Effect of controlled release of brain-derived neurotrophic factor and neurotrophin-3 from collagen gel on neural stem cells.

This study aimed to examine the effect of controlled release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from collagen gel on rat neural stem cells (NSCs). With three groups of collagen gel, BDNF/collagen gel, and NT-3/collagen gel as controls, BDNF and NT-3 were tested in the BDNF-NT-3/collagen gel group at different time points. The enzyme-linked immunosorbent assay results showed that BDNF and NT-3 were steadily released from collagen gels for 10 days. The cell viability test and the bromodeoxyuridine incorporation assay showed that BDNF-NT-3/collagen gel supported the survival and proliferation of NSCs. The results also showed that the length of processes was markedly longer and differentiation percentage from NSCs into neurons was much higher in the BDNF-NT-3/collagen gel group than those in the collagen gel, BDNF/collagen gel, and NT-3/collagen gel groups. These findings suggest that BDNF-NT-3/collagen gel could significantly improve the ability of NSCs proliferation and differentiation.

Controlled release of neurotrophin-3 from fibrin-based tissue engineering scaffolds enhances neural fiber sprouting following subacute spinal cord injury.

This study investigated whether delayed treatment of spinal cord injury with controlled release of neurotrophin-3 (NT-3) from fibrin scaffolds can stimulate enhanced neural fiber sprouting. Long Evans rats received a T9 dorsal hemisection spinal cord injury. Two weeks later, the injury site was re-exposed, and either a fibrin scaffold alone, a fibrin scaffold containing a heparin-based delivery system with different concentrations of NT-3 (500 and 1,000 ng/mL), or a fibrin scaffold containing 1,000 ng/mL of NT-3 (no delivery system) was implanted into the injury site. The injured spinal cords were evaluated for morphological differences using markers for neurons, astrocytes, and chondroitin sulfate proteoglycans 2 weeks after treatment. The addition of 500 ng/mL of NT-3 with the delivery system resulted in an increase in neural fiber density compared to fibrin alone. These results demonstrate that the controlled release of NT-3 from fibrin scaffolds can enhance neural fiber sprouting even when treatment is delayed 2 weeks following injury.

An injectable, biodegradable hydrogel for trophic factor delivery enhances axonal rewiring and improves performance after spinal cord injury.

The failure of long descending pathways to regenerate after spinal cord injury (SCI) is generally attributed to inhibitory proteins associated with the glial scar and myelin, or to the loss of neurons' intrinsic capacity to grow, or both. Here, we describe the use of hydrogels as a novel way to deliver molecules that promote axon growth in the injured CNS of adult rats. This method utilizes an injectable liquid polymer solution that crosslinks into a biodegradable, water-swollen hydrogel when photoactivated under visible light. Neurotrophin-3 (NT-3), a trophic factor known to act on corticospinal tract (CST) projection neurons, was used as a prototypic pro-regenerative molecule. Hydrogel release properties were established in vitro to ensure long-term, sustained NT-3 release over a 2-week period; this avoided the need for multiple injections or minipump implantation. Hydrogel/NT-3-treated animals showed improved recovery in the open-field BBB test and in a horizontal ladder walk test compared to controls implanted with hydrogel alone. At the anatomical level, hydrogel/NT-3-treated animals showed far greater axon growth than controls in two major descending pathways for motor control, the CST and the raphespinal tract. In the case of the CST, much of the NT-3-induced growth represented collateral branching from undamaged ventral CST fibers. These studies demonstrate the effectiveness of hydrogel technology as a clinically feasible delivery system to promote regeneration and enhance functional outcome after spinal cord injury.

Stimulation of neurite outgrowth by neurotrophins delivered from degradable hydrogels.

Degradable hydrogels are useful vehicles for the delivery of growth factors to promote the regeneration of diseased or damaged tissue. In the central nervous system, there are many instances where the delivery of neurotrophins has great potential in tissue repair, especially for treatment of spinal cord injury. In this work, hydrogels based on poly(ethylene glycol) that form via a photoinitiated polymerization were investigated for the delivery of neurotrophins. The release kinetics of these factors are controlled by changes in the network crosslinking density, which influences neurotrophin diffusion and subsequent release from the gels with total release times ranging from weeks to several months. The release and activity of one neurotrophic factor, ciliary-neurotrophic factor (CNTF), was assessed with a cell-based proliferation assay and an assay for neurite outgrowth from retinal explants. CNTF released from a degradable hydrogel above an explanted retina was able to stimulate outgrowth of a significantly higher number of neurites than controls without CNTF. Finally, unique microsphere/hydrogel composites were developed to simultaneously deliver multiple neurotrophins with individual release rates.

Delivery of neurotrophin-3 to the cochlea using alginate beads.

OBJECTIVE: The aim of this study was to design a novel cochlear neurotrophin (NT) delivery system for the rescue of auditory neurons after ototoxicity-induced deafening. BACKGROUND: NT-3 is a trophic growth factor that promotes the survival of the auditory nerve and may have a potential therapeutic role in slowing neuron loss in progressive deafness, especially as an adjunct to the current cochlear implant. Beads made from alginate are biodegradable, slow release substances that can be placed at the round window or inside the cochlea. This study investigated the loading properties, release kinetics, and implantation potential of alginate beads loaded with NT-3. METHODS: Alginate beads were prepared using an ionic gelation technique and postloaded with NT-3. Release of NT-3 was measured using enzyme-linked immunosorbent assay over 5 days. Alginate beads were implanted into deafened guinea pigs for 28 days, after which survival of auditory neurons was assessed. RESULTS: Enzyme-linked immunosorbent assay studies demonstrated a 98% to 99% loading of NT-3 with a slow, partial release over 5 days in Ringer's solution. Furthermore, the addition of heparin to the delivery system modulated NT-3 release to a steadier pattern. Implantation of alginate-heparin beads in guinea pig cochleae produced minimal local tissue reaction. NT-3 loaded beads implanted at both the round window and within the scala tympani of the basal turn provided auditory neurons significant protection from degradation and apoptosis compared with unloaded beads or untreated cochleae. CONCLUSIONS: This study demonstrates alginate beads to be a safe, biodegradable and effective delivery system for NT-3 to the cochlea.

Anterograde axonal transport of BDNF and NT-3 by retinal ganglion cells: roles of neurotrophin receptors.

Retinal ganglion cells (RGCs) transport exogenous neurotrophins anterogradely to the midbrain tectum/superior colliculus with significant downstream effects. We determined contributions of neurotrophin receptors for anterograde transport of intraocularly injected radiolabeled neurotrophins. In adult rodents, anterograde transport of brain-derived neurotrophic factor (BDNF) was receptor-mediated, and transport of exogenous BDNF and neurotrophin-3 (NT-3) was more efficient, per RGC, in rodents than chicks. RT-PCR and Western blot analysis of purified murine RGCs showed that adult RGCs express the p75 receptor. Anterograde transport of BDNF or NT-3 was not diminished in p75 knock-out mice (with unaltered final numbers of RGCs), but BDNF transport was substantially reduced by co-injected trkB antibodies. In chick embryos, however, p75 antisense or co-injected p75 antibodies significantly attenuated anterograde transport of NT-3 by RGCs. Thus, neither BDNF nor NT-3 utilizes p75 for anterograde transport in adult rodent RGCs, while anterograde NT-3 transport requires the p75 receptor in embryonic chicken RGCs.

Tracing neurotrophin-3 diffusion and uptake in the guinea pig cochlea.

Neurotrophin therapy in the cochlea can potentially slow or reverse the degeneration of the auditory nerve that occurs during progressive deafness. Studies were performed to trace the diffusion and uptake of neurotrophin-3 (NT-3) following infusion into the cochlea. NT-3 labeled with (125)I or coated onto fluorescent microspheres was introduced into the basal turn of normal hearing and deafened guinea pig cochleae via a single slow-rate injection. Cochleae were examined between 2 h and 28 days post-infusion by autoradiography or fluorescent microscopy to determine the number of turns labeled by NT-3, identify individual cells and tissues receiving NT-3 and quantify the proportion of signal in each tissue. In general, long-term infusions were required for all cochlear turns to receive NT-3. (125)I NT-3 signal was strongest in cells lining the perilymphatic space of the scala tympani, basilar membrane, osseous spiral lamina and spiral ligament. Signal in the peripheral nerve tract and Rosenthal's canal was only 1.3-2.1 times background levels of radiation. NT-3 microspheres were detected within neural areas of the cochlea (nerve tract and Rosenthal's canal) in all cases, but not within neuronal cell bodies. NT-3 microspheres remained in the cochlea for at least 28 days, suggesting a low clearance rate within cochlear tissues.

Controlled release of neurotrophin-3 from fibrin gels for spinal cord injury.

The goal of this work was to assess the feasibility of using affinity-based delivery systems to release neurotrophin-3 (NT-3) in a controlled manner from fibrin gels as a therapy for spinal cord injury. A heparin-based delivery system (HBDS) was used to immobilize NT-3 within fibrin gels via non-covalent interactions to slow diffusion-based release of NT-3, thus allowing cell-activated degradation of fibrin to mediate release. The HBDS consists of three components: immobilized linker peptide, heparin and NT-3. The linker peptide contained a Factor XIIIa substrate and was covalently cross-linked to fibrin during polymerization. This immobilized linker peptide sequesters heparin within fibrin gels, and sequestered heparin binds NT-3, preventing its diffusion. Mathematical modeling was performed to examine the effect of heparin concentration on the fraction of NT-3 initially bound to fibrin. In vitro release studies confirmed that heparin concentration modulates diffusion-based release of NT-3. Fibrin gels containing the HBDS and NT-3 stimulated neural outgrowth from chick dorsal root ganglia by up to 54% versus unmodified fibrin, demonstrating that the NT-3 released is biologically active. In a preliminary in vivo study, fibrin gels containing the HBDS and NT-3 showed increased neural fiber density in spinal cord lesions versus unmodified fibrin at 9 days.

Adenovirus vector-directed expression of the neurotrophin-3 receptor (TrkC) in mouse astrocytes.

In the present article we report the generation of a neurovirological reagent, an adenovirus vector that efficiently delivers the gene for the Neurotrophin-3 (NT-3) receptor, TrkC. Using this AdTrkC vector, we examined the induction of the expression of the above neurotrophin receptor in pure cultures of mouse astrocytes, a glial cell type that does not constitutively express this gene. Infection of astrocytes at an optimal dose of 100-200 plaque forming units (p.f.u.) per cell, induced expression of specific mRNA, as demonstrated by RT-PCR and Northern blot. This mRNA was translated to produce a mean of 20,157 biologically active receptor molecules per astrocyte with a Kd of 4.1 x 10(-11) M, as demonstrated by 125I-NT-3 binding. After 2D electrophoresis, the mature glycoprotein and some precursors were recognised by antibodies raised against the carboxy-terminal peptide of Trk. Binding of the ligand induced autophosphorylation ofTrkC and 3H-thymidine incorporation in transduced cells. These results demonstrate that our AdTrkC vector efficiently mediates the expression of high-levels of biologically active NT-3 receptors.

[A comparison of the kinetic characters of NT-3, NT-4 and BDNF retrogradely transported in facial nerve].

OBJECTIVE: To compare the kinetic characters of retrograde transport of neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) and brain-derived neurotrophin factor (BDNF) in facial nerve. METHODS: Radioactive tracer technique was used. After one lateral facial nerve trunk of adult rabbit was transected, a silicone chamber was inserted between the stumps, and 3.7 MBq of 125I-NT-3 or 125I-NT-4 or 125I-BDNF or 125I-HSA was administered into the chamber. At different time-points after injection, the facial nerve trunk and facial nerve motor neurone of brain-stem were collected and the uptake rates were measured. The kinetic parameters of each labeled compound were calculated using 3P87 program of kinetics. RESULTS: The transport amount of neurotrophin retrogradely transported by facial nerve is NT-3 > BDNF > NT-4 (P < 0.05), the transport rate is NT-4 > NT-3 > BDNF (P < 0.05). CONCLUSION: The findings could serve as the kinetic characters of retrograde transport of neurotrophins.

Tolerability of recombinant-methionyl human neurotrophin-3 (r-metHuNT3) in healthy subjects.

This phase I, double-blind, randomized, placebo-controlled study evaluated the safety of single and multiple (daily for 7 days) subcutaneous administrations of recombinant-methionyl human neurotrophin-3 (r-metHuNT3) in healthy human volunteers at seven doses, ranging from 3 to 500 microg/kg/day. No serious or life-threatening adverse events occurred. The most frequently recorded adverse effects were mild injection-site pain, diarrhea, and elevation of liver function tests. No change in neurologic function was noted with these dosing regimens. We conclude that r-metHuNT3 is safe and well tolerated in the dosages used in this study.

Neurotrophic factor regulation of developing avian oculomotor neurons: differential effects of BDNF and GDNF.

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target-derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) may be target-derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio-iodinated BDNF, NT-3, NT-4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT-3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT-3, and NT-4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75(NTR) and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target.