Postnatal expression of VEGF and its receptor flk-1 in peripheral ganglia.

The postnatal (P0-P12) and adult expression of vascular endothelial growth factor and its receptor flk-1 was investigated in superior cervical (SCG) and dorsal root ganglia (DRG) in mice by immunocytochemistry. At P0 all neurons in SCG and DRG contained VEGF. The number of VEGF-immunoreactive neurons in DRG but not in SCG, decreased postnatally and reached adult levels (34%) at P12. At P0 flk-1 was found in virtually all neurons in the SCG and in roughly half of the neurons in DRG. The number of flk-1 positive neurons then decreased and reached adult levels at P12. The findings demonstrate temporal changes in VEGF and flk-1 expression, suggesting developmental regulation of VEGF activity in peripheral ganglia.

Use of chemically extracted muscle grafts to repair extended nerve defects in rats.

Nerve regeneration, measured as axonal outgrowth, Schwann cell migration, macrophage invasion, and neovascularisation, was compared after repair of a 15 mm gap in rats' sciatic nerves using autologous muscle grafts made acellular either by freezing and thawing or by chemical extraction. Both extracted and freeze-thawed acellular muscle grafts could be used to bridge the defect. However, axons and Schwann cells, as shown by immunohistochemical staining for neurofilaments and S-100 protein, respectively, grew faster into the extracted muscle grafts than into the freeze-thawed acellular muscle grafts and somewhat more axons were observed in the former graft. There were no significant differences between the two graft types with respect to neovascularisation as showed by staining for endothelial alkaline phosphatase, and limited differences concerning invasion of macrophages (ED1 and ED2) as detected by immunocytochemistry. The results showed that chemically extracted muscle grafts could be used to bridge an extended nerve defect and that such grafts in some aspects were superior to freeze-thawed muscle grafts for extended gaps.

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.

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.

Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system.

Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells, and it promotes angiogenesis in vivo. Here we report that VEGF(165) has neurotrophic actions on cultured adult mouse superior cervical ganglia (SCG) and dorsal root ganglia (DRG), measured as axonal outgrowth. Maximal effect was observed at 10-50 ng/ml for SCG and 100 ng/ml for DRG. VEGF-induced axonal outgrowth was inhibited by the mitogen-activated protein kinase kinase inhibitor PD 98059 but not by the protein kinase inhibitor K252a. VEGF also increased survival of both neurons and satellite cells and the number of proliferating Schwann cells. Immunocytochemistry and immunoblotting revealed that VEGF was expressed in virtually all nerve cells in the SCG but only in a population of small-diameter (<35 micrometers) neurons representing approximately 30% of the neurons in DRG. Immunostaining showed that the VEGF receptor fetal liver kinase receptor (flk-1) was found on nerve cell bodies in DRG and to a lesser extent on neurons in SCG. Growth cones of regenerating axons from both types of ganglia exhibited flk-1 immunoreactivity, as did Schwann cells. We conclude that VEGF has both neurotrophic and mitogenic activity on cells in the peripheral nervous system.

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.

The insulin-like growth factors I and II stimulate proliferation of different types of Schwann cells.

A combination of immunocytochemistry for glial specific antigens and bromodeoxyuridine (BrdU) and teasing was used to identify proliferating cells in cultured rat sciatic nerve segments. The nerve segments were exposed to insulin, or the insulin-like growth factors IGF-I and IGF-II. Teasing in combination with BrdU immunocytochemistry showed that around 93% of the proliferating cells in the nerve segments were Schwann cells. Immunostaining for BrdU and GFAP (glial fibrillary acid protein) showed that IGF-II enhanced proliferation of Schwann cells surrounding unmyelinated nerve fibres. In contrast, truncated IGF-I promoted proliferation of Schwann cells of myelinated nerve fibres while insulin increased proliferation of both cell types.