Lev N. Novikov

Brain-derived neurotrophic factor promotes axonal regeneration and long-term survival of adult rat spinal motoneurons in vivo

This study shows that in adult rat spinal motoneurons brain-derived neurotrophic factor exerts a neuroprotective effect which extends several weeks beyond the duration of treatment. In addition, brain-derived neurotrophic factor strongly enhances regeneration of avulsed motor axons across the border between the central and peripheral nervous systems. Treatment with brain-derived neurotrophic factor is known to rescue adult rat spinal motoneurons from retrograde cell death induced by ventral root avulsion. The present experiments were designed to test whether this survival effect remains over an extended period of time following cessation of treatment and, also, whether brain-derived neurotrophic factor promotes regeneration of avulsed motor axons. After avulsion of a spinal ventral root, four weeks of treatment with brain-derived neurotrophic factor (10 microg/day) or vehicle was initiated. By using different retrograde tracers to obtain pre- and postoperative labelling of avulsed and regenerating motoneurons, respectively, the number of surviving motoneurons as well as the extent of motor axonal regeneration could be analysed. The expression of nitric oxide synthase in the lesioned motoneurons was also studied. In the vehicle-treated rats, only 10% of the avulsed motoneurons remained at 12 weeks postoperatively, 20-40% of which displayed nitric oxide synthase activity. Treatment with brain-derived neurotrophic factor during the initial four postoperative weeks resulted in 45% motoneuron survival and a complete blockage of nitric oxide synthase expression at 12 weeks postoperatively. Brain-derived neurotrophic factor also induced abundant regeneration of the avulsed motor axons, which formed extensive fibre bundles along the surface of the spinal cord and adjacent ventral roots. The long-term effect by brain-derived neurotrophic factor seemed to be even stronger on motor axonal regeneration than on motoneuron survival. The present results indicate a therapeutic potential for brain-derived neurotrophic factor in the early treatment of traumatic injuries to spinal nerves and roots.

Brain-derived neurotrophic factor promotes survival and blocks nitric oxide synthase expression in adult rat spinal motoneurons after ventral root avulsion ☆

In adult spinal motoneurons, retrograde cell death is induced by ventral root avulsion. A lethal effect of nitric oxide has been implicated, since nitric oxide synthase (NOS) is expressed in the motoneurons destined to die. Our study investigates the effects of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) on the retrograde cell death and NOS expression of adult rat spinal motoneurons. Following ventral root avulsion and 4 weeks of continuous treatment, BDNF, but not CNTF, was found to prevent cell death and NOS expression in the lesioned motoneurons. This suggests a therapeutic potential for BDNF in the adult nervous system, possibly through blockage of nitric oxide synthesis.

Persistent neuronal labeling by retrograde fluorescent tracers : a comparison between Fast Blue, Fluoro-Gold and various dextran conjugates

The permanence of retrograde neuronal labeling by the fluorescent tracers Fast Blue, Fluoro-Gold, Mini-Ruby, Fluoro-Ruby and Fluoro-Emerald was investigated in adult rat spinal motorneurons at 1, 4, 12 and 24 weeks after tracer application to a transected muscle nerve. After 1 week, the largest number of retrogradely labeled motoneurons was found with Mini-Ruby, Fluoro-Gold and Fluoro-Ruby, while Fluoro-Emerald yielded a smaller number of labeled cells. With increasing survival time, all of these tracers exhibited a marked decrease in the number of labeled neurons. Fast Blue also produced very efficient staining after 1 week and, in addition, the number of Fast Blue-labeled cells remained constant over the entire time period studied. Also in embryonic spinal cord tissue exposed to Fast Blue. the label persisted for at least 6 months after transplantation into adult spinal cord. Double-labeling experiments combining Fast Blue with Fluoro-Gold, Mini-Ruby, Fluoro-Ruby or Fluoro-Emerald showed that all these substances were non-toxic and that the time-related decrease in the number of neurons labeled by the latter tracers was due to degradation or leakage of the dyes. Thus, Fast Blue would be the tracer of choice for motoneuronal labeling in long-term experiments, whereas the usage of the other tracers should be restricted to experiments of limited duration.

A novel biodegradable implant for neuronal rescue and regeneration after spinal cord injury.

After spinal cord injury, the severed neuronal pathways fail to regenerate spontaneously. This study describes a biodegradable implant using poly-beta-hydroxybutyrate (PHB) fibers as carrier scaffold for matrix components and cell lines supporting neuronal survival and regeneration after spinal cord injury. After cervical spinal cord injury in adult rats, a graft consisting of PHB fibers coated with alginate hydrogel + fibronectin was implanted in the lesion cavity. In control groups, PHB was omitted and only alginate hydrogel or fibronectin, or their combination, were used for grafting. In addition, comparisons were made with animals treated intrathecally after spinal cord injury with the neurotrophic factors BDNF or NT-3. The neurons of the rubrospinal tract served as experimental model. In untreated animals, 45% of the injured rubrospinal neurons were lost at 8 weeks postoperatively. Implantation of the PHB graft reduced this cell loss by 50%, a rescuing effect similar to that obtained after treatment with BDNF or NT-3. In the absence of PHB support, implants of only alginate hydrogel or fibronectin, or their combination, had no effect on neuronal survival. After addition of neonatal Schwann cells to the PHB graft, regenerating axons were seen to enter the graft from both ends and to extend along its entire length. These results show that implants using PHB as carrier scaffold and containing alginate hydrogel, fibronectin and Schwann cells can support neuronal survival and regeneration after spinal cord injury

Survival effects of BDNF and NT-3 on axotomized rubrospinal neurons depend on the temporal pattern of neurotrophin administration

This study shows that both BDNF and NT‐3 can prevent cell death in axotomized adult rat rubrospinal neurons (RSNs), but that the efficacy of neuroprotection depends on the temporal pattern of treatment. At 8 weeks after cervical spinal cord injury, 51% of the RSNs had died. Subarachnoidal BDNF infusion into the cisterna magna for 4 weeks resulted in neuronal hypertrophy and 71% survival. Continuous infusion for 8 weeks into the lumbar subarachnoidal space with either BDNF or NT‐3 gave similar survival rates, while a combination of BDNF and NT‐3 resulted in 96% survival, although the cells were atrophic. When administration of either BDNF or NT‐3 was delayed and performed during postoperative weeks 5–8, the number of surviving neurons was increased compared to early treatment. Delayed treatment with a combination of BDNF and NT‐3 resulted in complete survival and a reduction in neuronal atrophy. A decreased expression of TrkB receptors and microtubule‐associated protein‐2 in the RSNs after axotomy was counteracted by BDNF and NT‐3. Microglial activity remained increased even when complete cell survival was achieved. Thus, the combination of neurotrophins as well as the temporal pattern of treatment need to be adequately defined to optimize survival of injured spinal tract neurons.

Characterisation of human mesenchymal stem cells following differentiation into Schwann cell-like cells.

Cell-based therapies provide a clinically applicable and available alternative to nerve autografts. Our previous studies have characterised rat-derived mesenchymal stem cells (MSC) and here we have investigated the phenotypic, molecular and functional characteristics of human-derived MSC (hMSC) differentiated along a Schwann cell lineage. The hMSC were isolated from healthy human donors and the identity of the undifferentiated hMSC was confirmed by the detection of MSC specific cells surface markers. The hMSC were differentiated along a glial cell lineage using an established cocktail of growth factors including glial growth factor-2. Following differentiation, the hMSC expressed the key Schwann cell (SC) markers at both the transcriptional and translational level. More importantly, we show the functional effect of hMSC on neurite outgrowth using an in vitro co-culture model system with rat-derived primary sensory neurons. The number of DRG sprouting neurites was significantly enhanced in the presence of differentiated hMSC; neurite length and density (branching) were also increased. These results provide evidence that hMSC can undergo molecular, morphological and functional changes to adopt a SC-like behaviour and, therefore, could be suitable as SC substitutes for nerve repair in clinical applications.

Differential effects of neurotrophins on neuronal survival and axonal regeneration after spinal cord injury in adult rats.

Spinal cord injury (SCI) induces retrograde cell death in descending pathways, which can be prevented by long‐term intrathecal infusion of neurotrophins (Novikova et al. [2000] Eur J Neurosci 12:776–780). The present study investigates whether the same treatment also leads to improved regeneration of the injured tracts. After cervical SCI in adult rats, a peripheral nerve graft was attached to the rostral wall of the lesion cavity. The animals were treated by local application into the cavity of Gelfoam soaked in (1) phosphate buffered saline (untreated controls) or (2) a mixture of the neurotrophins brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) (local treatment), or by intrathecal infusion of BDNF + NT‐3 for (3) 2 weeks (short‐term treatment) or (4) 5–8 weeks (long‐term treatment). Despite a very strong survival effect, long‐term treatment failed to stimulate ingrowth of descending tracts into the nerve graft. In comparison with untreated controls, the latter treatment also caused 35% reduction in axonal sprouting of descending pathways rostral to the lesion site and 72% reduction in the number of spinal cord neurons extending axons into the nerve graft. Local and short‐term treatments neither prevented retrograde cell death nor enhanced regeneration of descending tracts, but induced robust regeneration of spinal cord neurons into the nerve graft. These results indicate that the signal pathways promoting neuronal survival and axonal regeneration, respectively, in descending tracts after SCI respond differently to neurotrophic stimuli and that efficient rescue of axotomized tract neurons is not a sufficient prerequisite for regeneration. J. Comp. Neurol. 452:255–263, 2002.

Stimulating the neurotrophic and angiogenic properties of human adipose-derived stem cells enhances nerve repair.

In future, adipose-derived stem cells (ASC) might be used to treat neurological disorders. In this study, the neurotrophic and angiogenic properties of human ASC were evaluated, and their effects in a peripheral nerve injury model were determined. In vitro growth factor stimulation of the cells resulted in increased secretion of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor-A (VEGF-A), and angiopoietin-1 proteins. Conditioned medium from stimulated cells increased neurite outgrowth of dorsal root ganglia (DRG) neurons. Similarly, stimulated cells showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. ASC were seeded into a fibrin conduit that was used to bridge a 10 mm rat nerve gap. After 2 weeks, the animals treated with control or stimulated ASC showed an enhanced axon regeneration distance. Stimulated cells evoked more total axon growth. Analysis of regeneration and apoptosis-related gene expression showed that both ASC and stimulated ASC enhanced GAP-43 and activating transcription factor 3 (ATF-3) expression in the spinal cord and reduced c-jun expression in the DRG. Caspase-3 expression in the DRG was reduced by stimulated ASC. Both ASC and stimulated ASC also increased the vascularity of the fibrin nerve conduits. Thus, ASC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for nerve regeneration.

Effects of Neurotransplants and BDNF on the Survival and Regeneration of Injured Adult Spinal Motoneurons

We compared the effects of peripheral nerve grafts, embryonic spinal cord transplants and brain‐derived neurotrophic factor (BDNF) on the survival and axon regeneration of adult rat spinal motor neurons undergoing retrograde degeneration after ventral root avulsion. Following implantation into the dorsolateral funiculus of the injured spinal cord segment, neither a peripheral nerve graft nor a combination of peripheral nerve graft with embryonic spinal cord transplant could prevent the retrograde motor neuron degeneration induced by ventral root avulsion. However, intrathecal infusion of BDNF promoted long‐term survival of the lesioned motor neurons and induced abundant motor axon regeneration from the avulsion zone along the spinal cord surface towards the BDNF source. A combination of ventral root reconstitution and BDNF treatment might therefore be a promising means for the support of both motor neuron survival and guided motor axon regeneration after ventral root lesions.

Exogenous brain-derived neurotrophic factor regulates the synaptic composition of axonally lesioned and normal adult rat motoneurons

Brain-derived neurotrophic factor has previously been shown to promote survival and axonal regeneration in injured spinal motoneurons and, also, to modulate synaptic transmission and regulate the density of synaptic innervation in a variety of neurons. The present light and electron microscopic study demonstrates synaptotrophic effects of exogenously applied brain-derived neurotrophic factor on the synaptic composition of both normal and axonally lesioned adult rat spinal motoneurons. After L5-L6 ventral root avulsion, a massive loss of all types of boutons occurred on the somata of the lesioned motoneurons which persisted for at least 12 weeks postoperatively. We found that (i) intrathecal infusion of brain-derived neurotrophic factor during the first postoperative week did not prevent the synaptic detachment and activation of glial cells; (ii) prolonged treatment for four weeks restored synaptic covering and significantly reduced microglial reaction; (iii) the synaptotrophic effect remained significant for at least eight weeks after cessation of the treatment; (iv) brain-derived neurotrophic factor mainly supported F-type boutons with presumably inhibitory function, while it had little effect on S-type boutons associated with excitatory action; and (v) in normal unlesioned motoneurons, four weeks of treatment with brain-derived neurotrophic factor induced sprouting of F-type boutons, a loss of S-type boutons and motoneuron atrophy. The present data show that exogenous neurotrophins not only help to restore synaptic circuitry in axonally injured motoneurons, but also strongly influence the synaptic composition in normal motoneurons.