Nerve Express Download Fixed
Sherlene Hodson
Conduits for the repair of peripheral nerve gaps are a good alternative to autografts as they provide a protected environment and a physical guide for axonal re-growth. Conduits require colonization by cells involved in nerve regeneration (Schwann cells, fibroblasts, endothelial cells, macrophages) while in the autograft many cells are resident and just need to be activated. Since it is known that soluble Neuregulin1 (sNRG1) is released after injury and plays an important role activating Schwann cell dedifferentiation, its expression level was investigated in early regeneration steps (7, 14, 28 days) inside a 10 mm chitosan conduit used to repair median nerve gaps in Wistar rats. In vivo data show that sNRG1, mainly the isoform α, is highly expressed in the conduit, together with a fibroblast marker, while Schwann cell markers, including NRG1 receptors, were not. Primary culture analysis shows that nerve fibroblasts, unlike Schwann cells, express high NRG1α levels, while both express NRG1β. These data suggest that sNRG1 might be mainly expressed by fibroblasts colonizing nerve conduit before Schwann cells. Immunohistochemistry analysis confirmed NRG1 and fibroblast marker co-localization. These results suggest that fibroblasts, releasing sNRG1, might promote Schwann cell dedifferentiation to a "repair" phenotype, contributing to peripheral nerve regeneration.
The localization of the neural cell adhesion molecule (N-CAM) and its highly sialylated form, which is prevalent in young tissues and has therefore been called embryonic neural cell adhesion molecule, was studied in the developing and adult mouse optic nerve and retina immunohistologically and immunochemically. At embryonic and early postnatal ages, neuroblasts and young postmitotic neurons, Müller cells and astrocytes in the retina, and retinal ganglion cell axons and all glial cells in the optic nerve express highly sialylated neural cell adhesion molecule. Beginning with the third postnatal week, highly sialylated neural cell adhesion molecule disappears from retinal ganglion cell axons in the optic nerve and from neuronal cell bodies and processes in the retina. In addition, it is not detectable on oligodendrocytes in 3-week-old animals. However, highly sialylated neural cell adhesion molecule continues to be expressed in the adult optic nerve and retina by astrocytes and Müller cells. On these cells it is only absent from cell membranes contacting basal lamina. Weakly sialylated neural cell adhesion molecule, in contrast, is expressed by all cell types of retinal and optic nerve during development and in the adult. The loss of highly sialylated neural cell adhesion molecule from neurons and oligodendrocytes must therefore be considered as a cell type-specific conversion of the so-called embryonic to the adult form of neural cell adhesion molecule and does not simply reflect the disappearance of neural cell adhesion molecule from these cells. Weakly sialylated neural cell adhesion molecule, however, is absent from outer segments of photoreceptor cells and, as is the case for the highly sialylated form, from glial cell surfaces contacting basal lamina. Thus, the expression of highly sialylated neural cell adhesion molecule by pre- and postmitotic neurons and by oligodendrocytes is restricted mainly to the period of histogenetic events in retina and optic nerve, i.e. cell division, cell migration, dendritic and axonal growth and synaptogenesis. In addition to the observation that this form of neural cell adhesion molecule is less adhesive than the weakly sialylated, adult form, it is likely that highly sialylated neural cell adhesion molecule plays an important role during dynamic morphogenetic events. Furthermore, the expression of highly sialylated neural cell adhesion molecule by astrocytes and Müller cells in adult optic nerves and retinae suggests some histogenetically plastic functions for these cells in the adult mouse visual system.
Adult spinal cord motor and dorsal root ganglion (DRG) sensory neurons express multiple neuregulin-1 (NRG-1) isoforms that act as axon-associated factors promoting neuromuscular junction formation and Schwann cell proliferation and differentiation. NRG-1 isoforms are also expressed by muscle and Schwann cells, suggesting that motor and sensory neurons are themselves acted on by NRG-1 isoforms produced by their peripheral targets. To test this hypothesis, we examined the expression of the NRG-1 receptor subunits erbB2, erbB3, and erbB4 in rat lumbar DRG and spinal cord. All three erbB receptors are expressed in these tissues. Sciatic nerve transection, an injury that induces Schwann cell expression of NRG-1, alters erbB expression in DRG and cord. Virtually all DRG neurons are erbB2- and erbB3-immunoreactive, with erbB4 also detectable in many neurons. In spinal cord white matter, erbB2 and erbB4 antibodies produce dense punctate staining, whereas the erbB3 antibody primarily labels glial cell bodies. Spinal cord dorsal and ventral horn neurons, including alpha-motor neurons, exhibit erbB2, erbB3, and erbB4 immunoreactivity. Spinal cord ventral horn also contains a population of small erbB3+/S100beta+/GFAP- cells (GFAP-negative astrocytes or oligodendrocytes). We conclude that sensory and motor neurons projecting into sciatic nerve express multiple erbB receptors and are potentially NRG-1 responsive.
Purpose: : Fovin is expressed in the retina and is a member of the Tweety gene family. The Tweety gene products are proposed to be membrane channel proteins. In situ hybridization shows that fovin is expressed in the optic nerve. The goal of this study is to determine which cells in the optic nerve express fovin. This information will help us gain insight into fovin's function in the optic nerve.
Results: : Longitudinal sections of the optic nerve were viewed on an epifluorescence microscope. Parallel wavy lines of immunoreactive material were seen with the fovin antibody. No labeling was observed with the preimmune control serum. In situ hybridization experiments show fovin expression in rows of adjacent cells along the long axis of the optic nerve, while hybridization is not seen with the fovin sense probe. This same hybridization pattern is also seen when the PLP antisense probe is hybridized to the optic nerve sections.
Nerve growth factor (NGF) is a neurotrophin crucial for the development and survival of neurons. It also acts on cells of the immune system which express the NGF receptors TrkA and p75NTR and can be produced by them. However, mouse NK cells have not yet been studied in this context.
We used cell culture, flow cytometry, confocal microscopy and ELISA assays to investigate the expression of NGF receptors by NK cells and their secretion of NGF. We show that resting NK cells express TrkA and that the expression is different on NK cell subpopulations defined by the relative presence of CD27 and CD11b. Expression of TrkA is dramatically increased in IL-2-activated NK cells. The p75NTR is expressed only on a very low percentage of NK cells. Functionally, NGF moderately inhibits NK cell degranulation, but does not influence proliferation or cytokine production. NK cells do not produce NGF.
In this paper, we investigated if NGF is produced by NK cells and if NK cells express NGF receptors. We show for the first time that normal mouse NK cells can express TrkA and that this receptor is dynamically regulated on NK cells. In addition, we performed functional NK cell studies which revealed a tendency of NGF to negatively influence NK cell degranulation. NK cells do not produce NGF.
We investigated by flow cytometry the expression of NGF receptors on NK cells present within fresh splenocytes from B6 mice. Whereas p75NTR could be detected only on a very low number of NK cells (Fig. 1), TrkA was expressed by approximately 20% of the cells (Fig. 2A, Fig. 2B). The presence of TrkA in purified spleen NK cells was further confirmed by RT-PCR (data not shown).
Splenocytes were stained with anti-NK1.1, anti-CD3 and anti-p75NTR Ab or isotype control as described in Materials and Methods, and analyzed by flow cytometry. Dead cells were excluded by staining with Live Dead cell marker. A gate was set on NK cells (CD3-NK1.1+). Only a weak expression of p75NTR (grey lines) relative to isotype control (dashed lines) was observed at day 0 (left panel) and after 5 days of culture in the presence of IL-2 (right panel). Data shown are from one representative experiment out of three performed.
Splenocytes were stained with anti-NK1.1, anti-CD3 and anti-TrkA Ab or isotype control as described in Materials and Methods, and analyzed by flow cytometry. Dead cells were excluded by staining with Live Dead cell marker. A gate was set on NK cells (CD3-NK1.1+), and the percentage of TrkA+ NK cells was determined. A: kinetics of TrkA expression from day 0 to day 7 (culture in the presence of IL-2). The data shown are the means SEM of three experiments. *: p
When spleen NK cells were activated by culture in the presence of IL-2, expression of TrkA increased gradually and progressively. At day 2, half of the NK cells were TrkA+, and then the percentage increased to nearly 100% until day 5 (Fig. 2A, Fig. 2C). This maximal percentage did not change until day 7 (Fig. 2A). Expression of p75NTR was very moderately induced under these culture conditions (Fig. 1). Confocal microscopy confirmed the expression of TrkA by activated NK cells (Fig. 3). Despite the staining of a fraction of resting NK cells with the anti-TrkA Ab in flow cytometry, we could not detect TrkA-expressing fresh NK cells by confocal microscopy (Fig. 3). This suggests that the sensitivity of detection is higher in flow cytometry than in confocal microscopy.
Cells were prepared for confocal microscopy as described in Materials and Methods. TrkA, but not p75NTR, is expressed by activated NK cells (green staining). On fresh NK cells (NK Day 0), the expression level of TrkA is probably too low to be evidenced by confocal microscopy, in contrast to flow cytometry. PC12 is a rat pheochromocytoma cell line that serves as positive control, as it expresses both TrkA and p75NTR at high levels. The bar corresponds to 3 µm. Data shown are from one representative experiment out of three performed.
df19127ead