Neurotrophin-3 attenuates human peripheral blood T cell and monocyte activation status and cytokine production post stroke.

BACKGROUND AND PURPOSE: Stroke therapy still lacks successful measures to improve post stroke recovery. Neurotrophin-3 (NT-3) is one promising candidate which has proven therapeutic benefit in motor recovery in acute experimental stroke. Post stroke, the immune system has opposing pathophysiological roles: pro-inflammatory cascades and immune cell infiltration into the brain exacerbate cell death while the peripheral immune response has only limited capabilities to fight infections during the acute and subacute phase. With time, anti-inflammatory mechanisms are supposed to support recovery of the ischemic damage within the brain parenchyma. However, interestingly, NT-3 can improve recovery in chronic neurological injury when combined with the pro-inflammatory stimulus lipopolysaccharide (LPS). AIM: We elucidated the impact of NT-3 on human monocyte and T cell activation as well as cytokine production ex vivo after stroke. In addition, we investigated the age-dependent availability of the high affinity NT-3 receptor TrkC upon LPS stimulation. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from acute stroke patients and controls and incubated with different dosages of NT-3 (10 and 100 ng/mL) and with or without LPS or anti-CD3/CD28 for 48 h. Total TrkC expression and cell activation (CD25, CD69 and HLA-DR) were assessed by FACS staining. IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21 and IL-22 were quantified by cytometric bead array. RESULTS: Most monocytes and only a small proportion of T cells expressed TrkC in blood from humans without stroke. Activation of cells from young humans (without strokes) using anti-CD3/CD28 or LPS partially reduced the proportion of monocytes expressing TrkC whilst they increased the proportion of T cells expressing TrkC. In contrast, activation of cells from elderly humans (without strokes) did not affect the proportion of monocytes expressing TrkC and only anti-CD3/CD28 led to an increase in the proportion of CD4+ T cells expressing TrkC. In blood from stroke patients or controls, NT-3 treatment reduced the percentage of monocytes and CD4+ and CD8+ T cells that were activated and reduced all cytokines investigated besides IL-21. CONCLUSIONS: NT-3 attenuated immune responses in cells from stroke patients and controls. The mechanism whereby human immune cells respond to NT-3 may be via TrkC receptors whose levels are regulated by stimulation. Further work is required to determine whether the induction of sensorimotor recovery in rodents by NT-3 after CNS injury is caused by this attenuation of the immune response.

Therapeutic Development in Charcot Marie Tooth Type 1 Disease.

Charcot-Marie-Tooth disease (CMT) is the most frequent hereditary peripheral neuropathies. It is subdivided in two main groups, demyelinating (CMT1) and axonal (CMT2). CMT1 forms are the most frequent. The goal of this review is to present published data on 1-cellular and animal models having opened new potential therapeutic approaches. 2-exploration of these tracks, including clinical trials. The first conclusion is the great increase of publications on CMT1 subtypes since 2000. We discussed two points that should be considered in the therapeutic development toward a regulatory-approved therapy to be proposed to patients. The first point concerns long term safety if treatments will be a long-term process. The second point relates to the evaluation of treatment efficiency. Degradation of CMT clinical phenotype is not linear and progressive.

NT3P75-2 gene-modified bone mesenchymal stem cells improve neurological function recovery in mouse TBI model.

BACKGROUND: The attainment of extensive neurological function recovery remains the key challenge for the treatment of traumatic brain injury (TBI). Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) has been shown to improve neurological function recovery after TBI. However, the survival of BMSCs after transplantation in early-stage TBI is limited, and much is unknown about the mechanisms mediating this neurological function recovery. Secretion of neurotrophic factors, including neurotrophin 3 (NT3), is one of the critical factors mediating BMSC neurological function recovery. Gene mutation of NT3 (NT3P75-2) has been shown to enhance the biological function of NT3 via the reduction of the activation of the P75 signal pathway. Thus, we investigated whether NT3P75-2 gene-modified BMSCs could enhance the survival of BMSCs and further improve neurological function recovery after TBI. METHODS: The ability of NT3P75-2 induction to improve cell growth rate of NSC-34 and PC12 cells in vitro was first determined. BMSCs were then infected with three different lentiviruses (green fluorescent protein (GFP), GFP-NT3, or GFP-NT3P75-2), which stably express GFP, GFP-NT3, or GFP-NT3P75-2. At 24 h post-TBI induction in mice, GFP-labeled BMSCs were locally transplanted into the lesion site. Immunofluorescence and histopathology were performed at 1, 3, and/or 7 days after transplantation to evaluate the survival of BMSCs as well as the lesion volume. A modified neurological severity scoring system and the rotarod test were chosen to evaluate the functional recovery of the mice. Cell growth rate, glial activation, and signaling pathway analyses were performed to determine the potential mechanisms of NT3P75-2 in functional recovery after TBI. RESULTS: Overall, NT3P75-2 improved cell growth rate of NSC-34 and PC12 cells in vitro. In addition, NT3P75-2 significantly improved the survival of transplanted BMSCs and neurological function recovery after TBI. Overexpression of NT3P75-2 led to a significant reduction in the activation of glial cells, brain water content, and brain lesion volume after TBI. This was associated with a reduced activation of the p75 neurotrophin receptor (P75NTR) and the c-Jun N-terminal kinase (JNK) signal pathway due to the low affinity of NT3P75-2 for the receptor. CONCLUSIONS: Taken together, our results demonstrate that administration of NT3P75-2 gene-modified BMSCs dramatically improves neurological function recovery after TBI by increasing the survival of BMSCs and ameliorating the inflammatory environment, providing a new promising treatment strategy for TBI.

Effects of bioengineered scaffold loaded with neurotrophins and locomotor training in restoring H-reflex responses after spinal cord injury.

The combinational effects of a bioengineered scaffold loaded with neurotrophins and rehabilitation training on spasticity observed after spinal cord injury (SCI) has not been studied. We used an animal model of moderate contusion injury at T9/T10 that received bioengineered scaffold poly N-isopropylacrylamide-g-poly ethylene glycol (PNIPAAm-g-PEG) loaded with BDNF/NT3 followed by body weight supported treadmill training (BWSTT) and assessed the efficacy of the combinational bioengineered approaches in treating spasticity. Five animal groups were included: Group 1: Sham, Group 2: Injury (SCI), Group 3: SCI + BWSTT (BWSTT), Group 4: SCI + PNIPAAm-g-PEG loaded with BDNF/NT3 (Transplant), and Group 5: SCI + PNIPAAm-g-PEG loaded with BDNF/NT3 + BWSTT (Combinational). Results indicate no significant changes in the BBB scores of animals among various groups, however, a significant restoration in the rate depression property of H-reflex was observed in both BWSTT and Combinational animals. Transplant group reported no improvement in the rate depression property of H-reflex and were similar to SCI only group. Histological findings report restoration of the chloride cotransporter (KCC2) labeling in both BWSTT and Combinational animals and down-regulation of KCC2 in both SCI and Transplant only animals. Findings from this study confirm that rehabilitation training is critical in restoring H-reflex responses and transplantation therapies alone cannot restore these responses after SCI. Also, although no significant difference was observed between the BWSTT and Combinational animals, comparable improvements in the two groups does open new pathways to exploring unique tissue-engineering approaches with promising clinical application for individuals with SCI.

Neural repair by NT3-chitosan via enhancement of endogenous neurogenesis after adult focal aspiration brain injury.

The latent regenerative potential of endogenous neural stem/progenitor cells (NSCs) in the adult mammalian brain has been postulated as a likely source for neural repair. However, the inflammatory and inhibitory microenvironment after traumatic brain injury (TBI) prohibits NSCs from generating new functional neurons to restore brain function. Here we report a biodegradable material, chitosan, which, when loaded with neurotrophin-3 (NT3) and injected into the lesion site after TBI, effectively engaged endogenous NSCs to proliferate and migrate to the injury area. NSCs differentiate and mature into functional neurons, forming nascent neural networks that further integrate into existing neural circuits to restore brain function. Three main actions of NT3-chitosan, i.e., pro-neurogenesis, anti-inflammation, and pro-revascularization, elicit significant regeneration after TBI. Our study suggests that through creating an optimal microenvironment, endogenous NSCs are capable of executing neural repair, thus widening the therapeutic strategies to treat TBI and perhaps stroke or other neurological conditions.

The Promotion of Neural Regeneration in A Rat Facial Nerve Crush Injury Model Using Collagen-Binding NT-3.

Traumatic facial nerve injury, an important cause of facial paralysis, has a number of adverse effects, including facial muscle dysfunction and facial asymmetry. It has been demonstrated in our previous work that native human NT-3 fused with a collagen-binding domain (CBD-NT-3) could bind to collagen, specifically to exert neurotrophic effects, promoting axonal regeneration. To evaluate the effect of CBD-NT-3 in inducing facial nerve regeneration and functional recovery, the differing effects of CBD-NT-3 and native neurotrophin-3 (NAT-NT-3) were observed using the results of facial nerve functional recovery, electrophysiological testing, and axonal and myelin changes in a rat model of facial nerve crush injury. The rats were injected in the epineurium in crushed fibers of the facial nerve with CBD-NT-3, NAT-NT-3, and PBS respectively. After 4 weeks, the CBD-NT-3 group demonstrated significantly more ordered growth of axons and nerve functional recovery than the NAT-NT-3 group. The results suggest that CBD-NT-3 considerably enhances facial nerve regeneration and functional recovery.

Electrospun Fibers for Drug Delivery after Spinal Cord Injury and the Effects of Drug Incorporation on Fiber Properties.

There is currently no cure for individuals with spinal cord injury (SCI). While many promising approaches are being tested in clinical trials, the complexity of SCI limits several of these approaches from aiding complete functional recovery. Several different categories of biomaterials are investigated for their ability to guide axonal regeneration, to deliver proteins or small molecules locally, or to improve the viability of transplanted stem cells. The purpose of this study is to provide a brief overview of SCI, present the different categories of biomaterial scaffolds that direct and guide axonal regeneration, and then focus specifically on electrospun fiber guidance scaffolds. Much like other polymer guidance approaches, electrospun fibers can retain and deliver therapeutic drugs. The experimental section presents new data showing the incorporation of two therapeutic drugs into electrospun poly-L-lactic acid fibers. Two different concentrations of either riluzole or neurotrophin-3 were loaded into the electrospun fibers to examine the effect of drug concentration on the physical characteristics of the fibers (fiber alignment and fiber diameter). Overall, the drugs were successfully incorporated into the fibers and the release was related to the loading concentration. The fiber diameter decreased with the inclusion of the drug, and the decreased diameter was correlated with a decrease in fiber alignment. Subsequently, the study includes considerations for successful incorporation of a therapeutic drug without changing the physical properties of the fibers.

NT-3 promotes proprioceptive axon regeneration when combined with activation of the mTor intrinsic growth pathway but not with reduction of myelin extrinsic inhibitors.

Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons.

Graft of the NT-3 persistent delivery gelatin sponge scaffold promotes axon regeneration, attenuates inflammation, and induces cell migration in rat and canine with spinal cord injury.

Persistent neurotrophic factor delivery is crucial to create a microenvironment for cell survival and nerve regeneration in spinal cord injury (SCI). This study aimed to develop a NT-3/fibroin coated gelatin sponge scaffold (NF-GS) as a novel controlled artificial release therapy for SCI. In vitro, bone marrow-derived mesenchymal stem cells (MSCs) were planted into the NF-GS and release test showed that NF-GS was capable to generate a sustainable NT-3 release up to 28 days. MSCs in NF-GS had high cell activity with excellent cell distribution and phenotype. Then, the NF-GS was transplanted into the injury site of spinal cord of rat and canine in vivo, which exhibited strong biocompatibility during post-transplantation period. Four weeks following transplantation, the concentration of NT-3 was much higher than that in control groups. Cavity areas in the injury/graft site were significantly reduced due to tissue regeneration and axonal extensions associated with myelin sheath through the glial scar into the NF-GS. Additionally, the NF-GS decreased the inflammation by reducing the CD68 positive cells and TNF-α. A striking feature was the occurrence of some cells and myelin-like structure that appeared to traverse the NF-GS. The present results demonstrate that the NF-GS has the property to control the release of NT-3 from the NT-3/fibroin complex thus facilitating regeneration of injured spinal cord.

Delayed intramuscular human neurotrophin-3 improves recovery in adult and elderly rats after stroke.

There is an urgent need for a therapy that reverses disability after stroke when initiated in a time frame suitable for the majority of new victims. We show here that intramuscular delivery of neurotrophin-3 (NT3, encoded by NTF3) can induce sensorimotor recovery when treatment is initiated 24 h after stroke. Specifically, in two randomized, blinded preclinical trials, we show improved sensory and locomotor function in adult (6 months) and elderly (18 months) rats treated 24 h following cortical ischaemic stroke with human NT3 delivered using a clinically approved serotype of adeno-associated viral vector (AAV1). Importantly, AAV1-hNT3 was given in a clinically-feasible timeframe using a straightforward, targeted route (injections into disabled forelimb muscles). Magnetic resonance imaging and histology showed that recovery was not due to neuroprotection, as expected given the delayed treatment. Rather, treatment caused corticospinal axons from the less affected hemisphere to sprout in the spinal cord. This treatment is the first gene therapy that reverses disability after stroke when administered intramuscularly in an elderly body. Importantly, phase I and II clinical trials by others show that repeated, peripherally administered high doses of recombinant NT3 are safe and well tolerated in humans with other conditions. This paves the way for NT3 as a therapy for stroke.

Is there a role for neurotrophic factors and their receptors in augmenting the neuroprotective effect of (-)-epigallocatechin-3-gallate treatment of sciatic nerve crush injury?

This study analyzed and compared the effects of EGCG treatment on the expression of NTFs and NTF receptors expression in the sciatic nerve and the L3-L6 spinal cord segments at the early phase of regeneration following sciatic nerve crush injury. Analysis of BDNF, GDNF and NT3 neurotropic factors and Trk-B, Trk-C and NGFR-p75 receptors in neurons in the spinal cord of CRUSH and CRUSH + EGGC rats showed significant (p < 0.0001) decrease compared to NAÏVE and SHAM at day 1, 3, 7 and 14 after nerve injury. EGCG treatment significantly (p < 0.0001) increased the BDNF, GDN, NT3, Trk-B, Trk-C and NGFR-p75 immunostaining in the L3-L6 spinal cord compared to CRUSH animals. Also, EGCG treatment significantly increased the Trk-B protein concentration and Trk-B, NT3 and Trk-C gene expression in the spinal cords compared to CRUSH group. However, at day 1 and 3 post nerve injury, EGCG treatment significantly decreased the NGFR-p75 expression compared to CRUSH rats. In the sciatic nerve, EGCG treatment significantly (p < 0.01) increased the Trk-B and NGFR-p75 protein concentration in the controls. EGCG treatment significantly (p < 0.0001) increased the Trk-B, Trk-C and NGFR-p75 mRNA gene expressions in the sciatic nerves compared to CRUSH group. Only at day 1, CRUSH + EGCG animals displayed significant rise in the sciatic nerves NT3 gene expression compared to CRUSH group. Our data suggest that the EGCG neuroprotective effect on the spinal cord neurons may be mediated through the modulation of NTFs and NTF receptors following nerve crush injury in a rat model.

Spinal electro-magnetic stimulation combined with transgene delivery of neurotrophin NT-3 and exercise: novel combination therapy for spinal contusion injury.

Our recent terminal experiments revealed that administration of a single train of repetitive spinal electromagnetic stimulation (sEMS; 35 min) enhanced synaptic plasticity in spinal circuitry following lateral hemisection spinal cord injury. In the current study, we have examined effects of repetitive sEMS applied as a single train and chronically (5 wk, every other day) following thoracic T10 contusion. Chronic studies involved examination of systematic sEMS administration alone and combined with exercise training and transgene delivery of neurotrophin [adeno-associated virus 10-neurotrophin 3 (AAV10-NT3)]. Electrophysiological intracellular/extracellular recordings, immunohistochemistry, behavioral testing, and anatomical tracing were performed to assess effects of treatments. We found that administration of a single sEMS train induced transient facilitation of transmission through preserved lateral white matter to motoneurons and hindlimb muscles in chronically contused rats with effects lasting for at least 2 h. These physiological changes associated with increased immunoreactivity of GluR1 and GluR2/3 glutamate receptors in lumbar neurons. Systematic administration of sEMS alone for 5 wk, however, was unable to induce cumulative improvements of transmission in spinomuscular circuitry or improve impaired motor function following thoracic contusion. Encouragingly, chronic administration of sEMS, followed by exercise training (running in an exercise ball and swimming), induced the following: 1) sustained strengthening of transmission to lumbar motoneurons and hindlimb muscles, 2) better retrograde transport of anatomical tracer, and 3) improved locomotor function. Greatest improvements were seen in the group that received exercise combined with sEMS and AAV-NT3.

NT3-chitosan elicits robust endogenous neurogenesis to enable functional recovery after spinal cord injury.

Neural stem cells (NSCs) in the adult mammalian central nervous system (CNS) hold the key to neural regeneration through proper activation, differentiation, and maturation, to establish nascent neural networks, which can be integrated into damaged neural circuits to repair function. However, the CNS injury microenvironment is often inhibitory and inflammatory, limiting the ability of activated NSCs to differentiate into neurons and form nascent circuits. Here we report that neurotrophin-3 (NT3)-coupled chitosan biomaterial, when inserted into a 5-mm gap of completely transected and excised rat thoracic spinal cord, elicited robust activation of endogenous NSCs in the injured spinal cord. Through slow release of NT3, the biomaterial attracted NSCs to migrate into the lesion area, differentiate into neurons, and form functional neural networks, which interconnected severed ascending and descending axons, resulting in sensory and motor behavioral recovery. Our study suggests that enhancing endogenous neurogenesis could be a novel strategy for treatment of spinal cord injury.

Transcriptome analyses reveal molecular mechanisms underlying functional recovery after spinal cord injury.

Spinal cord injury (SCI) is considered incurable because axonal regeneration in the central nervous system (CNS) is extremely challenging, due to harsh CNS injury environment and weak intrinsic regeneration capability of CNS neurons. We discovered that neurotrophin-3 (NT3)-loaded chitosan provided an excellent microenvironment to facilitate nerve growth, new neurogenesis, and functional recovery of completely transected spinal cord in rats. To acquire mechanistic insight, we conducted a series of comprehensive transcriptome analyses of spinal cord segments at the lesion site, as well as regions immediately rostral and caudal to the lesion, over a period of 90 days after SCI. Using weighted gene coexpression network analysis (WGCNA), we established gene modules/programs corresponding to various pathological events at different times after SCI. These objective measures of gene module expression also revealed that enhanced new neurogenesis and angiogenesis, and reduced inflammatory responses were keys to conferring the effect of NT3-chitosan on regeneration.

Differential effects of AAV.BDNF and AAV.Ntf3 in the deafened adult guinea pig ear.

Cochlear hair cell loss results in secondary regression of peripheral auditory fibers (PAFs) and loss of spiral ganglion neurons (SGNs). The performance of cochlear implants (CI) in rehabilitating hearing depends on survival of SGNs. Here we compare the effects of adeno-associated virus vectors with neurotrophin gene inserts, AAV.BDNF and AAV.Ntf3, on guinea pig ears deafened systemically (kanamycin and furosemide) or locally (neomycin). AAV.BDNF or AAV.Ntf3 was delivered to the guinea pig cochlea one week following deafening and ears were assessed morphologically 3 months later. At that time, neurotrophins levels were not significantly elevated in the cochlear fluids, even though in vitro and shorter term in vivo experiments demonstrate robust elevation of neurotrophins with these viral vectors. Nevertheless, animals receiving these vectors exhibited considerable re-growth of PAFs in the basilar membrane area. In systemically deafened animals there was a negative correlation between the presence of differentiated supporting cells and PAFs, suggesting that supporting cells influence the outcome of neurotrophin over-expression aimed at enhancing the cochlear neural substrate. Counts of SGN in Rosenthal's canal indicate that BDNF was more effective than NT-3 in preserving SGNs. The results demonstrate that a transient elevation in neurotrophin levels can sustain the cochlear neural substrate in the long term.

Either brain-derived neurotrophic factor or neurotrophin-3 only neurotrophin-producing grafts promote locomotor recovery in untrained spinalized cats.

Background. Transplants of cellular grafts expressing a combination of 2 neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to promote and enhance locomotor recovery in untrained spinalized cats. Based on the time course of recovery and the absence of axonal growth through the transplants, we hypothesized that recovery was due to neurotrophin-mediated plasticity within the existing locomotor circuitry of the lumbar cord. Since BDNF and NT-3 have different effects on axonal sprouting and synaptic connectivity/strengthening, it becomes important to ascertain the contribution of each individual neurotrophins to recovery. Objective. We studied whether BDNF or NT-3 only producing cellular grafts would be equally effective at restoring locomotion in untrained spinal cats. Methods. Rat fibroblasts secreting one of the 2 neurotrophins were grafted into the T12 spinal transection site of adult cats. Four cats in each group (BDNF alone or NT-3 alone) were evaluated. Locomotor recovery was tested on a treadmill at 3 and 5 weeks post-transection/grafting. Results. Animals in both groups were capable of plantar weight-bearing stepping at speed up to 0.8 m/s as early as 3 weeks and locomotor capabilities were similar at 3 and 5 weeks for both types of graft. Conclusions. Even without locomotor training, either BDNF or NT-3 only producing grafts promote locomotor recovery in complete spinal animals. More clinically applicable delivery methods need to be developed.

The effects of controlled release of neurotrophin-3 from PCLA scaffolds on the survival and neuronal differentiation of transplanted neural stem cells in a rat spinal cord injury model.

Neural stem cells (NSCs) have emerged as a potential source for cell replacement therapy following spinal cord injury (SCI). However, poor survival and low neuronal differentiation remain major obstacles to the use of NSCs. Biomaterials with neurotrophic factors are promising strategies for promoting the proliferation and differentiation of NSCs. Silk fibroin (SF) matrices were demonstrated to successfully deliver growth factors and preserve their potency. In this study, by incorporating NT-3 into a SF coating, we successfully developed NT-3-immobilized scaffolds (membranes and conduits). Sustained release of bioactive NT-3 from the conduits for up to 8 weeks was achieved. Cell viability was confirmed using live/dead staining after 14 days in culture. The efficacy of the immobilized NT-3 was confirmed by assessing NSC neuronal differentiation in vitro. NSC neuronal differentiation was 55.2 ± 4.1% on the NT-3-immobilized membranes, which was significantly higher than that on the NT-3 free membrane. Furthermore, 8 weeks after the NSCs were seeded into conduits and implanted in rats with a transected SCI, the conduit+NT-3+NSCs group achieved higher NSC survival (75.8 ± 15.1%) and neuronal differentiation (21.5 ± 5.2%) compared with the conduit+NSCs group. The animals that received the conduit+NT-3+NSCs treatment also showed improved functional outcomes, as well as increased axonal regeneration. These results indicate the feasibility of fabricating NT-3-immobilized scaffolds using the adsorption of NT-3/SF coating method, as well as the potential of these scaffolds to induce SCI repair by promoting survival and neuronal differentiation of transplanted NSCs.

Reticulospinal plasticity after cervical spinal cord injury in the rat involves withdrawal of projections below the injury.

Restoring voluntary fine motor control of the arm and hand is one of the main goals following cervical spinal cord injury (SCI). Although the functional improvement achievable with rehabilitative training in rat models is frequently accompanied by corticospinal tract (CST) plasticity, CST rewiring alone seems insufficient to account for the observed recovery. Recent investigations in animal models of SCI have suggested that the reticulospinal tract (RtST) might contribute to mediating improved motor performance of the forelimb. Here we investigate whether the spared RtST can compensate for the loss of CST input and whether RtST projections rearrange in response to cervical SCI. Animals underwent unilateral ablation of the dorsal CST and rubrospinal tract at spinal level C4, while the ventral RtST projections were spared. At the end of the six-week recovery period, injured animals had made significant improvements in single pellet reaching. This was not accompanied by increased sprouting of the injured CST above the injury compared to uninjured control animals. Injury-induced changes in RtST fiber density within the gray matter, as well as in the number of RtST collaterals entering the gray matter or crossing the cord midline were minor above the injury. However, all analyses directly below the injured spinal level consistently point to a significant decrease of RtST projections. The mechanism and the functional relevance behind this new finding warrant further study. Our results also suggest that mechanisms other than anatomical plasticity, such as plastic changes on a cellular level, might be responsible for the observed spontaneous recovery.

Combination of hypoglossal-facial nerve surgical reconstruction and neurotrophin-3 gene therapy for facial palsy.

OBJECT: Facial nerve injury results in facial palsy that has great impact on the psychosocial conditions of affected patients. Reconstruction of the facial nerve to restore facial symmetry and expression is still a significant surgical challenge. In this study, the authors assessed a hypoglossal-facial nerve anastomosis method combined with neurotrophic factor gene therapy to treat facial palsy in adult rats after facial nerve injury. METHODS: Surgery consisted of the interposition of a predegenerated nerve graft (PNG) that was anastomosed with the hypoglossal and facial nerves at each of its extremities. The hypoglossal nerve was cut approximately 50% for this anastomosis to conserve partial hypoglossal function. Before their transplantation, the PNGs were genetically engineered using lentiviral vectors to induce overexpression of the neurotrophic factor neurotrophin-3 (NT-3) to improve axonal regrowth in the reconstructed nerve pathway. Reconstruction was performed after facial nerve injury, either immediately or after a delay of 9 weeks. The rats were followed up for 4 months postoperatively, and treatment outcomes were then assessed. RESULTS: Compared with the functional innervation in control rats that underwent facial nerve injury without subsequent treatment, functional innervation of the paralyzed whisker pad by hypoglossal motoneurons in rats treated 4 months after nerve reconstruction was evidenced by the retrograde transport of neuronal tracers, the recording of muscle action potentials conducted by the PNG, and the recovery of facial symmetry. Although a better outcome was observed when reconstruction was performed immediately after facial nerve injury, reconstruction with NT3-treated PNGs significantly improved functional reinnervation of the paralyzed whisker pad even when implantation occurred 9 weeks posttrauma. CONCLUSIONS: Results demonstrated that hypoglossal-facial nerve anastomosis facilitates innervation of paralyzed facial muscle via hypoglossal motoneurons without sacrificing ipsilateral hemitongue function. Neurotrophin-3 treatment through gene therapy could effectively improve such innervation, even after delayed reconstruction. These findings suggest that the combination of surgical reconstruction and NT-3 gene therapy is promising for its potential application in treating facial palsy in humans.

Synergistic effects of BDNF and rehabilitative training on recovery after cervical spinal cord injury.

Promoting the rewiring of lesioned motor tracts following a spinal cord injury is a promising strategy to restore motor function. For instance, axonal collaterals may connect to spared, lesion-bridging neurons, thereby establishing a detour for descending signals and thus promoting functional recovery. In our rat model of cervical spinal cord injury, we attempted to promote targeted rewiring of the unilaterally injured corticospinal tract (CST) via the spared reticulospinal tract (RtST). To promote new connections between the two tracts in the brainstem, we administered viral vectors producing two neurotrophins. Brain-derived neurotrophic factor (BDNF), a known promotor of collateral growth, was expressed in the motor cortex, and neurotrophin 3 (NT-3), which has chemoattractive properties, was expressed in the reticular formation. Because rehabilitative training has proven to be beneficial in promoting functionally meaningful plasticity following injury, we added training in a skilled reaching task. Different neurotrophin or control treatments with or without training were evaluated. As hypothesized, improvements of motor performance with the injured forelimb following neurotrophin treatment alone were absent or modest compared to untreated controls. In contrast, we found a significant synergistic effect on performance when BDNF treatment was combined with training. The mechanism of this recovery remains unidentified, as histological analyses of CST and RtST collateral projections did not reveal differences among treatment groups. In conclusion, we demonstrate that following a cervical spinal lesion, rehabilitative training is necessary to translate effects of BDNF into functional recovery by mechanisms which are likely independent of collateral sprouting of the CST or RtST into the gray matter.