Neurotrophin-3 Enhances the Effectiveness of Cell Therapy in Chronic Spinal Cord Injuries.

Neurotrophin-3 enhances the effectiveness of human olfactory ensheathing cells in improving hind limb mobility in rats with post-traumatic cysts of the spinal cord. Transplantation of olfactory ensheathing cells into spinal cord cysts reduced their size; neurotrophin-3 did not modulate this effect. Combined preparation of human olfactory ensheathing cells and neurotrophin- 3 can be used in neurosurgery for the treatment of patients with spinal cord injuries.

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.

Distinct Effects of BDNF and NT-3 on the Dendrites and Presynaptic Boutons of Developing Olfactory Bulb GABAergic Interneurons In Vitro.

Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) are known to regulate neuronal morphology and the formation of neural circuits, yet the neuronal targets of each neurotrophin are still to be defined. To address how these neurotrophins regulate the morphological and synaptic differentiation of developing olfactory bulb (OB) GABAergic interneurons, we analyzed the effect of BDNF and NT-3 on GABA+-neurons and on different subtypes of these neurons: tyrosine hydroxylase (TH+); calretinin (Calr+); calbindin (Calb+); and parvalbumin (PVA+). These cells were generated from cultured embryonic mouse olfactory bulb neural stem cells (eOBNSCs) and after 14 days in vitro (DIV), when the neurons expressed TrkB and/or TrkC receptors, BDNF and NT-3 did not significantly change the number of neurons. However, long-term BDNF treatment did produce a longer total dendrite length and/or more dendritic branches in all the interneuron populations studied, except for PVA+-neurons. Similarly, BDNF caused an increase in the cell body perimeter in all the interneuron populations analyzed, except for PVA+-neurons. GABA+- and TH+-neurons were also studied at 21 DIV, when BDNF produced significantly longer neurites with no clear change in their number. Notably, these neurons developed synaptophysin+ boutons at 21 DIV, the size of which augmented significantly following exposure to either BDNF or NT-3. Our results show that in conditions that maintain neuronal survival, BDNF but not NT-3 promotes the morphological differentiation of developing OB interneurons in a cell-type-specific manner. In addition, our findings suggest that BDNF and NT-3 may promote synapse maturation by enhancing the size of synaptic boutons.

Surface modification of neurotrophin-3 loaded PCL/chitosan nanofiber/net by alginate hydrogel microlayer for enhanced biocompatibility in neural tissue engineering.

This study prepared a novel three-dimensional nanocomposite scaffold by the surface modification of PCL/chitosan nanofiber/net with alginate hydrogel microlayer, hoping to have the privilege of both nanofibers and hydrogels simultaneously. Bead free randomly oriented nanofiber/net (NFN) structure composed of chitosan and polycaprolactone (PCL) was fabricated by electrospinning method. The low surface roughness, good hydrophilicity, and high porosity were obtained from the NFN structure. Then, the PCL/chitosan nanofiber/net was coated with a microlayer of alginate containing neurotrophin-3 (NT-3) and conjunctiva mesenchymal stem cells (CJMSCs) as a new stem cell source. According to the cross-sectional FESEM, the scaffold shows a two-layer structure with interconnected pores in the range of 20 µm diameter. The finding revealed that the surface modification of nanofiber/net by alginate hydrogel microlayer caused lower inflammatory response and higher proliferation of CJMSCs than the unmodified scaffold. The initial burst release of NT-3 was 69% in 3 days which followed by a sustained release up to 21 days. The RT-PCR analysis showed that the expression of Nestin, MAP-2, and ß-tubulin III genes were increased 6, 5.4, and 8.8-fold, respectively. The results revealed that the surface-modified biomimetic scaffold possesses enhanced biocompatibility and could successfully differentiate CJMSCs to the neuron-like cells.

Select neurotrophins promote oligodendrocyte progenitor cell process outgrowth in the presence of chondroitin sulfate proteoglycans.

Axonal damage and the subsequent interruption of intact neuronal pathways in the spinal cord are largely responsible for the loss of motor function after injury. Further exacerbating this loss is the demyelination of neighboring uninjured axons. The post-injury environment is hostile to repair, with inflammation, a high expression of chondroitin sulfate proteoglycans (CSPGs) around the glial scar, and myelin breakdown. Numerous studies have demonstrated that treatment with the enzyme chondroitinase ABC (cABC) creates a permissive environment around a spinal lesion that permits axonal regeneration. Neurotrophic factors like brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factor-3 (NT-3), and ciliary neurotrophic factor (CNTF) have been used to promote neuronal survival and stimulate axonal growth. CSPGs expressed near a lesion also inhibit migration and differentiation of endogenous oligodendrocyte progenitor cells (OPCs) in the spinal cord, and cABC treatment can neutralize this inhibition. This study examined the neurotrophins commonly used to stimulate axonal regeneration after injury and their potential effects on OPCs cultured in the presence of CSPGs. The results reveal differential effects on OPCs, with BDNF and GDNF promoting process outgrowth and NT-3 stimulating differentiation of OPCs, while CNTF appears to have no observable effect. This finding suggests that certain neurotrophic agents commonly utilized to stimulate axonal regeneration after a spinal injury may also have a beneficial effect on the endogenous oligodendroglial cells as well.

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

Neurotrophin-3 (NT-3) acts as an important growth factor to stimulate and control tissue development. The NT-3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT-3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT-3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT-3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle-stimulating hormone levels. NT-3 increased CYP11A1-positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT-3 significantly down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT-3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT-3 dose-dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT-3 antagonist Celitinib can antagonize NT-3 action in vitro. In conclusion, the present study demonstrates that NT-3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.

Construction of a NT-3 sustained-release system cross-linked with an acellular spinal cord scaffold and its effects on differentiation of cultured bone marrow mesenchymal stem cells.

OBJECTIVE: This study sought to promote the adhesion, proliferation and differentiation of rat bone marrow mesenchymal stem cells by constructing a neurotrophin-3 (NT-3) sustained-release system cross-linked with an acellular spinal cord scaffold. METHODS: 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) chemistry combined with chemical extraction was used to construct an acellular spinal cord scaffold. The decellularization completion was validated. An EDC cross-linking method was used to construct the NT-3 cross-linked acellular spinal scaffold. ELISA was used to verify sustained release of NT-3; the dorsal root ganglion method was used to verify the biological activity of the sustained-release NT-3. DAPI staining was used to confirm the adhesion of the cultured rat bone marrow mesenchymal stem cells (P3) to the NT-3 scaffold, and cell counting kit-8 (CCK-8) analysis was used to verify the cellular proliferation after 24 h and 48 h of culture. Immunohistochemistry was used to confirm the differentiation of the bone marrow cells into neuron-like cells. RESULTS: An NT-3 sustained-release system cross-linked to an acellular spinal cord scaffold was successfully constructed. Sustained-release NT-3 could persist for 35 days and had biological activity for at least 21 days. It could promote the adhesion, proliferation and differentiation of rat bone marrow mesenchymal stem cells. CONCLUSION: As a composite scaffold, an NT-3 sustained-release system cross-linked with an acellular spinal cord scaffold has potential applications for tissue engineering.

Copper complexes of synthetic peptides mimicking neurotrophin-3 enhance neurite outgrowth and CREB phosphorylation.

In this work we report on the synthesis and physiochemical/biological characterization of a peptide encompassing the first thirteen residues of neurotrophin-3 (NT-3). The protein capability to promote neurite outgrowth and axonal branching by a downstream mechanism that involves the increase of the cAMP response element-binding level (CREB) was found for the NT3(1-13) peptide, thus validating its protein mimetic behaviour. Since copper ions are also involved in neurotransmission and their internalization may be an essential step in neuron differentiation and CREB phosphorylation, the peptide and its copper complexes were characterized by potentiometric and spectroscopic techniques, including UV-visible, CD and EPR. To have a detailed picture of the coordination features of the copper complexes with NT3(1-13), we also scrutinized the two peptide fragments encompassing the shorter sequences 1-5 and 5-13, respectively, showing that the amino group is the main anchoring site for Cu(ii) at physiological pH. The peptide activity increased in the presence of copper ions. The effect of copper(ii) addition is more marked for NT3(1-13) than the other two peptide fragments, in agreement with its higher affinity for metal ions. Confocal microscopy measurements carried out on fluorescently labelled NT3(1-13) indicated that copper ions increase peptide internalization.

Neurotrophin-3 acts on the endothelial-mesenchymal transition of heterotopic ossification in rats.

Despite the fact that extensive studies have focused on heterotopic ossification (HO), its molecular mechanism remains unclear. The endothelial-mesenchymal transition (EndMT), which may be partially modulated by neuroendocrine cytokines is thought to play a major role in HO. Neurotrophin-3 (NT-3), which has neuroendocrine characteristics is believed to promote skeletal remodeling. Herein, we suggest that that NT-3 may promote HO formation through regulation of EndMT. Here, we used an in vivo model of HO and an in vitro model of EndMT induction to elucidate the effect and underlying mechanism of NT-3 on EndMT in HO. Our results showed that heterotopic bone and cartilage arose from EndMT and NT-3 promoted HO formation in vivo. Our in vitro results showed that NT-3 up-regulated mesenchymal markers (FSP-1, α-SMA and N-cadherin) and mesenchymal stem cell (MSC) markers (STRO-1, CD44 and CD90) and down-regulated endothelial markers (Tie-1, VE-cadherin and CD31). Moreover, NT-3 enhanced a chondrogenesis marker (Sox9) and osteogenesis markers (OCN and Runx2) via activation of EndMT. However, both EndMT specific inhibitor and tropomyosin-related kinase C (TrkC) specific inhibitor rescued NT-3-induced HO formation and EndMT induction in vivo and in vitro. In conclusion, our findings demonstrate that NT-3 promotes HO formation via modulation of EndMT both in vivo and in vitro, which offers a new potential target for the prevention and therapy of HO.

Silver nanoparticles conjugated with Neurotrophin 3 upregulate myelin gene transcription pathway.

Mathematical modeling is the art of converting problems from the biological area into handy mathematical formulations whose theoretical and numerical analysis provides understandings about the directions and solutions to the particular problem. Recently, the combination therapy treatments have been revealed exceptionally fruitful by using mathematical modeling technique. The human nervous system is composed of axons, covered by the myelin sheath. Axons carry signals and promote myelin development. The abnormalities in myelination formation due to mutations in myelin gene result in memory disorders and impaired cognitive activities. The ERBb gene family is responsible for causing abnormalities in myelin gene. Using this knowledge, the pathway of mutated myelin gene was retrieved and its model was developed. Modeling and simulation analysis was performed to determine the level of expression of several genes. The Neurotrophin 3 ligand-coated with silver nanoparticle was induced in the model to normalize the transcription of myelin gene. It was observed that the myelin gene expression level increases from 0 after two days of NT3 induction and reaches to the maximum level on the 10th day of drug induction along with an increase in ERBb expression. This research work can be used in the future as a part of drug discovery and formulation.

Neurotrophin-3 improves fracture healing in rats.

OBJECTIVE: This study aimed at investigating the role of Neurotrophin-3 (NT-3) in the bone fracture healing of rats and to provide a reference for clinical treatment. MATERIALS AND METHODS: 40 Sprague-Dawley (SD) rats were randomly divided into control group and NT-3 group. The tibia fracture model was made in NT-3 group, and the tibia bone mineral density (BMD) was measured before and after the surgery. The biomechanics indexes were inspected after the surgery, including elasticity modulus, max load, and bending rigidity. The levels of bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-ß1 in serum were examined by enzyme-linked immunosorbent assay (ELISA). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the levels of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) mRNA expression in callus tissue. The pathological profile of tibia fracture healing was characterized after the surgery. RESULTS: The levels of BMD in NT-3 group were significantly higher than that in control group after the surgery (p < 0.05). The levels of elasticity modulus, maximum load, stiffness of shinbone, BMP-2 and TGF-ß1 were significantly higher in NT-3 group than those in control group after the surgery (p < 0.05). Compared with the control group, the expression of HIF-1α mRNA was significantly lower and the expression of VEGF mRNA was significantly higher in NT-3 group after the surgery (p < 0.05). Histological study showed that the periosteal reaction, capillary proliferation, cartilage cells production and ossification were happened after treating NT-3 for tibia fracture model rats. CONCLUSIONS: NT-3 can significantly improve fracture healing in rats.

NT3-chitosan enables de novo regeneration and functional recovery in monkeys after spinal cord injury.

Spinal cord injury (SCI) often leads to permanent loss of motor, sensory, and autonomic functions. We have previously shown that neurotrophin3 (NT3)-loaded chitosan biodegradable material allowed for prolonged slow release of NT3 for 14 weeks under physiological conditions. Here we report that NT3-loaded chitosan, when inserted into a 1-cm gap of hemisectioned and excised adult rhesus monkey thoracic spinal cord, elicited robust axonal regeneration. Labeling of cortical motor neurons indicated motor axons in the corticospinal tract not only entered the injury site within the biomaterial but also grew across the 1-cm-long lesion area and into the distal spinal cord. Through a combination of magnetic resonance diffusion tensor imaging, functional MRI, electrophysiology, and kinematics-based quantitative walking behavioral analyses, we demonstrated that NT3-chitosan enabled robust neural regeneration accompanied by motor and sensory functional recovery. Given that monkeys and humans share similar genetics and physiology, our method is likely translatable to human SCI repair.

Neurotrophin-3 released from implant of tissue-engineered fibroin scaffolds inhibits inflammation, enhances nerve fiber regeneration, and improves motor function in canine spinal cord injury.

Spinal cord injury (SCI) normally results in cell death, scarring, cavitation, inhibitory molecules release, etc., which are regarded as a huge obstacle to reconnect the injured neuronal circuits because of the lack of effective stimulus. In this study, a functional gelatin sponge scaffold was used to inhibit local inflammation, enhance nerve fiber regeneration, and improve neural conduction in the canine. This scaffold had good porosity and modified with neurotrophin-3 (NT-3)/fibroin complex, which showed sustained release in vitro. After the scaffold was transplanted into canine spinal cord hemisection model, hindlimb movement, and neural conduction were improved evidently. Migrating host cells, newly formed neurons with associated synaptic structures together with functional blood vessels with intact endothelium in the regenerating tissue were identified. Taken together, the results demonstrated that using bioactive scaffold could establish effective microenvironment stimuli for endogenous regeneration, providing a potential and practical strategy for treatment of spinal cord injury. © 2018 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2158-2170, 2018.

[Neurotrophin 3 promotes osteogenic differentiation of human dental follicle cells].

OBJECTIVE: This study aims to investigate the effect of neurotrophin 3 (NT-3) on the osteogenic differentiation of human dental follicle cells (hDFCs). METHODS: hDFCs were isolated and cultured in vitro. Immunocytochemical staining was used to identify the origin of hDFCs. The effects of different NT-3 concentrations on hDFCs proliferation were detected by using CCK-8 assay. The alkaline phosphatase (ALP) activities and mRNA expression levels of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) were determined to investigate the effects of NT-3 on hDFCs osteogenesis. The difference in the number of mineralized nodules was detected using alizarin red staining. RESULTS: Vimentin and cytokeratin staining results showed that hDFCs originated from the mesenchymal cells. NT-3 exerted no evident effect on hDFCs proliferation. The ALP activity and the BMP-2 and OCN mRNA expression levels of hDFCs were significantly improved under treatment with different NT-3 concentrations (25, 50, and 100 ng·mL ⁻¹) compared with those in the control group. BMP-2 and OCN mRNA relative expression levels of hDFCs reached the highest when the NT-3 concentration was 100 ng·mL ⁻¹. The number of mineralized nodules reached the maximum when the hDFCs were treated with 50 and 100 ng·mL ⁻¹ NT-3. CONCLUSIONS: Appropriate mass concentration of NT-3 can promote the osteogenic differentiation of hDFCs.

[Neurotrophin-3 enhances the osteogenesis ability of human bone marrow mesenchymal stem cells stimulated by lipopolysaccharide].

Objective To investigate the protective effect of neurotrophin-3 (NT-3) on human bone marrow mesenchymal stem cells (hBMSCs) and its ability to promote the differentiation of hBMSCs into osteoblasts in the inflammatory environment. Methods The cell inflammation model was established by lipopolysaccharide (LPS). The hBMSCs without any stimulation was defined as the inflammatory control group; the hBMSCs stimulated by 100 ng/mL human NT-3 recombinant protein as the NT-3 group; the hBMSCs stimulated by 100 mmol/L pyrvinium pamoate (PP) for 12 hours and then stimulated by 100 ng/mL human NT-3 recombinant protein as the Wnt inhibitor group; the normal cultured hBMSCs as the normal control group. We performed the experiment of osteoblast induction on all groups. CCK-8 assay was used to detect the proliferation of hBMSCs; the fluorescein isothiocyanate labeled annexin V/propidium iodide (Annexin V-FITC/PI) double labeling combined with flow cytometry was used to detect the apoptosis of hBMSCs; ELISA was used to detect the protein levels of runt-related transcription factor 2 (RUNX2) protein and alkaline phosphatase (ALP); and the alizarin red staining experiment was conducted to detect the ability of calcium nodule formation. Results Compared with the inflammatory control group, the proliferative activity of hBMSCs in NT-3 group significantly increased, the apoptosis obviously decreased, and the contents of RUNX2 and ALP, as well as the intensity of alizarin red staining in NT-3 group evidently rose. Compared with the NT-3 group, the proliferative activity of hBMSCs in the Wnt inhibitor group was inhibited, the cell apoptosis was promoted, and the contents of RUNX2 and ALP, as well as the intensity of alizarin red staining in the Wnt inhibitor group were reduced remarkably. Conclusion NT-3 can protect hBMSCs from anti-inflammatory damage and promote the differentiation of hBMSCs into osteoblasts.

Comparison of the Pharmacological Effects of Dimeric Dipeptide Nerve Growth Factor Mimetic GK-2 and Mexidol on the Model of Ischemic Stroke in Rats.

We compared the effects of GK-2 (dimeric dipeptide mimetic of nerve growth factor) and Mexidol (standard preparation for the therapy of stroke) on rat model of transient occlusion of the middle cerebral artery. GK-2 and Mexidol were administered intraperitoneally in the most active doses (1 and 100 mg/kg, respectively) 6 h after surgery and then once a day for 6 days. The preparations reduced the volume of cerebral infarction (by 60 and 30%, respectively). At the same time, GK-2 had a pronounced and statistically more reliable effect in a dose that is lower by two orders of magnitude. In addition, GK-2 significantly reduced the neurological deficit in the limb placement test, while Mexidol was ineffective in this test.

Neurotrophin 3 upregulates proliferation and collagen production in human aortic valve interstitial cells: a potential role in aortic valve sclerosis.

Calcific aortic valve disease (CAVD) is a leading cardiovascular disorder in the elderly. Diseased aortic valves are characterized by sclerosis (fibrosis) and nodular calcification. Sclerosis, an early pathological change, is caused by aortic valve interstitial cell (AVIC) proliferation and overproduction of extracellular matrix (ECM) proteins. However, the mechanism of aortic valve sclerosis remains unclear. Recently, we observed that diseased human aortic valves overexpress growth factor neurotrophin 3 (NT3). In the present study, we tested the hypothesis that NT3 is a profibrogenic factor to human AVICs. AVICs isolated from normal human aortic valves were cultured in M199 growth medium and treated with recombinant human NT3 (0.10 µg/ml). An exposure to NT3 induced AVIC proliferation, upregulated the production of collagen and matrix metalloproteinase (MMP), and augmented collagen deposition. These changes were abolished by inhibition of the Trk receptors. NT3 induced Akt phosphorylation and increased cyclin D1 protein levels in a Trk receptor-dependent fashion. Inhibition of Akt abrogated the effect of NT3 on cyclin D1 production. Furthermore, inhibition of either Akt or cyclin D1 suppressed NT3-induced cellular proliferation and MMP-9 and collagen production, as well as collagen deposition. Thus, NT3 upregulates cellular proliferation, ECM protein production, and collagen deposition in human AVICs. It exerts these effects through the Trk-Akt-cyclin D1 cascade. NT3 is a profibrogenic mediator in human aortic valve, and overproduction of NT3 by aortic valve tissue may contribute to the mechanism of valvular sclerosis.

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.

Applying Neurotrophins to the Round Window Rescues Auditory Function and Reduces Inner Hair Cell Synaptopathy After Noise-induced Hearing Loss.

HYPOTHESIS: Applying neurotrophins to the round window immediately after a single noise exposure will prevent noise-induced hidden hearing loss. BACKGROUND: Loud noise can eliminate neural connections between inner hair cells and their afferent neurons (thereby diminishing sound perception) without causing a detectable change on audiogram. This phenomenon is termed hidden hearing loss. METHODS: Guinea pigs were exposed for 2 hours to 4 to 8 kHz noise at either 95 or 105 dB SPL. Immediately afterward a 4 µl bolus of neurotrophins (brain-derived neurotrophic factor 1 µg/µl, and neurotrophin-3 1 µg/µl) was delivered to the round window of one ear, and saline to the other. Auditory brainstem responses to pure-tone pips were acquired preoperatively, and at 1 and 2 weeks' postexposure. Cochleae were removed and whole mounted for immunohistochemical analysis, with presynaptic ribbons of inner hair cells and associated postsynaptic glutamatergic AMPA receptors identified using CtBP2 and GluA2 antibodies respectively. RESULTS: After exposure to 105 dB noise, threshold did not change, but the amplitude growth of the auditory brainstem response was significantly reduced in control ears in response to 16 and 32 kHz tones. The amplitude growth was also reduced neurotrophin ears, but to a lesser degree and the reduction was not significant. Similar results were obtained from control ears exposed to 95 dB, but amplitude growth recovered in neurotrophin-treated ears, this reaching statistical significance in response to 16 kHz tones. There were significantly more presynaptic ribbons, postsynaptic glutamate receptors, and colocalized ribbons after neurotrophin treatment. CONCLUSION: A single dose of neurotrophins delivered to the round window reduced synaptopathy and recovered high-frequency hearing in ears exposed to 95 dB noise. These findings suggest that hidden hearing loss may be reduced by providing trophic support to the cochlea after injury.

Local Delivery of Neurotrophin-3 and Anti-NogoA Promotes Repair After Spinal Cord Injury.

Tissue and functional repair after spinal cord injury (SCI) continue to elude researchers. Neurotrophin-3 (NT-3) and anti-NogoA have been shown to promote axonal regeneration in animal models of SCI; however, localized and sustained delivery to the central nervous system (CNS) remains a critical challenge for these and other macromolecular therapeutics. An injectable drug delivery system (DDS) has previously been developed, which can provide safe local delivery to the spinal cord. This DDS, composed of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (nps) dispersed in a hyaluronan methylcellulose hydrogel, was adapted for the tunable bioactive delivery of NT-3 and anti-NogoA. Furthermore, the combined delivery of NT-3 and anti-NogoA from the DDS in an impact/compression model of SCI increases axon density and improves locomotor function. The benefits of this np/hydrogel DDS observed for NT-3 and anti-NogoA demonstrate the utility of the DDS as a local delivery strategy for protein therapeutics to the CNS.