as a neuroprotective strategy that improves SCI outcomes. However, it has not been tested in a larger animal yet, a necessary step before conducting a human clinical trial. METHODS: SCI was induced using a compression/contusion weight drop model in three groups of pigs with 2 animals per group. Group A: control, Group B: treated with nonselective gap junctions blocker: Carbenoxolone, Group C: treated with connexin-43 memetic peptide: Gap26. All medications were delivered intrathecally at the time of injury. We assessed the locomotor development of the animals over 11 weeks. After which the animals were euthanized and their spinal cords were harvested for histological and immunofluorescence assessment. RESULTS: Treatment with intrathecal connexins inhibitors improved SCI outcomes. In terms of locomotion recovery, Groups B and C regained the stepping ability in their hind limbs, whereas the control group did not. Similarly, groups B and C exhibited a decreased level of astrocytes activation at the injury site, and their PGE2 serum levels remained low in the two treated groups. The histological damage was particularly limited in Gap 26-treated group (C). CONCLUSION: We translated the positive neuroprotective effect of a connexin-43 mimetic peptide and a gap junction blocker in a porcine SCI model. This study supports the potential role of these agents in improving SCI outcome.

Targeted activation of midbrain neurons restores locomotor function in mouse models of parkinsonism

The pedunculopontine nucleus (PPN) is a locomotor command area containing glutamatergic neurons that control locomotor initiation and maintenance. These motor actions are deficient in Parkinson’s disease (PD), where dopaminergic neurodegeneration alters basal ganglia activity. Being downstream of the basal ganglia, the PPN may be a suitable target for ameliorating parkinsonian motor symptoms. Here, we use in vivo cell-type specific PPN activation to restore motor function in two mouse models of parkinsonism made by acute pharmacological blockage of dopamine transmission. With a combination of chemo- and opto-genetics, we show that excitation of caudal glutamatergic PPN neurons can normalize the otherwise severe locomotor deficit in PD, whereas targeting the local GABAergic population only leads to recovery of slow locomotion. The motor rescue driven by glutamatergic PPN activation is independent of activity in nearby locomotor promoting glutamatergic Cuneiform neurons. Our observations point to caudal glutamatergic PPN neurons as a potential target for neuromodulatory restoration of locomotor function in PD.

Neuroprotective effect of aldose reductase knockout in a mouse model of spinal cord injury involves NF-κB pathway.

The inflammatory response following spinal cord injury (SCI) involves the activation of resident microglia and the infiltration of macrophages. Activated microglia/macrophages have either detrimental or beneficial effects on neural regeneration based on their functional polarized M1/M2 subsets. Aldose reductase (AR) has recently been shown to be a key component of the innate immune response. However, the mechanisms involved in AR and innate immune response remain unclear. In this study, wild-type (WT) or AR-deficiency (KO) mice were subjected to SCI by a spinal crush injury model. AR KO mice showed better locomotor recovery and smaller injury lesion areas after spinal cord crushing compared with WT mice. Here, we first demonstrated that AR deficiency repressed the expression level of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide (LPS) in vitro via the activation of autophagy. AR deficiency caused 4-hydroxy-2-(E)-nonenal (4-HNE) accumulation in LPS-induced macrophages. We also found that exogenous addition of low concentrations of 4-HNE in LPS-induced macrophages had the effect of promoting further activation of NF-κB pathway, whereas high concentrations of 4-HNE had inhibitory effects. Together, these results indicated that autophagy as a mechanism underlying AR and 4-HNE in LPS-induced macrophages.

Restoring Function After Severe Spinal Cord Injury Through BioLuminescent-OptoGenetics.

The ability to manipulate specific neuronal populations of the spinal cord following spinal cord injury (SCI) could prove highly beneficial for rehabilitation in patients through maintaining and strengthening still existing neuronal connections and/or facilitating the formation of new connections. A non-invasive and highly specific approach to neuronal stimulation is bioluminescent-optogenetics (BL-OG), where genetically expressed light emitting luciferases are tethered to light sensitive channelrhodopsins (luminopsins, LMO); neurons are activated by the addition of the luciferase substrate coelenterazine (CTZ). This approach utilizes ion channels for current conduction while activating the channels through the application of a small chemical compound, thus allowing non-invasive stimulation and recruitment of all targeted neurons. Rats were transduced in the lumbar spinal cord with AAV2/9 to express the excitatory LMO3 under control of a pan-neuronal or motor neuron-specific promoter. A day after contusion injury of the thoracic spine, rats received either CTZ or vehicle every other day for 2 weeks. Activation of either neuron population below the level of injury significantly improved locomotor recovery lasting beyond the treatment window. Utilizing histological and gene expression methods we identified neuronal plasticity as a likely mechanism underlying the functional recovery. These findings provide a foundation for a rational approach to spinal cord injury rehabilitation, thereby advancing approaches for functional recovery after SCI.SummaryBioluminescent optogenetic activation of spinal neurons results in accelerated and enhanced locomotor recovery after spinal cord injury in rats.

L-Arginine Depletion Improves Spinal Cord Injury via Immunomodulation and Nitric Oxide Reduction.

Background: Spinal cord injury (SCI) elicits robust neuroinflammation that eventually exacerbates the initial damage to the spinal cord. L-arginine is critical for the responsiveness of T cells, which are important contributors to neuroinflammation after SCI. Furthermore, L-arginine is the substrate for nitric oxide (NO) production, which is a known inducer of secondary damage. Methods: To accomplish systemic L-arginine depletion, repetitive injections of recombinant arginase-1 (rArg-I) were performed. Functional recovery and histopathological parameters were analyzed. Splenic immune responses were evaluated by flow cytometry. Pro-inflammatory gene expression and nitrite concentrations were measured. Results: We show for the first time that systemic L-arginine depletion improves locomotor recovery. Flow cytometry and immunohistological analysis showed that intraspinal T-cell infiltration was reduced by 65%, and peripheral numbers of Th1 and Th17 cells were suppressed. Moreover, rArg-I treatment reduced the intraspinal NO production by 40%. Histopathological analyses revealed a 37% and 36% decrease in the number of apoptotic neurons and neuron-macrophage/microglia contacts in the spinal cord, respectively. Conclusions: Targeting detrimental T-cell responses and NO-production via rArg-I led to a reduced neuronal cell death and an improved functional recovery. These findings indicate that L-arginine depletion holds promise as a therapeutic strategy after SCI.

Ac-SDKP peptide improves functional recovery following spinal cord injury in a preclinical model

Damage to the spinal cord triggers a local complex inflammatory reaction that results in irreversible impairments or complete loss of motor function. The evidence suggested that inhibiting the pro-inflammatory macrophage/microglia (M1 subsets) and stimulating the anti-inflammatory macrophage/microglia (M2 subsets) are potential strategies for the treatment of neuroinflammation-related diseases. We evaluated the potentially protective effect of Ac-SDKP as an endogenous tetrapeptide on rat spinal cord injury (SCI). Wistar rats were subjected to a weight-drop contusion model and were treated with Ac-SDKP (0.8 mg/kg) given subcutaneously once a day for 7 days starting at two clinically relevant times, at 2 h or 6 h post-injury. The effect of Ac-SDKP was assessed by motor functional analysis, real-time PCR (CD86 and CD206 mRNA), western blot (caspase-3), ELISA (TNF-a, IL-10), and histological analysis (toluidine blue staining). Ac-SDKP improved locomotor recovery and rescue motor neuron loss after SCI. Moreover, a decreased in TNF-a level as well as caspase 3 protein levels occurred in the lesion epicenter of the spinal cord following treatment. In addition, CD206 mRNA expression level increased significantly in Ac-SDKP treated rats compared with SCI. Together these data suggest that Ac-SDKP might be a novel immunomodulatory drug. It may be beneficial for the treatment of SCI with regards to increasing CD206 gene expression and suppress inflammatory cytokine to improve motor function and reducing histopathological lesion.