Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H]thymidine incorporation through their respective receptors

The factors that control proliferation of Schwann cells during peripheral nerve regeneration are not yet known. In this study we investigated the effects of insulin, insulin-like growth factor I and II (IGF-I and IGF-II), IGF-I analogues, and factors that interfere with their respective receptors, on [³H]thymidine incorporation into cultured nerve segments from the rat sciatic nerve. Segments cultured in nM (0.1–1.7 nM) concentrations of insulin, truncated IGF-I (tIGF-I), long R³IGF-I, or IGF-II exhibited an increase in [³H]thymidine incorporation compared with control segments. IGF-II was most potent. JB1, an IGF-I antagonist, counteracted the effects of tIGF-I and insulin. The results suggest that non-neuronal cells in the nerve segment, probably Schwann cells, possess distinct receptors for insulin, IGF-I, and IGF-II and that these receptors may be involved in the control of Schwann cell proliferation during peripheral nerve regeneration. © 1996 Wiley-Liss, Inc.     

Evidence that 4S RNA is axonally transported in normal and regenerating rat sciatic nerves

Studies in regenerating goldfish optic nerves indicate that RNA may be axonally transported during optic nerve regeneration^{14, 18, 19}. The present study was performed to determine if the axonal migration of RNA could be demonstrated during regeneration of the rat sciatic nerve.Rats, which had only the left sciatic nerve crushed 10 days earlier, were injected bilaterally with [³H]uridine into the spinal cord at segmental levels L₅ and L₆, thus labeling ventral horn cells giving rise to the sciatic nerve. Six, 14 and 20 days later rats were sacrificed by cardiac perfusion of saline followed by 10% formaldehyde. Formaldehyde-precipitable radioactivity, identified as [³H]RNA, was 4–5 times greater in the regenerating sciatic nerve compared to the normal nerve and moved without impediment beyond the point of the crush into the regenerating portion of the nerve.The axonal migration of free unincorporated labeled RNA precursors was also demonstrated, raising the possibility that the distribution of [³H]RNA along the sciatic nerve might be entirely extra-axonal; i.e., free [³H]uridine is taken up by Schwann cells from the axon where it is incorporated into [³H]RNA. This interpretation of the data would also result in the appearance of a proximodistal distribution of RNA associated radioactivity. To determine whether any sciatic nerve [³H]RNA was due to axonal transport, rats which had only the left sciatic nerve crushed 10 days earlier were injected bilaterally with [³H]uridine into the spinal cord. Fourteen days after injection, rats were sacrificed and radioactivity present in the nerve was confirmed as RNA by SDS polyacrylamide gel electrophoresis. Radioactivity in the various RNA species 14 days after intraspinal injection showed the following distribution: 28 + 18S RNA — normal39.3%±2.1; regenerating45.4%±1.6; 4S RNA — normal43.0%±1.3; regenerating46.8%±2.7. Similar characterization of sciatic nerve RNA 1 or 3 days following the intravenous administration of [³H]uridine gave the following distribution: 28 + 18S RNA — normal72.4%±3.0; regenerating75.0%±3.6; 4S RNA — normal7.7%±1.3; regenerating10.7%±0.8.The intraspinal injection of [³H]uridine would label Schwann cell RNA and, in addition, any species of intra-axonal RNA, while intravenous injections would label Schwann cell RNA and not axonal RNA. If 4S RNA is in the axon, one would predict relatively more labeled 4S RNA following intraspinal injections than following intravenous injections. The data demonstrate an enrichment of 4S RNA in both normal and regenerating rat sciatic nerve following the intraspinal but not following the intravenous injection of labeled precursor. Therefore, we suggest that 4S RNA migrates axonally in both normal and regenerating sciatic nerves of rats.     

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.     

Regulation of protease nexin-1 and angiotensin II receptor subtype 1 expression: Inverse relationship in experimental models of nerve injury

The up-regulation of PN-1 following nerve lesion has been investigated in vitro in cultures of dorsal root ganglion (DRG) explants, sciatic nerve segments, and isolated Schwann cells. In the first culture model, Schwann cells associated with neuronal processes synthesized small amounts of PN-1. Injury of the neurites emerging from the DRGs led to enhanced levels of PN-1 in Schwann cells located distal to the lesion site where degeneration of neuronal processes took place. In cultured sciatic nerve segments, PN-1 synthesis increased with a time-course comparable to that in ganglion explants following lesion. In the third model, PN-1 levels gradually rose in isolated Schwann cells during the first 3-8 days in culture. Dissociation of Schwann cells from the sciatic nerve therefore causes an effect similar to nerve damage. Impairment of Schwann cell-neuron interactions was followed by a reduction in the expression levels of the angiotensin II (Ang II) receptor subtype AT₁ in all three systems studied. Since the neuropeptide Ang II is able to repress PN-1 synthesis in cultured Schwann cells, loss of neuronal contact might decrease their responsiveness to Ang II, thus resulting in PN-1 upregulation by default. © 1995 Wiley-Liss, Inc.     

Sciatic conditioned medium increases survival, proliferation and differentiation of retinal cells in culture

Many evidences clearly demonstrate that Schwann cells provide trophic support for neurons. Different cytokines, including neurotrophins (NTs), are produced and released by Schwann cells. These trophic molecules play an important role on neuronal survival either during the development or during adult life. Cytokines have also a pivotal role on neuronal regeneration after lesions occurring during pathological conditions. The aim of this work was to study the effect of sciatic conditioned medium (SCM) on rat retinal cells maintained in culture. Our results show that treatment with SCM obtained after 14 days in vitro (SCM 14 day) induced a three-fold increase in protein content of the culture after 48 h in vitro and this value remained equally high up to 72 h. This effect was totally blocked either by addition of 30 microM BAPTA-AM, an intracellular calcium chelator, 15 microM fluorodeoxyuridine, an inhibitor of cell division, or 10 microM genistein (geni) plus 1.25 microM chelerythrine chloride (CC), the two last ones inhibitors of tyrosine kinases and protein kinase C, respectively. SCM induced an increase in [(3)H]-choline uptake and [(3)H]-thymidine incorporation of retinal cells. SCM also stimulated an increase in cytoplasmic processes outgrowth of retinal cells and survival of retinal ganglion cells. Our results clearly suggest that soluble molecules released by sciatic nerve fragments are able to increase the proliferation and survival of retinal cells in culture.     

Synthesis of progesterone in Schwann cells: regulation by sensory neurons

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3 beta-hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3 beta-hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3 beta-hydroxysteroid dehydrogenase were induced. Regulation of 3 beta-hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3 beta-hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re-expressed. After nerve transection, which does not allow axonal regeneration, 3 beta-hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3 beta-hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.     

Serotonin receptors expressed by myelinating Schwann cells in rat sciatic nerve

We have previously reported that Schwann cells cultured from rat sciatic nerves express 5-HT_2A receptors. In this study we extend these in vitro observations to Schwann cells in situ. Since the serotonin (5-HT) levels in rat sciatic nerve are elevated following nerve injury, we examined Schwann cells in healthy and injured adult rat sciatic nerves. These nerves were double-labeled immunohistochemically with an anti-idiotypic antibody that recognizes 5-HT_1B, 5-HT_2A, and 5-HT_2C receptors and an antibody against S100β, a Schwann cell marker. 5-HT receptor labeling was observed in Schwann cells of healthy and regenerating nerves, but not of degenerating nerves, while S100β labeling was observed in the Schwann cells of all nerves examined. The 5-HT receptor immunolabeling was cytoplasmic, as with the cultured Schwann cells. While staining was observed at the nodes of Ranvier, it was not restricted to these locations. These results suggest that myelinating rat Schwann cells normally express 5-HT receptors in vivo, and that receptor expression is reduced during times when 5-HT levels are elevated in the sciatic endoneurium. © 1997 Elsevier Science B.V. All rights reserved.     

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.     

Effects of protein kinase inhibitors on regeneration in vitro of adult frog sciatic sensory axons.

The effects of protein kinase inhibitors on regeneration in vitro of adult frog sciatic sensory axons were tested. Regeneration of crush-injured nerves for 8 days in serum-free medium was inhibited by staurosporine (100 nM) and H-7 (100 microM), which are both known to inhibit protein kinase C. With the use of a compartmented culture system it could be shown that H-7 exerted both local (outgrowth region) and central (ganglia) effects, the latter being more pronounced. The local effects could be due to reduction of Schwann cell proliferation by H-7. Immunohistochemistry demonstrated the presence of protein kinase C in neuronal cell bodies but not in axonal processes. Proliferation of Schwann cells was accompanied by increased protein kinase C immunoreactivity at the site of injury. H-7 caused a selective inhibition in the incorporation of radioactive phosphate into one 74 kDa protein of both ganglia and nerve but also a more general decrease in protein labelling. The results show that protein phosphorylations, possibly mediated by protein kinase C, are involved in regeneration-related mechanisms operating at both local and central levels in the adult frog sciatic sensory axons.     

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.     

Morphological evidence for a transport of ribosomes from Schwann cells to regenerating axons

Recently, we showed that Schwann cells transfer ribosomes to injured axons. Here, we demonstrate that Schwann cells transfer ribosomes to regenerating axons in vivo. For this, we used lentiviral vector-mediated expression of ribosomal protein L4 and eGFP to label ribosomes in Schwann cells. Two approaches were followed. First, we transduced Schwann cells in vivo in the distal trunk of the sciatic nerve after a nerve crush. Seven days after the crush, 12% of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was inserted into the sciatic nerve. Fluorescent ribosomes were detected in regenerating axons up to 8 weeks after graft insertion. Together, these data indicate that regenerating axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein synthesis by transferring protein synthetic machinery and mRNAs to these axons.     

Adenosine inhibition of the regeneration in vitro of adult frog sciatic sensory axons

The sensory axons of the adult frog sciatic nerve have earlier been shown to regenerate in vitro. If a local test crush is made at the initiation of culturing, regeneration starts after 3.4 days and proceeds at a rate of about 0.8–0.9 mm/day for several days. In the present experiments regeneration was inhibited by adenosine in a reversible and dose-dependent fashion. Similarly, both an adenosine analogue, 2-chloroadenosine (2-CA), and a non-hydrolyzable ATP analogue, AMP-PNP, reduced the outgrowth of sensory axons. The effect of adenosine was partially antagonized by theophylline at a critical concentration. Using a compartmental system, it could clearly be shown that adenosine exerted its effects at the outgrowth region. Adenosine, 2-CA, and AMP-PNP were also found to inhibit the proliferation of Schwann cells in the regenerating nerve. Various experiments showed that the latter can not explain the outgrowth inhibitory effects, which could be mediated by adenosine receptors associated with the elongating axons.     

Alteration of axonal microfilaments alters Schwann cell lipid metabolism

To investigate the importance of the neuronal cytoskeleton in Schwann cell metabolism, three agents acting on the microfilaments (cytochalasin D, brevine, and phalloïdin) were injected into the endoneurium of rat sciatic nerve. Sciatic nerves were removed 24 h later and separated into two pieces: the first one was the injection site and the second was from nerves located distal to the injection site. The pieces of nerve were incubated with [¹⁴C] galactose for 3 h. At the site of injection, [¹⁴C] incorporation into monogalactosyldiacylglycerol (MGDG) was perturbed by the three agents, whereas in the distal part, sulfatides and phosphatidylserine were affected by cytochalasin D and brevine. These results show that the three compounds acting on microfilaments have a different effect on Schwann cell metabolism, depending on whether incubated Schwamn cells were directly in contact with the toxin or they were only in contact with the axons affected by the toxin. In the latter case, axonal microfilaments seem to be involved in the regulation of Schwann cell metabolism.     

Isolation of activated adult Schwann cells and a spontaneously immortal Schwann cell clone.

Successful mammalian peripheral nerve regeneration is dependent on activated Schwann cells. Schwann cells facilitate neuronal regrowth through the production of tropic cell membrane molecules, neurotrophins, and extracellular matrix components. To better understand Schwann cell function in the regenerating nerve, we have designed a method of isolating proliferating adult Schwann cells from the injured rat sciatic nerve. Relying on the mitotic signal that is present after a crush injury, we can obtain sufficient numbers of dividing Schwann cells within one week of initial culture. A spontaneously immortal Schwann cell clone (iSC) was observed in and isolated from one of these primary cultures. These cells were transformed at a time of maximal Schwann cell activation in response to injury. Both the primary Schwann cells and the iSC have been characterized as Schwann cells by morphology, immunohistochemistry and gene expression.     

Axon-Schwann cell interactions regulate the expression of fibroblast growth factor-5 (FGF-5)

We screened for genes whose expression is significantly up- or downregulated during Wallerian degeneration in adult rat sciatic nerve with cDNA arrays. Fibroblast growth factor-5 (FGF-5) mRNA seemed to be induced. This was confirmed by northern blotting and in situ hybridization, as well as Western blotting for FGF-5 in axotomized nerve. Axon-Schwann cell interactions decreased the steady-state level of FGF-5 mRNA in regenerating sciatic nerves, and forskolin diminished its expression in cultured Schwann cells. We conclude that denervated Schwann cells synthesize FGF-5, which is a secreted, neuronotrophic member of the FGF family.     

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.     

Induction of myelin genes during peripheral nerve remyelination requires a continuous signal from the ingrowing axon

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were (1) down-regulated to basal levels (PO and MBP mRNAs), (2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), (3) upregulated (mRNAs encoding 2′3′-cyclic nucleotide phosphodiesterase and β-actin), or (4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons. © 1993 Wiley-Liss, Inc.     

The effects of acute morphine treatment on the incorporation of [H] l-lysine by normal and regenerating facial nucleus neurons

Although opiates significantly alter RNA and protein synthesis in a variety of neuronal cell types, their effect on the biosynthetic activity of regenerating neurons has not been investigated. In the present study, the effect of morphine on the incorporation of [³H] l-lysine into proteins of facial nucleus neurons was examined by light microscopic radioautography. Silver grains present within various compartments of normal and regenerating (3-, 7-, 14- and 21 days post-axotomy) neurons from saline-treated Wistar rats were compared with the amount present in similar cells from animals receiving 40 mg/kg morphine sulfate i.v.At 14- and 21-days post-axotomy, regenerating neurons were larger and the grain count in the emulsion over these cells was greater than that observed in normal (unoperated) neurons. In normal facial neurons, the accumulation of lysine into the nucleus and nucleolus was significantly lower 60 min after morphine administration. However, morphine's inhibition of lysine incorporation was even more pronounced in regenerating neurons. In these cells, nuclear lysine uptake was depressed at 3 and 7 days, while maximum inhibition of cytoplasmic incorporation occurred at 14-days post-axotomy. Morphine administration decreased nucleolar lysine incorporation at all survival intervals.