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Objective-The embryonic morphogen sonic hedgehog (SHh) has been shown to induce neovascularization of ischemic tissue but has not been shown to play a role in regulating vascular nerve supply. Accordingly, we investigated the hypothesis that systemic injection of SHh protein could improve nerve blood flow and function in diabetic neuropathy (DN). Methods and Results-Twelve weeks after induction of diabetes with streptozotocin, motor and sensory nerve conduction velocities (MCV and SCV) of the sciatic nerves were significantly reduced in diabetic rats. SHh-treated diabetic rats demonstrated marked improvement of both MCV and SCV (PϽ0.05). Laser Doppler perfusion imaging showed that nerve blood flow was significantly reduced in the diabetic rats but was restored in SHh-treated diabetic rats (PϽ0.05 versus diabetic saline-treated rats) to levels similar to those achieved with vascular endothelial growth factor-2 (VEGF-2) gene therapy. In vivo perfusion of Bandeuraea simplicifolia (BS)-1 lectin showed marked reduction in the vasa nervora in diabetic sciatic nerves but restoration of nerve vasculature to nondiabetic levels in the SHh-treated and plasmid DNA encoding human VEGF-2 (phVEGF-2)-treated diabetic nerves. Interestingly, the SHh-induced vasculature was characterized by larger diameter and more smooth muscle cell-containing vessels, compared with VEGF-2 gene-treated diabetic rats. Conclusions-These data indicate that Shh induces arteriogenesis and restores nerve function in DN. (Arterioscler ThrombVasc Biol. 2004;24:2102-2107.)Key Words: angiogenesis Ⅲ diabetes mellitus Ⅲ cytokine Ⅲ microcirculation Ⅲ peripheral vasculature I n the United States alone, Ͼ18 million people have diabetes. 1 Diabetic neuropathy (DN) is a frequent complication of diabetes, affecting 1 to 7 million people, including 7% within 1 year of diagnosis and 50% of patients after 25 years. It has also been reported that up to 90% of patients have subclinical levels of neuropathy. 2 Although several factors have been reported to contribute to diabetic polyneuropathy, 3-9 the pathogenic basis has remained uncertain. 10 An association between changes in the vasa nervorum and DN has been noted in multiple previous reports; [11][12][13][14][15][16][17] however, the pathophysiologic importance of these observations remains uncertain. The possibility that attenuation of the vasa nervorum might be a major factor in the development of DN is suggested by several recent studies. Impaired ischemiainduced angiogenesis was noted in animal models of diabetes, 18 and more recently we have reported that both ischemic 19 and DN 20 are associated with attenuation of the vasa nervorum and that local delivery of naked DNA encoding for vascular endothelial growth factor (VEGF-1 and VEGF-2) restores the vascular supply and has a favorable effect on the nerve conduction velocities. These observations, documenting the loss of vasa nervorum in diabetic animals, and restoration of neural vascularity by VEGF, associated with a return of nerve function, suggested that t...

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Neurotrophins play important roles in the survival, differentiation, and maintenance of CNS neurons. To begin to investigate specific roles for these factors in the mammalian visual system, we have examined the cellular localization of the neurotrophin receptor trkB within the developing cerebral cortex and thalamus of the ferret using extracellular domain-specific antibodies. At prenatal ages (gestation is 41 d), trkB-immunostained fibers were observed in the internal capsule and as two distinct fascicles within the intermediate zone of the cerebral cortex. The staining of these fiber tracts declined with increasing age, whereas soma and dendrite staining of cortical neurons was first evident in early postnatal life and increased during subsequent development. Staining of subplate neurons [by prenatal day 5 (P5)] was followed by staining of cortical layer 5 neurons (at P10). By P31, trkB immunoreactivity was particularly prominent in layers 3 and 5 but was absent from subplate neurons. Staining included cells, especially pyramidal neurons, in all cortical layers by P45, and this pattern was maintained into adulthood. The optic tract and fibers within the lateral geniculate nucleus (LGN) were also strongly trkB immunoreactive at prenatal ages. Cellular staining of a subset of LGN neurons, those within the C-layers and perigeniculate nucleus, was apparent by P10 and maintained until P45, when the adult pattern of highly trkB-immunoreactive neurons in all layers of the LGN first appeared. The pattern of trkB immunoreactivity suggests that specific subsets of cortical and thalamic neurons may respond to neurotrophins such as brain-derived neurotrophic factor and/or NT-4/5 at discrete developmental times and locations. The appearance of trkB on axon fibers early in development and then on cell bodies and dendritic processes later is consistent with roles for both long-range and local, including autocrine and/or paracrine, delivery of neurotrophins in cell survival and maturation.

IntroductionThe mammalian hedgehog (Hh) protein family (sonic, desert, and indian) are relatives of a Drosophila hedgehog protein that regulates segmental polarity during development and are expressed at many sites in the developing mouse embryo (1). Within the nervous system, sonic hedgehog protein (SHh) is associated with development and patterning of the central nervous system (2-5). In contrast, the morphogenic effects of desert hedgehog protein (DHh) are restricted to the peripheral nervous system and appear to focus specifically on cellular elements of the epi-and perineurial sheaths (6). Deletion of the dhh gene that codes for DHh in mice results in disruption of the fascicular structure of peripheral nerves, with development of a thin and disorganized perineurial sheath and an increase in blood-nerve barrier permeability that is associated with decreased connexin-43 expression by perineurial cells (6). A similar modification of nerve structure was associated with a homozygous missense mutation of the DHH gene in a human diagnosed with "minifascicular neuropathy" (7). These findings point to a role for Hh proteins in the development of the peripheral nervous system.Recent findings in acute peripheral-nerve injury models have revealed a novel function for hedgehog proteins in adult nerve regeneration and repair. The shh mRNA was strikingly upregulated in the peripheral nerve after crush injury (8), and exogenous SHh protein administration enhanced the speed of nerve recovery (9). The Hh signaling pathway is also upregulated soon after injury in a model of surgically induced hind limb ischemia, and exogenous SHh protein administration augments blood-flow recovery and limb salvage (10). Emerging evidence therefore suggests that signaling pathways initiated by Hh proteins are involved in the response to a range of peripheral-nerve lesions and participate in the repair and recovery process. Hedgehog proteins modulate development and patterning of the embryonic nervous system. As expression of desert hedgehog and the hedgehog receptor, patched-1, persist in the postnatal and adult peripheral nerves, the hedgehog pathway may have a role in maturation and maintenance of the peripheral nervous system in normal and disease states. We measured desert hedgehog expression in the peripheral nerve of maturing diabetic rats and found that diabetes caused a significant reduction in desert hedgehog mRNA. Treating diabetic rats with a sonic hedgehog-IgG fusion protein fully restored motor-and sensory-nerve conduction velocities and maintained the axonal caliber of large myelinated fibers. Diabetes-induced deficits in retrograde transport of nerve growth factor and sciatic-nerve levels of calcitonin gene-related product and neuropeptide Y were also ameliorated by treatment with the sonic hedgehog-IgG fusion protein, as was thermal hypoalgesia in the paw. These studies implicate disruption of normal hedgehog function in the etiology of diabetes-induced peripheral-nerve dysfunction and indicate that delivery of exogenous hedge...

Nerve growth factor receptor immunoreactivity is transiently associated with the subplate neurons of the mammalian cerebral cortex ( Communicated by Gunther S. Stent, October 13, 1989 ABSTRACT Nerve growth factor and its receptor (NGFR) are known to be present in diverse embryonic and neonatal central nervous system tissues, including the cerebral cortex. However, the identity of the cortical cells expressing NGFR immunoreactivity has not been established. We have used immunolabeling coupled with [3HJthymidine autoradiography to identify such cells in ferret and cat brain. Polyclonal antibodies raised against a synthetic peptide corresponding to a conserved amino acid sequence of the NGFR were used for this purpose. Western (immunologic) blot analyses show that these antibodies specifically recognize NGFR and precursor proteins. In both species, NGFR immunoreactivity is primarily associated with the early generated and transient subplate neuron population of the developing neocortex, as indicated by the following evidence: the immunoreactive cells (i) are located directly beneath the developing cortical plate, (a) frequently have the inverted pyramid shape characteristic of subplate neurons, and (iii) can be labeled by an injection of[3H]thymidine on embryonic day (E) 28, a time when only subplate neurons are being generated. Intense NGFR immunostaining is seen on the cell bodies of these neurons as early as E30, several days after their last round of cell division, and this immunostaining remains strong for -3 weeks. The NGFR

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