Martin Kanje

Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor

Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates axonal outgrowth. Here we used in situ hybridization and immunocytochemistry to study the VEGF receptor flk‐1 in cultured superior cervical ganglia (SCG) and dorsal root ganglia (DRG) from adult mice, and also the effects of VEGF on regeneration in vitro. Neurons in both ganglia contained the flk‐1 receptor and showed an increased mRNA expression and immunoreactivity for flk‐1 after 48 h in culture. In SCG, but not in DRG, double immunostaining for flk‐1 and VEGF revealed coexpression in many neurons, implying that VEGF may exert both autocrine and paracrine actions. One proportion of the flk‐1‐positive neurons in DRG stained positive for the large neuron marker RT97 and another proportion expressed calcitonin gene‐related peptide (CGRP). Small IB4‐positive neurons were devoid of flk‐1 immunoreactivity. Most flk‐1‐positive neurons in the DRG, but not in the SCG, were also immunoreactive to neuropilin‐1. VEGF was found to stimulate axonal outgrowth from DRG, both by an action on the growing axons and the nerve cell bodies. The latter effect could be mediated by retrograde axonal transport as revealed by the use of a two compartment system to assay axonal outgrowth. We also found that the VEGF‐induced axonal outgrowth was blocked by the flk‐1 inhibitor SU5416. The results strongly suggest that VEGF acts as a neurotrophic factor and plays an important role during the regeneration of peripheral nerves.

Insulin-like growth factor I (IGF-I) stimulates regeneration of the rat sciatic nerve

The effect of insulin-like growth factor I (IGF-I) was tested on regeneration of the rat sciatic nerve after a crush lesion. IGF-I was administered via miniosmotic pumps to the dorsal root ganglia or locally around the crush lesion. Regeneration of sensory fibers was measured after 3 or 4 days superfusion by pinching. IGF-I stimulated regeneration in both administration paradigms. Regeneration was inhibited if the nerve was perfused with specific antibodies to native IGF-I. The results suggest that endogenous extracellular IGF-I plays an important role during regeneration of peripheral nerve fibers.

Can sensory and motor collateral sprouting be induced from intact peripheral nerve by end-to-side anastomosis?

The possibility that collateral sprouting could occur from intact axons in au undamaged sciatic nerve was studied in the rat by suturing either a 7-day predegenerated or a fresh nerve segment in an end-to-side fashion to the sciatic nerve proper. Following a 14- or 35-day recovery period, the pinch reflex test was performed on the transplanted segment to demonstrate the presence of sensory axons. The majority of cases, using a predegenerated nerve segment hut not a fresh segment, responded positively. Neurofilament staining and histological examination confirmed the presence of axons in the attached nerve segment. In another series of experiments, the proximal peroneal fascicle was ligated and cut. Following a 7-day predegeneration period the distal stump was sutured end-to-side to the ipsilateral tibia1 fascicle. After 90 days, stimulation of the tibia1 nerve proximal to the attached site induced substantial contraction in both the native gastrocnemius muscle and the foreign tibialis anterior muscle. These findings suggest that collateral sprouting may occur from intact axons, perhaps induced by factors emanating from the attached nerve segment, and subsequently make functional peripheral connections.

Regeneration of the rat sciatic nerve into allografts made acellular through chemical extraction

The aim of this study was to develop a procedure by which myelin and Schwann cells could be removed from a peripheral nerve while the basal lamina tubes, remained intact, and to test if such preparations could be used as allografts for the repair of a gap in the continuity of the rat sciatic nerve. We found that extraction with the detergents Triton X-100 and deoxycholate resulted in acellular nerve segments with preserved basal lamina tubes, here defined as the tubes which surrounds the axon/Schwann cell units. The morphology of the acellular nerve segments was revealed by scanning electron microscopy, teasing, immunohistochemistry and electrophoresis. Such grafts when allografted between two outbred rat strains, were found to support outgrowth of axons and migration of Schwann cells, which reoccupied the empty basal lamina tubes without excessive signs of inflammation. This new paradigm offers a possible solution to the major shortcomings of autologous nerve grafts, i.e., the requirement to sacrifice a healthy nerve and the shortage of graft material available for repair.

Vascular endothelial growth factor stimulates Schwann cell invasion and neovascularization of acellular nerve grafts

The aim of this study was to investigate the effects of vascular endothelial growth factor (VEGF) on regeneration of the rat sciatic nerve in vivo. To that end we used 10-mm long cell-free nerve grafts to bridge a gap in the sciatic nerve. The grafts were pretreated with either VEGF (50, 100 or 250 ng/ml), nerve growth factor (NGF, 100 ng/ml) or laminin (100 ng/ml) before implantation. Outgrowth of axons, Schwann cells, blood vessels and macrophages were studied 10 days post-implantation by the use of immunocytochemistry and histochemistry. Grafts pretreated with VEGF stimulated the outgrowth of Schwann cells and blood vessels but not axons. In such grafts, the Schwann cells also exhibited a dramatic change in morphology and became filled with large lipid-containing vacuoles. These cells also showed an intense immunoreactivity for the VEGF receptor flk-1. Neither pretreatment with laminin nor NGF affected the outgrowth of Schwann cells. However, NGF treatment increased the number of axons in the graft but was not able to counteract injury-induced downregulation of substance P in the dorsal root ganglia. The results show that local application of VEGF promotes at least two events, invasion of Schwann cells and neovascularization, which are important during nerve regeneration. The findings suggest that the effects of the pretreatment by the growth factors is local and limited to the graft, whereas central events like neuropeptide synthesis is not affected.

Insulin-like growth factor (IGF-1) as a stimulator of regeneration in the freeze-injured rat sciatic nerve

The effect of insulin-like growth factor (IGF-1) on the ability of the rat sciatic nerve to regenerate into a freeze-injured nerve segment was investigated. The freeze-injured segment was perfused for 6 days with Ringer solution and different concentrations of IGF-1, dispensed by a subcutaneously implanted osmotic minipump. At a pump concentration of 50, 100 and 200 micrograms IGF-1/ml the regeneration length increased with 14, 25 and 26%, respectively, as measured by the pinch test and by immunocytochemical staining for neurofilaments (NF) in the growing neurites. Schwann cells invading the freeze-injured segment were visualized by immunostaining for S-100 protein. In nerves perfused with Ringer solution alone the Schwann cells were present as far as the neurites had regenerated, while neurites seemed to grow slightly ahead of the Schwann cells in the nerves perfused with IGF-1. Incorporation of [3H]thymidine increased in IGF-1-treated nerves. However, IGF-1 perfusion did not increase thymidine incorporation when outgrowth of neurites was detained by a transection proximal to the freeze-injured area. The results suggest that IGF-1 affects regeneration by local stimulation of the growing neurites and that IGF-1 stimulates the proliferation of non-neuronal cells indirectly.

Inhibition of c-Jun phosphorylation reduces axonal outgrowth of adult rat nodose ganglia and dorsal root ganglia sensory neurons.

The role of c-Jun activation for survival and regeneration of sensory neurons is unclear. Here we report that c-Jun N-terminal kinase (JNK)-mediated c-Jun activation is important for axonal outgrowth of sensory neurons in rat nodose and dorsal root ganglia (DRG). Peripheral severance of the vagus or the sciatic nerve resulted in a massive and rapid, but transient increase of the activated JNK (p-JNK) in neuronal nuclei, followed by c-Jun phosphorylation and activating transcription factor-3 (ATF3) induction. JNK inhibition by the selective JNK inhibitors SP600125 and (D)-JNKI1 did not affect neuronal survival in explanted or dissociated ganglia, but dramatically reduced axonal outgrowth, c-Jun activation, and ATF3 induction. Using retrograde labeling, we demonstrated that activated c-Jun (p-c-Jun) and ATF3 were associated with regenerative neurons. Taken together, our results suggest that JNK-mediated c-Jun activation is one of the first cell body reactions in response to nerve injury and that this activation and subsequent ATF3 induction are associated with axonal outgrowth.

5-HT1B and 5-HT1D receptors in the human trigeminal ganglion: co-localization with calcitonin gene-related peptide, substance P and nitric oxide synthase

Abstract 5-Hydroxytryptamine (5-HT) is implicated in migraine and agonist directed aganist 5-HT 1B and 5-HT 1D receptors are commonly used as effective therapies. The antimigraine mechanisms involve the inhibition of intracranial sensory neuropeptide release. In order to determine which 5-HT 1 receptor subtypes are involved we have by immunocytochemistry examined the distribution of 5-HT 1B and 5-HT 1D receptors in the human trigeminal ganglia, and addressed which of them colocalize with calcitonin gene-related peptide (CGRP), substance P (SP) or nitric oxide synthase (NOS). We detected that 5-HT 1D receptor immunoreactivity (i.r.) was predominantly expressed in medium-sized cells (86% of positive cells, 30–60 μm). About 9% of the 5-HT 1D receptor i.r. cells were large in size (>60 μm) and 5% were small in size ( 1B receptor i.r. was mainly expressed in medium-sized cells (81% in 30–60 μm, 15% in >60 μm and 4% in 1B or 5-HT 1D receptor immunoreactive cells co-localized with either CGRP, SP or NOS. Thus, 89% of the CGRP i.r. cells expressed 5-HT 1D receptor i.r. and 65% of the CGRP positive cells were 5-HT 1B receptor positive. Most of the 5-HT 1D (95%) and the 5-HT 1B (94%) receptor i.r. cells showed SP immunostaining and 83% of 5-HT 1D receptor and 86% of 5-HT 1B receptor i.r. cells contained NOS. In conclusion, both 5-HT 1B and 5-HT 1D receptors are expressed in the human trigeminal ganglion and they are mainly localized in medium-sized cells and they seem to colocalize with CGRP, SP and NOS.

A New Type of “Bioartificial” Nerve Graft for Bridging Extended Defects in Nerves

In the rat sciatic nerve, a gap of around 10 mm in nerve continuity seems to be the maximal distance which can be successfully repaired by silicone tubes. In this study we tested if a new artificial nerve graft, composed of eight polyamide filaments (diameter 250 μm) placed inside silicone tubes (1.8 mm inner diameter), could be used to bridge an extended gap (15 mm) in rat sciatic nerve. Silicone tubes containing eight polyamide sutures were found to support regeneration across such a gap. After 4 weeks sensory fibres had bridged the gap and grown into the distal nerve segment as revealed by a positive pinch reflex test as well as positive staining for neurofilaments in the distal nerve segment. Myelinated axons could be observed in the tissue matrix formed in between and peripheral to the synthetic filaments along the whole length of the tube. In contrast, when silicone tubes without filaments were used to bridge the 15 mm gap, the tubes contained only fluid or in two cases a thin tissue strand. No positive pinch reflex response was elicited in the nerve segment distal to such a tube. We conclude that the new artificial nerve graft can be used to support regeneration across extended gaps in nerves.

Retrograde axonal transport of JNK signaling molecules influence injury induced nuclear changes in p-c-Jun and ATF3 in adult rat sensory neurons

In the present study, we investigated if the previously observed JNK-mediated activation of c-Jun and induction of ATF3 could be ascribed to axonal transport of JNK signaling components, or if axonal transport of the transcription factors themselves contributes to the nuclear changes in injured sensory neurons. We observed retrograde axonal transport of a number of JNK upstream kinases in ligated rat sciatic nerve. In these preparations, axonal transport of JNK/p-JNK, the JNK scaffolding protein JIP, and the transcription factors ATF3 and ATF2/p-ATF2 was also found. No or little retrograde transport of c-Jun and p-c-Jun was found, whereas an anterograde transport of Hsp27, a protein previously reported in the context of p-c-Jun and ATF3, was observed. In separate experiments, we found that in vitro inhibition of axonal transport or axonal inhibition of JNK reduced the number of p-c-Jun- and ATF3-positive neuronal nuclei. The results suggest that retrograde axonal transport of JNK signaling components contributes to the injury induced c-Jun phosphorylation and ATF3 induction.