Jan-Olof Kellerth

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.

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.

A Quantitative morphological study of HRP-labelled cat α-motoneurones supplying different hindlimb muscles

Cat alpha-motoneurones supplying the quadriceps (Q), posterior biceps (PB), gastrocnemius (G), soleus (SOL) and short intrinsic plantar foot (SP) muscles were studied after retrograde or intracellular labelling with HRP. The average soma sizes were rather similar for the different pools, the SOL cells being the smallest. The median number of first-order dendrites ranged from 10 (PB) to 12 (SOL). The median diameters of the first-order dendrites ranged from 6 (SOL) to 8.5 (PB, G) micrometer. The dendritic projection patterns were rather similar for the different motoneurone groups, except for a prominent dorsomedial projection of SP dendrites. A considerable fraction of the dendrites extended into the white matter. The diameter of the first-order dendrite correlated positively to the number of end branches as well as to the combined length, surface area and volume of the whole dendrite. These relations appeared to be independent of motoneurone group and dendritic orientation. The combined diameter of the first-order dendrites, which reflects the total dendritic size of a motoneurone, exhibited median values between 82 micrometers (SOL) and 112 micrometers (Q). With respect to the relative scaling of soma and dendrites, motoneurones with large somas tended to have proportionally larger dendritic trees. The distribution of dendritic diameters, number of branches, dendritic surface area and volume, and the combined dendritic parameter (epsilon d3/2) at various distances from the soma were quite similar for the different motoneurone groups.

Electron microscopic observations on the synaptic contacts of group Ia muscle spindle afferents in the cat lumbosacral spinal cord

After intra-axonal injection of horseradish peroxidase (HRP) into afferent fibers originating from muscle spindle primary endings of the cat gastrocnemius, group Ia boutons located in the ventral horn of the spinal cord were identified and studied electron microscopically. The Ia boutons were invariably found to contain spherical synaptic vesicles (S-type boutons), and a number of them were also postsynaptic to smaller P-type boutons (large S-type boutons with axo-axonic contacts). None of the present Ia-boutons belonged to the previously described M-type. The vast majority of the studied boutons were considered to be located at less than 500 microns distance from the alpha-motoneuron soma. The results are discussed in relation to previous light and electron microscopic data.

Changes in synaptology of adult cat spinal α-motoneurons after axotomy

The aim of this electron-microscopic study was to analyze the distribution of synaptic contacts on the cell bodies and dendrites of permanently axotomized adult cat spinal α-motoneurons. Following transection and ligation of the medial gastrocnemius nerve, the synaptic covering of the cell bodies and three different dendritic compartments of homonymous α-motoneurons was analyzed quantitatively at 3, 6, and 12 weeks postoperatively. The synaptic boutons were classified according to their size and the shape of their synaptic vesicles. On the soma, a transient increase in the number of boutons was noted at 3 weeks and 6 weeks postoperatively, while after 12 weeks the bouton number had decreased to half of its normal value. The transient increase was mainly due to an increase in the number of F-type boutons. At 12 weeks postoperatively, the synaptic covering was reduced by 83% on the soma and by 57% on the proximal dendrites. In the distal dendritic regions, the values for synaptic covering remained largely unchanged. In summary, axotomized motoneurons exhibit a reduction in synaptic covering which is maximal on the cell body and becomes less pronounced centrifugally along the dendrites. However, if also taking into account the loss of distal dendritic branches that occurs in axotomized motoneurons, the total loss of boutons is several times larger in the dendrites than on the soma.

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.

Sensory neuroprotection, mitochondrial preservation, and therapeutic potential of N-acetyl-cysteine after nerve injury

Neuronal death is a major factor in many neuropathologies, particularly traumatic, and yet no neuroprotective therapies are currently available clinically, although antioxidants and mitochondrial protection appear to be fruitful avenues of research. The simplest system involving neuronal death is that of the dorsal root ganglion after peripheral nerve trauma, where the loss of approximately 40% of primary sensory neurons is a major factor in the overwhelmingly poor clinical outcome of the several million nerve injuries that occur each year worldwide. N-acetyl-cysteine (NAC) is a glutathione substrate which is neuroprotective in a variety of in vitro models of neuronal death, and which may enhance mitochondrial protection. Using TdT uptake nick-end labelling (TUNEL), optical disection, and morphological studies, the effect of systemic NAC treatment upon L4 and 5 primary sensory neuronal death after sciatic nerve transection was investigated. NAC (150 mg/kg/day) almost totally eliminated the extensive neuronal loss found in controls both 2 weeks (no treatment 21% loss, NAC 3%, P=0.03) and 2 months after axotomy (no treatment 35% loss, NAC 3%, P=0.002). Glial cell death was reduced (mean number TUNEL positive cells 2 months after axotomy: no treatment 51/ganglion pair, NAC 16/ganglion pair), and mitochondrial architecture was preserved. The effects were less profound when a lower dose was examined (30 mg/kg/day), although significant neuroprotection still occurred. This provides evidence of the importance of mitochondrial dysregulation in axotomy-induced neuronal death in the peripheral nervous system, and suggests that NAC merits investigation in CNS trauma. NAC is already in widespread clinical use for applications outside the nervous system; it therefore has immediate clinical potential in the prevention of primary sensory neuronal death, and has therapeutic potential in other neuropathological systems.

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.