Adipose tissue-derived neurotrophic factor 3 regulates sympathetic innervation and thermogenesis in adipose tissue.

Activation of brown fat thermogenesis increases energy expenditure and alleviates obesity. Sympathetic nervous system (SNS) is important in brown/beige adipocyte thermogenesis. Here we discover a fat-derived "adipokine" neurotrophic factor neurotrophin 3 (NT-3) and its receptor Tropomyosin receptor kinase C (TRKC) as key regulators of SNS growth and innervation in adipose tissue. NT-3 is highly expressed in brown/beige adipocytes, and potently stimulates sympathetic neuron neurite growth. NT-3/TRKC regulates a plethora of pathways in neuronal axonal growth and elongation. Adipose tissue sympathetic innervation is significantly increased in mice with adipocyte-specific NT-3 overexpression, but profoundly reduced in mice with TRKC haploinsufficiency (TRKC +/-). Increasing NT-3 via pharmacological or genetic approach promotes beige adipocyte development, enhances cold-induced thermogenesis and protects against diet-induced obesity (DIO); whereas TRKC + /- or SNS TRKC deficient mice are cold intolerant and prone to DIO. Thus, NT-3 is a fat-derived neurotrophic factor that regulates SNS innervation, energy metabolism and obesity.

Opposite Roles of NT-3 and BDNF in Synaptic Remodeling of the Inner Ear Induced by Electrical Stimulation.

With the development of neural prostheses, neural plasticity including synaptic remodeling under electrical stimulation is drawing more and more attention. Indeed, intracochlear electrical stimulation used to restore hearing in deaf can induce the loss of residual hearing and synapses of the inner hair cells (IHCs). However, the mechanism under this process is largely unknown. Considering that the guinea pig is always a suitable and convenient choice for the animal model of cochlea implant (CI), in the present study, normal-hearing guinea pigs were implanted with CIs. Four-hour electrical stimulation with the intensity of 6 dB above electrically evoked compound action potential (ECAP) threshold (which can decrease the quantity of IHC synapses and the excitability of the auditory nerve) resulted in the upregulation of Bdnf (p < 0.0001) and downregulation of Nt-3 (p < 0.05). Intracochlear perfusion of exogenous NT-3 or TrkC/Fc (which blocks NT-3) can, respectively, resist or aggravate the synaptic loss induced by electrical stimulation. In contrast, local delivery of exogenous BDNF or TrkB/Fc (which blocks BDNF) to the cochlea, respectively, exacerbated or protected against the synaptic loss caused by electrical stimulation. Notably, the synaptic changes were only observed in the basal and middle halves of the cochlea. All the findings above suggested that NT-3 and BDNF may play opposite roles in the remodeling of IHC synapses induced by intracochlear electrical stimulation, i.e. NT-3 and BDNF promoted the regeneration and degeneration of IHC synapses, respectively.

Protective effect of brain-derived neurotrophic factor and neurotrophin-3 overexpression by adipose-derived stem cells combined with silk fibroin/chitosan scaffold in spinal cord injury.

Objectives: Spinal cord injury (SCI) is a most debilitating traumatic injury, and cytotherapy is a promising alternative treatment strategy. Here we investigated the effect and mechanism of adipose-derived stem/stromal cells (ASCs) with overexpressing brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) (BDNF-NT3) in combination with silk fibroin/chitosan scaffold (SFCS) in SCI.Methods: Female Sprague-Dawley rats were used as an SCI model. SFCS,SFCS and ASCs, or ASCs overexpressing NT3, BDNF, and BDNF-NT3 were implanted into SCI rats. Basso, Beattie, and Bresnahan score, pathological changes, and spinal cord tissue and nerve fiber morphology were observed and assayed. GAP-43, GFAP, and caspase-3 expression was determined using immunohistochemistry and western blotting.Results: Smoother spinal cords, less scar tissue, and lower inflammatory activity were found in the SFCS, SFCS and ASCs, ASCs with NT3, BDNF, and BDNF-NT3 overexpression treatment than in the untreated SCI rat groups. Increasing formation of nerve fibers was observed in the above groups in order. GAP-43 expression significantly increased, while GFAP and caspase-3 expression significantly decreased. These results indicated obvious alleviation in pathological changes and BDNF-NT3 overexpression in ASCs combined with SFCS treatment in SCI rats.Conclusion: Thus, BDNF-NT3 overexpression from ASCs with SFCS had synergistic neuroprotective effects on SCI and may be a treatment option for SCI.

NT-3 Promotes Oligodendrocyte Proliferation and Nerve Function Recovery After Spinal Cord Injury by Inhibiting Autophagy Pathway.

BACKGROUND: Spinal cord injury (SCI) is a serious medical problem, leading to lifelong disability and increasing the health burden worldwide. Traditional treatments have limited effects on neuronal function recovery. Previous studies showed that neurotrophin-3 (NT-3) promoted oligodendrocyte survival and improved neuronal functional recovery after SCI. However, the mechanism by which NT-3 promotes oligodendrocyte survival after SCI remains unclear, which limits its application. MATERIALS AND METHODS: A total of 75 female Sprague-Dawley rats were randomly divided into three groups: the NS group, NT-3 group, and NT-3 + rapamycin group. After successful modeling, the spinal cord specimens were taken at the corresponding time points. Western blot was used to detect autophagy-related proteins and Olig1 protein expression and combined with pathology, immunohistochemistry, flow cytometry, and other methods to detect the proliferation of oligodendrocytes after NT-3 application. RESULTS: NT-3 was found to significantly promote the recovery of motor function by Basso-Beattie-Bresnahan scores analysis in the rat SCI model. Furthermore, intraspinal administration of NT-3 could downregulate the expression of Beclin-1 in oligodendrocytes, indicating that NT-3 could inhibit excessive autophagy of oligodendrocytes after SCI. The effects of NT-3 on oligodendrocyte survival could be blocked by an autophagy activator rapamycin. CONCLUSIONS: This study found that NT-3 could promote the recovery of motor function after SCI in rats. The underlying reason may be that NT-3 inhibits the expression of autophagy proteins in oligodendrocytes and promotes oligodendrocyte proliferation. This study provided evidence for the future clinical application of NT-3 in SCI patients.

The opposing contribution of neurotrophin-3 and nerve growth factor to orofacial heat hyperalgesia in rats.

It has been proposed that neurotrophin-3 acts in a manner that is opposed to nerve growth factor, especially in the modulation of heat hyperalgesia. Injury to the constriction of the infraorbital nerve (CION) is a well-established model of trigeminal neuropathic pain that leads to robust heat, cold, and mechanical hyperalgesia. Here, we assessed the effect of local neurotrophin-3 treatment on CION-induced hyperalgesia, and we examined some mechanisms related to the effect of neurotrophin-3. Neurotrophin-3 (1 µg/50 µl) injected into the upper lip of CION rats caused a significant and long-lasting reduction of CION-induced heat hyperalgesia, but failed to affect cold and mechanical hyperalgesia. Increased levels of neurotrophin-3 were detected in the injured nerve at the time point that represents the peak of heat hyperalgesia. The anti-hyperalgesic effect of neurotrophin-3 was markedly reduced in the presence of an antagonist of TrkA receptors (K-252a, 1 µg/50 µl). Moreover, association of lower doses of neurotrophin-3 with an antibody anti-nerve growth factor resulted in a synergistic anti-hyperalgesic effect in CION rats. Local injection of nerve growth factor (3 µg/50 µl) or the TRPV1 agonist capsaicin (1 µg/50 µl), but not neurotrophin-3 injection (1 µg/50 µl), resulted in long-lasting facial heat hyperalgesia, which was both significantly reduced by previous neurotrophin-3 local treatment. In conclusion, we suggest that neurotrophin-3 is a potent modulator of facial heat hyperalgesia, which may exert an inhibitory influence on the trkA pathway. Neurotrophin-3 treatment may represent a promising approach, especially in pain conditions associated with increased levels of nerve growth factor.

Pre-Clinical Evaluation of CBD-NT3 Modified Collagen Scaffolds in Completely Spinal Cord Transected Non-Human Primates.

Spinal cord injury (SCI) repair is one of the most desirable but extremely challenging clinical problems. Developing suitable animal models and validating the therapeutic interventions in these models is the prerequisite for SCI repair improvement. Non-human primates, closer to humans than other species, are considered to be ideal models for translating laboratory discoveries into human clinical trials. In this study, the acute thoracic (T9) complete transection model in rhesus monkeys was established to evaluate the effects of linear-ordered collagen scaffold (LOCS) and LOCS combined with collagen binding neurotrophin-3 (CBD-NT3), which has been demonstrated to promote axonal regrowth and functional restoration in rodent models. After 10 months post-surgery, the grafted groups dramatically reduced cystic cavity formation and chondroitin sulfate proteoglycans (CSPGs) deposition and facilitated the ingrowth of axonal fibers at the lesion site. Further, the grafted groups displayed more regenerated fibers, exhibiting remyelination and synapse formation. Notably, the LOCS+CBD-NT3 group showed significant locomotor and electrophysiological recovery compared with the Control and LOCS groups. Therefore, LOCS+CBD-NT3 transplantation represents an effective strategy to promote spinal cord repair in non-human primates. More importantly, this complete transection model in non-human primate will contribute to effectively evaluating the potential interventions and accelerating clinical transformation in the future.

Validation study of neurotrophin-3-releasing chitosan facilitation of neural tissue generation in the severely injured adult rat spinal cord.

It was previously reported that a tube holding chitosan carriers loaded with neurotrophin-3 (NT-3), after insertion into a 5 mm long transection gap in the adult rat spinal cord, triggered de novo neural tissue generation and functional recovery. Here, we report an effort to validate these findings using stringent blinding methodologies, which are crucial for robustness in reproducing biomedical studies. Radio frequency identification (RFID) chips were utilized to label rats that were randomly assigned into three experimental groups: transection with chitosan-NT-3 implant (C-NT3), transection only (T-controls), and laminectomy only (S-controls), blinding the experimenters to the treatments. Three months after surgery, animals only known by their RFID were functionally, electrophysiologically, and anatomically assessed. The data were then collected into the proper groups and statistically analyzed. Neural tissue with nestin-, Tuj1-, and NeuN-positive cells was found bridging the transection gap in C-NT3 rats, but not in T-controls. Motor- and somatosensory-evoked potentials were detected in C-NT3 rats and S-controls, but not in T-controls. Hind limb movement was significantly better in C-NT3 rats compared with T-controls. Our validation study indicates that C-NT3 implants facilitate neural tissue generation, at least in part, by eliciting endogenous neurogenesis. Our data support the use of C-NT3 implants for tissue remodeling in the injured spinal cord.

Stroke Recovery in Rats after 24-Hour-Delayed Intramuscular Neurotrophin-3 Infusion.

OBJECTIVE: Neurotrophin-3 (NT3) plays a key role in the development and function of locomotor circuits including descending serotonergic and corticospinal tract axons and afferents from muscle and skin. We have previously shown that gene therapy delivery of human NT3 into affected forelimb muscles improves sensorimotor recovery after stroke in adult and elderly rats. Here, to move toward the clinic, we tested the hypothesis that intramuscular infusion of NT3 protein could improve sensorimotor recovery after stroke. METHODS: Rats received unilateral ischemic stroke in sensorimotor cortex. To simulate a clinically feasible time to treatment, 24 hours later rats were randomized to receive NT3 or vehicle by infusion into affected triceps brachii for 4 weeks using implanted catheters and minipumps. RESULTS: Radiolabeled NT3 crossed from the bloodstream into the brain and spinal cord in rodents with or without strokes. NT3 increased the accuracy of forelimb placement during walking on a horizontal ladder and increased use of the affected arm for lateral support during rearing. NT3 also reversed sensory impairment of the affected wrist. Functional magnetic resonance imaging during stimulation of the affected wrist showed spontaneous recovery of peri-infarct blood oxygenation level-dependent signal that NT3 did not further enhance. Rather, NT3 induced neuroplasticity of the spared corticospinal and serotonergic pathways. INTERPRETATION: Our results show that delayed, peripheral infusion of NT3 can improve sensorimotor function after ischemic stroke. Phase I and II clinical trials of NT3 (for constipation and neuropathy) have shown that peripheral high doses are safe and well tolerated, which paves the way for NT3 as a therapy for stroke. ANN NEUROL 2019;85:32-46.

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.

Neurotrophin-3 provides neuroprotection via TrkC receptor dependent pErk5 activation in a rat surgical brain injury model.

BACKGROUND: Surgical brain injury (SBI) which occurs due to the inadvertent injury inflicted to surrounding brain tissue during neurosurgical procedures can potentiate blood brain barrier (BBB) permeability, brain edema and neurological deficits. This study investigated the role of neurotrophin 3 (NT-3) and tropomyosin related kinase receptor C (TrkC) against brain edema and neurological deficits in a rat SBI model. METHODS: SBI was induced in male Sprague Dawley rats by partial right frontal lobe resection. Temporal expression of endogenous NT-3 and TrkC was evaluated at 6, 12, 24 and 72 h after SBI. SBI rats received recombinant NT-3 which was directly applied to the brain surgical injury site using gelfoam. Brain edema and neurological function was evaluated at 24 and 72 h after SBI. Small interfering RNA (siRNA) for TrkC and Rap1 was administered via intracerebroventricular injection 24 h before SBI. BBB permeability assay and western blot was performed at 24 h after SBI. RESULTS: Endogenous NT-3 was decreased and TrkC expression increased after SBI. Topical administration of recombinant NT-3 reduced brain edema, BBB permeability and improved neurological function after SBI. Recombinant NT-3 administration increased the expression of phosphorylated Rap1 and Erk5. The protective effect of NT-3 was reversed with TrkC siRNA but not Rap1 siRNA. CONCLUSIONS: Topical application of NT-3 reduced brain edema, BBB permeability and improved neurological function after SBI. The protective effect of NT-3 was possibly mediated via TrkC dependent activation of Erk5.

AAV-mediated NT-3 overexpression protects cochleae against noise-induced synaptopathy.

The synapse between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) has been identified as a sensitive structure to noise-induced damage in the mammalian cochlea. Since this synapse provides the major information pathway from the cochlea to the auditory brain, it is important to maintain its integrity. Neurotrophin-3 (NT-3) has been known to play an important role in the development and the functional maintenance of this synapse. Application of exogenous NT-3, or overexpression of this gene in a transgenic animal model, have shown the value to protect this synapse from noise-induced damage. In the present study, NT-3 overexpression was induced by cochlear gene transfection before noise exposure via the use of an adeno-associated viral (AAV) vector. We found that such an overexpression provided a significant synaptic protection against a noise exposure that caused massive damage to the synapses, likely due to it promoting the repair of the synapse after the initial damage.

Neurotrophin-3 restores synaptic plasticity in the striatum of a mouse model of Huntington's disease.

AIMS: Neurotrophin-3 (NT-3) is expressed in the mouse striatum; however, it is not clear the NT-3 role in striatal physiology. The expression levels of mRNAs and immune localization of the NT-3 protein and its receptor TrkC are altered in the striatum following damage induced by an in vivo treatment with 3-nitropropionic acid (3-NP), a mitochondrial toxin used to mimic the histopathological hallmarks of Huntington's disease (HD). The aim of this study was to evaluate the role of NT-3 on corticostriatal synaptic transmission and its plasticity in both the control and damaged striatum. METHODS: Corticostriatal population spikes were electrophysiologically recorded and striatal synaptic plasticity was induced by high-frequency stimulation. Further, the phosphorylation status of Trk receptors was tested under conditions that imitated electrophysiological experiments. RESULTS: NT-3 modulates both synaptic transmission and plasticity in the striatum; nonetheless, synaptic plasticity was modified by the 3-NP treatment, where instead of producing striatal long-term depression (LTD), long-term potentiation (LTP) was obtained. Moreover, the administration of NT-3 in the recording bath restored the plasticity observed under control conditions (LTD) in this model of striatal degeneration. CONCLUSION: NT-3 modulates corticostriatal transmission through TrkB stimulation and restores striatal LTD by signaling through its TrkC receptor.

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.

Neurite outgrowth in cultured mouse pelvic ganglia - Effects of neurotrophins and bladder tissue.

Neurotrophic factors regulate survival and growth of neurons. The urinary bladder is innervated via both sympathetic and parasympathetic neurons located in the major pelvic ganglion. The aim of the present study was to characterize the effects of the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) on the sprouting rate of sympathetic and parasympathetic neurites from the female mouse ganglion. The pelvic ganglion was dissected out and attached to a petri dish and cultured in vitro. All three factors (BDNF, NT-3 and NGF) stimulated neurite outgrowth of both sympathetic and parasympathetic neurites although BDNF and NT-3 had a higher stimulatory effect on parasympathetic ganglion cells. The neurotrophin receptors TrkA, TrkB and TrkC were all expressed in neurons of the ganglia. Co-culture of ganglia with urinary bladder tissue, but not diaphragm tissue, increased the sprouting rate of neurites. Active forms of BDNF and NT-3 were detected in urinary bladder tissue using western blotting whereas tissue from the diaphragm expressed NGF. Neurite outgrowth from the pelvic ganglion was inhibited by a TrkB receptor antagonist. We therefore suggest that the urinary bladder releases trophic factors, including BDNF and NT-3, which regulate neurite outgrowth via activation of neuronal Trk-receptors. These findings could influence future strategies for developing pharmaceuticals to improve re-innervation due to bladder pathologies.

Transplants of Neurotrophin-Producing Autologous Fibroblasts Promote Recovery of Treadmill Stepping in the Acute, Sub-Chronic, and Chronic Spinal Cat.

Adult cats show limited spontaneous locomotor capabilities following spinal transection, but recover treadmill stepping with body-weight-supported training. Delivery of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT-3) can substitute for body-weight-supported training, and promotes a similar recovery in a shorter period of time. Autologous cell grafts would negate the need for the immunosuppressive agents currently used with most grafts, but have not shown functional benefits in incomplete spinal cord injury models and have never been tested in complete transection or chronic injury models. In this study, we explored the effects of autologous fibroblasts, prepared from the individual cats and modified to produce BDNF and NT-3, on the recovery of locomotion in acute, sub-chronic and chronic full-transection models of spinal injury. Fourteen female cats underwent complete spinal transection at T11/T12. Cats were separated into four groups: sham graft at the time of injury, and BDNF and NT-3 producing autologous fibroblasts grafted at the time of injury, 2 weeks after injury, or 6 weeks after injury. Kinematics were recorded 3 and 5 weeks after cell graft. Additional kinematic recordings were taken for some cats until 12 weeks post-graft. Eleven of 12 cats with neurotrophin-producing grafts recovered plantar weight-bearing stepping at treadmill speeds from 0.3 to 0.8 m/sec within 5 weeks of grafting, whereas control cats recovered poor quality stepping at low speeds only (≤ 0.4 m/sec). Further, kinematic measures in cats with grafts were closer to pre-transection values than those for controls, and recovery was maintained up to 12 weeks post-grafting. Our results show that not only are autologous neurotrophin-producing grafts effective at promoting recovery of locomotion, but that delayed delivery of neurotrophins does not diminish the therapeutic effect, and may improve outcome.

Infralimbic Neurotrophin-3 Infusion Rescues Fear Extinction Impairment in a Mouse Model of Pathological Fear.

The inability to properly extinguish fear memories constitutes the foundation of several anxiety disorders, including panic disorder. Recent findings show that boosting prefrontal cortex synaptic plasticity potentiates fear extinction, suggesting that therapies that augment synaptic plasticity could prove useful in rescue of fear extinction impairments in this group of disorders. Previously, we reported that mice with selective deregulation of neurotrophic tyrosine kinase receptor, type 3 expression (TgNTRK3) exhibit increased fear memories accompanied by impaired extinction, congruent with an altered activation pattern of the amygdala-hippocampus-medial prefrontal cortex fear circuit. Here we explore the specific role of neurotrophin 3 and its cognate receptor in the medial prefrontal cortex, and its involvement in fear extinction in a pathological context. In this study we combined molecular, behavioral, in vivo pharmacology and ex vivo electrophysiological recordings in TgNTRK3 animals during contextual fear extinction processes. We show that neurotrophin 3 protein levels are increased upon contextual fear extinction in wild-type animals but not in TgNTRK3 mice, which present deficits in infralimbic long-term potentiation. Importantly, infusion of neurotrophin 3 to the medial prefrontal cortex of TgNTRK3 mice rescues contextual fear extinction and ex vivo local application improves medial prefrontal cortex synaptic plasticity. This effect is blocked by inhibition of extracellular signal-regulated kinase phosphorylation through peripheral administration of SL327, suggesting that rescue occurs via this pathway. Our results suggest that stimulating neurotrophin 3-dependent medial prefrontal cortex plasticity could restore contextual fear extinction deficit in pathological fear and could constitute an effective treatment for fear-related disorders.

Round-window delivery of neurotrophin 3 regenerates cochlear synapses after acoustic overexposure.

In acquired sensorineural hearing loss, such as that produced by noise or aging, there can be massive loss of the synaptic connections between cochlear sensory cells and primary sensory neurons, without loss of the sensory cells themselves. Because the cell bodies and central projections of these cochlear neurons survive for months to years, there is a long therapeutic window in which to re-establish functional connections and improve hearing ability. Here we show in noise-exposed mice that local delivery of neurotrophin-3 (NT-3) to the round window niche, 24 hours after an exposure that causes an immediate loss of up to 50% loss of synapses in the cochlear basal region, can regenerate pre- and post-synaptic elements at the hair cell / cochlear nerve interface. This synaptic regeneration, as documented by confocal microscopy of immunostained cochlear sensory epithelia, was coupled with a corresponding functional recovery, as seen in the suprathreshold amplitude of auditory brainstem response Wave 1. Cochlear delivery of neurotrophins in humans is likely achievable as an office procedure via transtympanic injection, making our results highly significant in a translational context.

Sustained release of neurotrophin-3 via calcium phosphate-coated sutures promotes axonal regeneration after spinal cord injury.

Because of the dynamics of spinal cord injury (SCI), the optimal treatment will almost certainly be a combination approach to control the environment and promote axonal growth. This study uses peripheral nerve grafts (PNGs) as scaffolds for axonal growth while delivering neurotrophin-3 (NT-3) via calcium phosphate (CaP) coatings on surgical sutures. CaP coating was grown on sutures, and NT-3 binding and release were characterized in vitro. Then, the NT-3-loaded sutures were tested in a complete SCI model. Rats were analyzed for functional improvement and axonal growth into the grafts. The CaP-coated sutures exhibited a burst release of NT-3, followed by a sustained release for at least 20 days. Functionally, the rats with PNGs + NT-3-loaded sutures and the rats treated with PNGs scored significantly higher than controls on day 56 postoperatively. However, functional scores in rats treated with PNGs + NT-3-loaded suture were not significantly different from those of rats treated with PNGs alone. Cholera toxin subunit B (CTB) labeling rostral to the graft was not observed in any controls, but CTB labeling rostral to the graft was observed in almost all rats that had had a PNG. Neurofilament labeling on transverse sections of the graft revealed that the rats treated with the NT-3-loaded sutures had significantly more axons per graft than rats treated with an NT-3 injection and rats without NT-3. These data demonstrate that PNGs serve as scaffolds for axonal growth after SCI and that CaP-coated sutures can efficiently release NT-3 to increase axonal regeneration. © 2016 Wiley Periodicals, Inc.

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.