Estrogen and progesterone stimulate Schwann cell proliferation in a sex- and age-dependent manner.

The effects of estrogen and progesterone on Schwann cell proliferation were studied in cultured segments of the rat sciatic nerve from adult male, female, and newborn rats, by measurement of [3H thymidine incorporation or bromo-deoxy-uridine- (BrdU)-labelling and immunocytochemistry. Estrogen (100 nM-500 nM) enhanced [3H] thymidine incorporation in segments from male and newborn rats, while it had no effect on segments from female rats. Progesterone stimulated thymidine incorporation in segments from female and newborn rats (100 nM-500 nM), but caused only a small proliferative response in Schwann cells from male rats at high concentrations. The proliferative effects of estrogen and progesterone were blocked when the segments were cultured in the presence of inhibitors of their respective receptors, ICI 128 780 and zk 112994. The data suggest that Schwann cells possess distinct receptors for estrogen and progesterone and that these receptors may be involved in the control of Schwann cell proliferation. It also shows that the response of Schwann cells to sex hormones varies with sex and perhaps also with age.     

Time-dependent effects of insulin on Schwann cell proliferation in the in vitro regenerating adult frog sciatic nerve

The present study showed that insulin (0.01 μg/ml, ≈? 2 nM) inhibited [³H]-thymidine incorporation in support cells, most likely Schwann cells, of the cultured frog sciatic nerve. A 25–35% inhibition took place in regenerating nerve preparations as well as in preparations devoid of neuronal protein synthesis, i.e., in isolated 5 mm nerve segments and in gangliectomized nerves, suggesting that the effect was direct and not mediated via the neuronal cells. The inhibition by insulin was time-dependent in that an effect was seen after 4 days but not at shorter or at longer periods of culturing. In separate experiments biotinylated insulin was shown to be taken up by Schwann cells in the regenerating nerve. Addition of serum increased the [³H]-thymidine incorporation severalfold and abolished the inhibitory action of insulin. Our results suggest that insulin, at a certain stage of the regeneration programme, exerts a direct, inhibitory effect on the proliferation of the Schwann cells in the cultured frog sciatic nerve. © 1993 Wiley-Liss, Inc.     

Insulin-like growth factor I: A mitogen for rat schwann cells in the presence of elevated levels of cyclic AMP

To develop effective procedures for improving the regeneration of peripheral nerves and for preventing the formation of neurofibromas, it is necessary to identify the different mitogens that stimulate the proliferation of Schwann cells. Insulinlike growth factor I (IGF-I), which is a potent autocrine growth factor in many tissues, is synthesized by proliferating Schwann cells. However, the role of IGF-I in stimulating their division is still uncertain. Here we show that nanomolar concentrations of IGF-I stimulate the growth of Schwann cells in primary culture. IGF-I alone was uneffective but in the presence of forskolin (5 μM) or dibutyryl cyclic AMP (dbcAMP, 10 μM), it became a potent mitogen. Neither IGF-II nor epidermal growth factor (EGF) were effective, even in the presence of forskolin. Insulin also stimulated Schwann cell proliferation in the presence of forskolin, but only at micromolar concentration. Receptors for IGF-I were visualized on the Schwann cell surface by indirect immunofluorescence staining using anti-human IGF-I receptor antibodies. Their presence was also assessed by binding assays using [¹²⁵I]-IGF-I as a ligand. Scatchard analysis showed a single class of high-affinity receptors (K_d = 1.5 nM). Competition studies with unlabeled IGF-I or insulin indicated a half-maximal displacement of [¹²⁵I]-IGF-I by IGF-I at about 5 nM, while insulin was about 500-fold less effective. The number of binding sites for IGF-I was increased by exposing cells for 3 days to forskolin (- forskolin: about 5,100; + forskolin: about 12,200 binding sites/cell). These results suggest that forskolin increases available receptors for IGF-I, which is consistent with the synergism between cAMP and IGF-I in stimulating Schwann cell growth. © 1993 Wiley-Liss, Inc.     

Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

Schwann cell proliferation was studied in cultured segments of the rat sciatic nerve by measurement of [³H] thymidine incorporation or through bromodeoxyuridine-(BrdU)-labelling and immunocytochemistry. The aim was to delineate mechanisms involved in the injury-induced proliferative response of Schwann cells. Removal of extracellular Ca²⁺ by addition of EGTA to the culture medium suppressed [³H] thymidine incorporation as did the calmodulin inhibitor 48/80. The Ca²⁺ ionophore A23187 increased incorporation. Staurosporin, an inhibitor of protein kinase C (PKC), suppressed [³H] thymidine incorporation while phorbol-12-myristate-13-acetate (PMA) enhanced incorporation. Manipulation of the cAMP system showed that increased cAMP levels inhibited proliferation. Inhibition of protein kinase A by HA 1004 increased the incorporation of [³H] thymidine. Immunostaining for BrdU and glial specific markers together with morphological evaluation of myelin association showed that proliferation occurred in Schwann cells. The results are consistent with a model in which Schwann cell proliferation is enhanced by Ca²⁺ through activation of calmodulin-dependent and/or PKCdependent mechanisms. Inhibition is achieved through the cAMP system. Together, these results show that Schwann cells regulate proliferation differently in an integrated environment, e.g. the nerve structure, than in isolation as primary monocultures. J. Neurosci. Res. 52:530–537, 1998. © 1998 Wiley-Liss, Inc.     

Insulin and IGF-II, but not IGF-I, stimulate the in vitro regeneration of adult frog sciatic sensory axons

We used the in vitro regenerating frog sciatic nerve to look for effects of insulin and insulin-like growth factors I and II (IGF-I, IGF-II) on regeneration of sensory axons and on injury induced support cell proliferation in the outgrowth region. In nerves cultured for 11 days, a physiological dose ( 10ng/ml, ≈ 2 nM) of insulin or IGF-II increased ganglionic protein synthesis (by 20% and 50%, respectively) as well as the level of newly formed, radiolabelled axonal material distal to a crush injury (both by 80%), compared to untreated, paired controls. In addition, insulin increased the outgrowth distance of the furthest regenerating sensory axons by 10%. The preparation was particularly sensitive to insulin during the first 5 days of culturing. Furthermore, both insulin and IGF-II were found to inhibit proliferation of support cells in the outgrowth region in a manner suggesting effects via their individual receptors. The inhibition, about 30%, was observable after 4 but not 11 days in culture. It is not clear if this reflects a stimulated differentiation of some cells. By contrast, IGF-I lacked effects on both regeneration and proliferation. In conclusion, the results suggest that insulin and IGF-II are involved in the regulation of peripheral nerve regeneration.     

Chromaffin cells express two types of insulin-like growth factor receptors

The receptor binding, internalization and tyrosine kinase activation of insulin-like growth factors, IGF-I and IGF-II have been investigated in cultured adult bovine chromaffin cells. IGF-I receptor alpha-subunits (Mr approximately 130,000) bound IGF-I and IGF-II with identical affinity (Kd approximately 1 nM) and insulin with about 1000 times lower affinity. IGF-II receptors (Mr approximately 250,000) bound IGF-II with a Kd of 0.5 nM, IGF-I with about 10 times lower affinity and insulin with greater than 10,000 times lower affinity. The amounts of IGF-I and IGF-II receptors on the cell surface were 8 x 10(4) and 4 x 10(4) sites per cell, respectively. Insulin bound to a specific receptor with Kd approximately 2 nM and the amount of receptors was 1.5 x 10(4) sites per cell. IGF-I and IGF-II stimulated tyrosine kinase activity and autophosphorylation of the IGF-I receptor beta-subunit (Mr approximately 94,000) with equal potency (ED50 approximately 1 nM), whereas insulin was approximately 5 times less potent. Both IGF-I and IGF-II were internalized after their binding to cell surface receptors. Mannose-6-phosphate, which binds to the IGF-II receptor, did not alter the binding or internalization of IGF-II. It is concluded that IGF-I and IGF-II can exert their biological effects in chromaffin cells by activation of the IGF-I receptor tyrosine kinase or by interaction with the IGF-II receptor.     

Muscle regeneration

Summary Against the background of the importance of growth hormone (GH) for normal muscle growth, a study was performed to investigate whether lack of GH after hypophysectomy affects the cell proliferation and the local production of insulin-like growth factor-I (IGF-I) in the early stages of muscle regeneration in adult rats. The level of IGF-I in the serum of hypophysectomised rats was reduced to about 30% of that of controls. The incorporation of [methyl-³H]thymidine into the regenerating muscle showed a peak 6 days after the operation and then gradually declined to the end of the period of study 30 days after initiation of regeneration by ischemic necrosis. The DNA content rose to a maximum level after 6–8 days, and remained high after 30 days. There was no major difference in the incorporation of [³H]thymidine in regenerating muscle of hypophysectomised and control rats, but the DNA concentration in the regenerating muscles of hypophysectomised rats was significantly reduced after 30 days. There was a corresponding reduction in the number of nuclei per muscle fibre, indicating that hypophysectomy has a small effect on the cell proliferation during the early stages of muscle regeneration. Immunohistochemical demonstration of IGF-I in the regenerating muscle revealed the transient presence of immunoreactive material in satellite cells and myotubes after 6 to 8 days of regeneration but no immunoreactivity after 30 days. No obvious difference was observed between hypophysectomised and control rats, indicating that the endogenous production of IGF-I in regenerating skeletal muscle can occur independently of GH.Supported by the Swedish Medical Research Council (Project no. 7122 and 7120)     

Influence of non-neuronal cells on regeneration of the rat sciatic nerve

The ability of the rat sciatic nerve to regenerate into a previously frozen distal nerve segment was studied and compared to regeneration after a crush lesion. The regeneration rate in the frozen segment was 1.9 mm/day, which was approximately half of that observed after a crush lesion (3.3 mm/day). If an unfrozen nerve segment was left intact beyond the frozen section, the rate of regeneration increased to 3.2 mm/day. However, a fresh nerve segment sutured along the frozen segment did not significantly affect the rate of regeneration. Incorporation of [3H]thymidine in the regenerating nerve, analyzed after 1, 3 and 6 days, showed an increased labelling in the frozen segment. This increase spread from the proximal nerve segment into the frozen section. In nerves where a segment was left intact beyond the frozen section, [3H]thymidine incorporation was seen to enter the frozen section from both sides. The spreading of [3H]thymidine incorporation appeared to correlate with the rate of regeneration. However, the same pattern of incorporation could be observed in nerves where regeneration was detained by a transection. The results suggest that Schwann and/or other cells which invade the frozen nerve segment affect the rate of axonal elongation, and that the migration of these cells occurs independently of regenerating fibers.     

Binding of []insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain

The occurrence of insuling receptors and biological responses to insulin has been investigated in trypsin-dissociated fetal rat brain cells maintained in culture for 8 days. Binding of [¹²⁵]insulin to brain cells in culture was time- and pH-dependent and 85–90% specific. Porcine insulin competed for [¹²⁵]insulin binding in a dose-dependent manner. Unrelated polypeptides, including angiotensin II, glucagon, bovine growth hormone, and bovine prolactin did not compete for [¹²⁵]insulin binding. The half-life of [¹²⁵]insulin dissociation from receptors at 24°C was 15 min and a plot of ln[B/Bo] vs time suggested two dissociation rate constants of2.7 × 10⁻⁴ sec⁻¹ and5.0 × 10⁻⁵ sec⁻¹. Scatchard analysis of the binding data gave a curvelinear plot which may indicate negative cooperativity or the occurrence of both high affinity(K_a = 2 × 10¹¹M⁻¹) and low affinity(K_a = 4 × 10¹⁰M⁻¹) sites. Of the estimated total of 4.9 × 10⁴ binding sites per cell, 28–30% appear to be high affinity sites.Incubation of cultures with insuling caused a time- and dose-dependent stimulation of [³H]thymidine and [³H]uridine incorporation into TCA-precipitable material. Maximum stimulation of thymidine incorporation (2–5-fold) occured 11 h after incubation with 167 nM insulin. The same concentration of insulin caused a 2.2-fold increase in [³H]uridine incorporation in 2 h. These results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.     

Axolemma is a mitogen for human Schwann cells

The mechanisms responsible for the induction of Schwann cell proliferation in peripheral nerves undergoing wallerian degeneration and segmental demyelination are not understood. To determine whether contact with axolemma stimulates mitosis of human Schwann cells, cultured Schwann cells from spinal roots obtained postmortem and from sural nerve biopsy specimens were incubated with axolemmal fractions prepared from human spinal cord or from adult rat central nervous system. Schwann cell proliferation was estimated by autoradiographic assay of tritiated thymidine incorporation. Schwann cell labeling indices after exposure to human or rat axolemmal fractions ranged from 26.7 to 59.9%; labeling indices of Schwann cells cultured without axolemmal fraction were 9.8 to 22.4%. The stimulation index, or ratio of Schwann cell labeling index with axolemmal fraction to that without axolemmal fraction, ranged from 1.97 to 3.40. This study demonstrates that both human and rat axolemma are capable of stimulating human Schwann cell replication in vitro.     

Nerve growth factor receptors on cultured rat Schwann cells

Neonatal rat Schwann cells were grown in tissue culture and assayed for NGF receptors with time in culture. NGF receptor levels on freshly prepared Schwann cells (day 0) were low but increased dramatically during the first week in culture. Characterization of 125I-NGF binding to resuspended cells grown for 4 d in culture revealed that binding was not saturable at high ligand concentrations (50-70 nM) and that a high-capacity, low-affinity NGF binding component existed on these cells as compared to PC12 cells. The monoclonal antibody, 192-IgG, which recognizes the rat NGF receptor, was used as an immunohistochemical tool to verify the presence of NGF receptors on the cultured rat Schwann cells. In contrast to radiolabeled NGF, 125I-192-IgG demonstrated saturable binding to Schwann cells in suspension, with Kd and Bmax values of 4 nM and 115 fmol/10(6) cells, respectively. Schwann cells showed no evidence of slow dissociation or internalization of NGF binding at any of several NGF concentrations. 192-IgG was used to immunoprecipitate 125I-NGF chemically crosslinked to cell membranes. SDS-PAGE and subsequent autoradiography of the immunoprecipitated NGF receptors revealed that 2 species of NGF receptors were precipitated from Schwann cells and PC12 cells. In PC12 cells, 2 bands with molecular weights of 90 and 210 kDa were identified. The Schwann cell NGF receptor species migrated slower on the gels, with apparent molecular weights of 95 and 220 kDa. Further analysis of glial cell NGF receptors showed that Schwann cells isolated from the vagus nerve of neonatal rats also expressed NGF receptors in culture; however, astrocytes cultured from neonatal rat cerebral cortex, cultured under conditions reported here, were devoid of detectable NGF receptors. These results show that NGF receptor levels on Schwann cells increase with time in culture, and this resembles what is observed in Schwann cells in vivo when adult peripheral nerve is injured. The data are discussed in terms of a supportive role for the Schwann cell in facilitating peripheral nerve development and regeneration.     

Stimulation of DNA synthesis in Balbc 3T3 cells by peripheral nerve degenerating in vitro

The release of mitogenic substances from degenerating peripheral nerves was detected and characterized in vitro. Cultures of serum-starved, subconfluent $\text{Balb}\text{c}$ 3T3 cells were exposed 24 h to myelinated peripheral nerve fascicles, with [³H]thymidine added during the last 3 h. Cells exposed to peripheral nerves incorporated twice as much [³H]thymidine as control cultures without nerves (P < 0.005). Autoradiography showed a graded decrease in labeling index with increasing distance from nerves. The mitogenic response varied in a dose-dependent manner with increasing nerve length. Also, the response varied according to the degree of myelination. Myelinated sciatic nerve fascicles caused greater incorporation of [³H]thymidine (P < 0.005) than unmyelinated abdominal vagus nerves of similar size, suggesting myelin-derived growth factor activity. Evidence from other laboratories has led to the hypothesis that during peripheral nerve injury, myelin proteins are degraded by lysosome-derived acid proteinases yielding mitogenic polypeptide fragments. We report that the addition of the acid proteinase inhibitor, pepstatin, to the culture media caused a small but significant decrease (P < 0.05) in the mitogenic effect of peripheral nerves. The work supports the concept that the cell proliferation accompanying Wallerian degeneration is stimulated by mitogens released by the injured nerve.     

Neuronal stimulation of [3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-³H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [³H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [³H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [³H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [³H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [³H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.     

MMP‐9 controls Schwann cell proliferation and phenotypic remodeling via IGF‐1 and ErbB receptor‐mediated activation of MEK/ERK pathway

Phenotypic remodeling of Schwann cells is required to ensure successful regeneration of damaged peripheral axons. After nerve damage, Schwann cells produce an over 100‐fold increase in metalloproteinase‐9 (MMP‐9), and therapy with an MMP inhibitor increases the number of resident (but not infiltrating) cells in injured nerve. Here, we demonstrate that MMP‐9 regulates proliferation and trophic signaling of Schwann cells. Using in vivo BrdU incorporation studies of axotomized sciatic nerves of MMP‐9?/? mice, we found increased Schwann cell mitosis in regenerating (proximal) stump relative to wild‐type mice. Treatment of cultured primary Schwann cells with recombinant MMP‐9 suppressed their growth, mitogenic activity, and produced a dose‐dependent, biphasic, and selective activation of ERK1/2, but not JNK and p38 MAPK. MMP‐9 induced ERK1/2 signaling in both undifferentiated and differentiated (using dbcAMP) Schwann cells. Using inhibitors to MEK and trophic tyrosine kinase receptors, we established that MMP‐9 regulates Ras/Raf/MEK—ERK pathways through IGF‐1, ErbB, and PDGF receptors. We also report on the early changes of MMP‐9 mRNA expression (within 24 h) after axotomy. These studies establish that MMP‐9 controls critical trophic signal transduction pathways and phenotypic remodeling of Schwann cells. © 2009 Wiley‐Liss, Inc.     

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.     

Specific and potent mitogenic effect of axolemmal fraction on schwann cells from rat sciatic nerves in serum-containing and defined media

Using cultures of Schwann cells from neonatal rat sciatic nerves, we examined the mitogenic activity of an axolemmal fraction from adult rat CNS. Axolemmal fraction proved a potent mitogen, stimulating [³H]thymidine incorporation into Schwann cell DNA 13.5 fold over control values when axolemmal fraction equivalent to 16 μg of protein per culture microwell or more was added. Half maximal stimulation was obtained with addition of axolemmal fraction equivalent to 4 μg of protein. The concentration-dependence and magnitude of the mitogenic response of the cultured cells were nearly identical whether they were maintained in vitro for 1 day or for 2 weeks prior to addition of the axolemmal fraction. A study of the time-course of the effect of axolemmal fraction on Schwann cell mitosis showed that maximal [³H]thymidine incorporation took place during the fifth day after addition of axolemmal fraction. Axolemmal fraction also produced stimulation of [³H]thymidine incorporation into Schwann cells, seeded and cultured in a serum-free defined medium. Though the concentration-dependence of the mitogenic effect in the absence of serum was similar to that in a serum-containing medium, maximal stimulation in the defined medium was only 2.8-fold. The mitogenic activity of axolemmal fraction was rapidly and almost totally inactivated by sonication or homogenization, and was partially lost after exposure to heat. The mitogenic activities of plasma membrane fragments from rat skeletal muscle or rat erythrocytes, andof mitochondrial fragments (the major contaminant of the axolemmal fraction) were one-tenth that of axolemmal fraction or less. In contrast to glial growth factor prepared from bovine pituitaries (GGF-BP), which stimulates proliferation of both fibroblasts and Schwann cells, axolemmal fraction induced proliferation of Schwann cells but not of endoneurial fibroblasts; cultures treated with axolemmal fraction demonstrated a 3-fold increase in Schwann cell population in 10 days without detectable increase in number of fibroblasts. Also in contrast to GGF-BP, the mitogenic effect of which is considerably enhanced by simultaneous addition of cholera toxin to the medium, cholera toxin had no effect on the Schwann cell proliferative response to axolemmal fraction.     

Galectin-3 inhibits Schwann cell proliferation in cultured sciatic nerve

The production of galectin-3, a carbohydrate-binding mammalian lectin, is upregulated in Schwann cells after peripheral nerve injury in areas where Schwann cells proliferate. Here we tested if galectin-3 affected proliferation of Schwann cells in cultured sciatic nerve segments. Galectin-3 significantly decreased the number of bromodeoxyuridine-labelled Schwann cell nuclei. Neither lactose nor a synthetic inhibitor directed against the carbohydrate-binding region abolished the effects of galectin-3. In addition, a mutant galectin-3 unable to bind endogenous carbohydrates had similar effects as normal galectin-3. We conclude that galectin-3 reduces proliferation of Schwann cells in cultured sciatic nerve segments by a mechanism which is independent of its carbohydrate-binding moiety.     

Diabetic Schwann cells suffer from nerve growth factor and neurotrophin‐3 underproduction and poor associability with axons

Schwann cells (SCs) are integral to peripheral nerve biology, contributing to saltatory conduction along axons, nerve and axon development, and axonal regeneration. SCs also provide a microenvironment favoring neural regeneration partially due to production of several neurotrophic factors. Dysfunction of SCs may also play an important role in the pathogenesis of peripheral nerve diseases such as diabetic peripheral neuropathy where hyperglycemia is often considered pathogenic. In order to study the impact of diabetes mellitus (DM) upon the regenerative capacity of adult SCs, we investigated the differential production of the neurotrophic factors nerve growth factor (NGF) and neurotrophin‐3 (NT3) by SCs harvested from the sciatic nerves of murine models of type 1 DM (streptozotocin treated C57BL/6J mice) and type 2 DM (LepR^?/? or db/db mice) or non‐diabetic cohorts. In vitro, SCs from diabetic and control mice were maintained under similar hyperglycemic and euglycemic conditions respectively. Mature SCs from diabetic mice produced lower levels of NGF and NT3 under hyperglycemic conditions when compared to SCs in euglycemia. In addition, SCs from both DM and non‐DM mice appear to be incapable of insulin production, but responded to exogenous insulin with greater proliferation and heightened myelination potentiation. Moreover, SCs from diabetic animals showed poorer association with co‐cultured axons. Hyperglycemia had significant impact upon SCs, potentially contributing to the pathogenesis of diabetic peripheral neuropathy. GLIA 2013;61:1990–1999     

Effects of insulin, insulin-like growth factor-II and nerve growth factor on neurite outgrowth in cultured human neuroblastoma cells

The identification of biologically important and chemically well-defined substances that can promote axon and dendrite formation would improve present understanding of the development of the nervous system. Physiological concentrations of insulin and insulin-like growth factor-II (IGF-II) reversibly enhanced neurite outgrowth (NTO) in human neuroblastoma SH-SY5Y cells cultured in media with and without serum. Nerve growth factor (NGF), in contrast, did not enhance NTO in serum-free media. Furthermore, anti-NGF antiserum inhibited NGF but not insulin-enhanced NTO. Insulin increased [3H]leucine and [3H]uridine uptake. These increases, together with increased NTO, were inhibited by cycloheximide and actinomycin D, respectively. The inhibition of NTO by cycloheximide was reversible. Human neuroblastoma cell lines that were responsive by NTO to NGF were also responsive to insulin, with the exception of line CHP-270. Moreover, cell lines unresponsive by NTO to NGF, and to tumor promoters, were uniformly unresponsive to insulin. These findings suggest that there are common defects in distal sites, because specific NGF and tumor promotor receptors are present in these lines. Insulin increased [3H]thymidine uptake in SH-SY5Y and CHP-100 cells. However, the enhancement of NTO by insulin and IGF-II in SH-SY5Y cells was independent of the cellular proliferation rate. Our results, together with the observations of others, suggest that insulin and IGF-II may modulate NTO in the nervous system.     

Identification of key genes involved in axon regeneration and Wallerian degeneration by weighted gene co-expression network analysis

Peripheral nerve injury repair requires a certain degree of cooperation between axon regeneration and Wallerian degeneration.Therefore,investigating how axon regeneration and degeneration work together to repair peripheral nerve injury may uncover the molecular mechanisms and signal cascades underlying peripheral nerve repair and provide potential strategies for improving the low axon regeneration capacity of the central nervous system.In this study,we applied weighted gene co-expression network analysis to identify differentially expressed genes in proximal and distal sciatic nerve segments from rats with sciatic nerve injury.We identified 31 and 15 co-expression modules from the proximal and distal sciatic nerve segments,respectively.Functional enrichment analysis revealed that the differentially expressed genes in proximal modules promoted regeneration,while the differentially expressed genes in distal modules promoted neurodegeneration.Next,we constructed hub gene networks for selected modules and identified a key hub gene,Kif22,which was up-regulated in both nerve segments.In vitro experiments confirmed that Kif22 knockdown inhibited proliferation and migration of Schwann cells by modulating the activity of the extracellular signal-regulated kinase signaling pathway.Collectively,our findings provide a comparative framework of gene modules that are co-expressed in injured proximal and distal sciatic nerve segments,and identify Kif22 as a potential therapeutic target for promoting peripheral nerve injury repair via Schwann cell proliferation and migration.All animal experiments were approved by the Institutional Animal Ethics Committee of Nantong University,China(approval No.S20210322-008)on March 22,2021.