Skeletal muscle regeneration in young rats is dependent on growth hormone.

Skeletal muscle fibres have a well known ability to regenerate after different kinds of injury. This study was undertaken to establish if regenerating skeletal muscle is dependent on growth hormone (GH) in the same manner as normal, growing skeletal muscle in young rats. Muscle regeneration was achieved by injection of notexin into the soleus muscle. Initial necrosis, which included all muscle fibres, was followed by a rapid and uniform regeneration throughout the muscle. Cell proliferation was estimated by scintillation counting and autoradiography of incorporated [3H]thymidine, injected intravenously 1 h before killing, 7 or 27 days after the initiation of regeneration. GH deficiency was accomplished by hypophysectomy 4 days before the [3H]thymidine injection. Cell proliferation was diminished in both regenerating and normal muscle of the hypophysectomized rats compared to control and GH-substituted rats. After 7 days of regeneration the reduction of cell proliferation seen in hypophysectomized rats was less pronounced in the regenerating than in the normal muscle. These findings demonstrate that GH plays an important role for muscle regeneration in young rats, although other substances appear to be of greater importance during the early stages of regeneration.     

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.     

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.     

Muscle regeneration following injury can be modified in vivo by immune neutralization of basic fibroblast growth factor, transforming growth factor β1 or insulin-like growth factor I

Previous in vitro studies pointed out the role played by several growth factors (basic fibroblast growth factor or bFGF, transforming growth factor beta-1 or TGFβ1, insulin-like growth factor-I or IGF-I) on the proliferation, the differentiation and the fusion of myogenic precursor cells. We attempted to modify the muscle regeneration which follows the denervation-devascularization of extensor digitorum longus in mice, by acting on the growth factors which are possibly involved in this process. The injection of neutralizing antibodies against either bFGF or IGF-I into the muscle at the time of lesion reduced the number and diameter of regenerating myofibres, suggesting a delay in proliferation and/or fusion of activated satellite cells. The neutralization of TGFβ1 led to an increased number of small regenerating myofibres, which would be due to the promoting effects of the remaining growth factors (i.e. bFGF and IGF-I) on myoblast proliferation. These contrasted results strongly suggest that the growth factors regulate in vivo muscle regeneration and would be accessible tools for future therapy of muscular disorders.     

Heat‐Stress effects on the myosin heavy chain phenotype of rat soleus fibers during the early stages of regeneration

Introduction: We investigated heat‐stress effects on the adult myosin heavy chain (MyHC) profile of soleus muscle fibers at an early stage of regeneration. Methods: Regenerating fibers in adult rats were analyzed 2, 4, or 6 days after bupivacaine injection. Rats were heat stressed by immersion in water (42 ± 1°C) for 30 minutes 24 hours after bupivacaine injection and every other day thereafter. Results: No adult MyHC isoforms were observed after 2 days, whereas some fibers expressed only fast MyHC after 4 days. Heat stress increased fast and slow MyHC in regenerating fibers after 6 days. Regenerating fibers expressing only slow MyHC were observed only in heat‐stressed muscles. Bupivacaine injection increased the number of Pax7⁺ and MyoD⁺ satellite cells in regenerating fibers, more so in heat‐stressed rats. Conclusion: The results indicate that heat stress accelerates fast‐to‐slow MyHC phenotype conversion in regenerating fibers via activation of satellite cells. Muscle Nerve 52 : 1047–1056, 2015     

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.     

The regeneration of a hormone-sensitive muscle (levator ani) in the rat

In 1-month-old male rats the levator ani muscle was crushed and allowed to regenerate. The main questions were whether or not the course of regeneration is similar to that of limb muscles and whether or not the regenerating levator ani muscle is sensitive to the effects of testosterone. Regeneration was followed in three groups of rats: castrated, normal, and testosterone-treated. Histological analysis revealed no detectable differences between regeneration of the levator ani and other muscles. By 31 days both the gross weight and mean cross-sectional area of the muscle fibers in castrated rats were much less than in the other two groups. Testosterone-treated muscles were larger, but not significantly so, than normal regenerating muscles. Twitch and tetanic tensions were lowest in regenerating muscles from castrated rats and highest in those from hormone-treated rats. Both the contraction (time to peak) and half-relaxation times at 7 and 14 days were fastest in hormone-treated muscles and were very slow in castrated rats. By 30 days, the differences between normal and hormone-treated muscles had disappeared. It is concluded that the regenerating levator ani muscle is sensitive to the effects of testosterone.     

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.     

Higher content of insulin-like growth factor-I in dystrophic mdx mouse: potential role in the spontaneous regeneration through an electrophysiological investigation of muscle function

Insulin-like growth factor-I (IGF-I) is known to promote proliferation and differentiation of muscle cells during growth and regeneration. Both these conditions are characterized by acquisition of specialized muscle functions, such as a large macroscopic chloride conductance (GCl), a parameter that is a target of growth hormone (GH)/IGF-I axis action on skeletal muscle. The present study has been aimed at evaluating the role of IGF-I in the spontaneous regeneration occurring in hind limb muscle of dystrophic mdx mouse. IGF-I levels have been measured in hind limb muscles, plasma and liver of mdx and control mice of 8-10 weeks and 5 months of age by radioimmunoassay. In parallel the biophysical and pharmacological properties of muscle chloride channels of extensor digitorum longus (EDL) muscle fibers of mice belonging to the same age-group have been measured electrophysiologically in vitro. At 8-10 weeks of age, significantly greater amounts of IGF-I were found in plasma and hind limb muscles of mdx mice with respect to controls. Such a difference was only just detectable and no longer statistically significant at 5 months of age. No differences were found in hepatic IGF-I levels at either age. The EDL muscle fibers of mdx mice at 8-10 weeks of age were characterized by higher GCl values and by a different pharmacological sensitivity to the enantiomers of 2-(p-chlorophenoxy)-propionic acid (CPP), specific chloride channel ligands, with respect to age-matched controls. However, these differences were no longer detected at 5 months of age. Our results suggest a role of IGF-I in the high regenerative potential of muscles from mdx mice and support the hypothesis that the biophysical and pharmacological properties of chloride channels of EDL muscle fibers are sensitive indices of the action of regeneration-promoting factors on muscle function.     

Expression of Insulin-like Growth Factor-I and Related Peptides during Motoneuron Regeneration

The regulation of insulin-like growth factor-I (IGF-I) and related peptides during motoneuron regeneration was examined in the facial nerve following facial nerve transection. One to 39 days after axotomy, the mRNAs and peptides of IGF-I, type-I insulin-like growth factor receptor (IGFR), insulin-like growth factor binding proteins 1-5 (IGFBP-1-5), and glial fibrillary acidic protein (GFAP) were assayed in brain stem sections by in situ hybridization and immunohistochemistry. Relative mRNA levels of IGF-I, IGFR, IGFBP-2, and GFAP in the ipsilateral facial nucleus were highest 4-7 days after transection and declined thereafter. Double immunostaining experiments showed that both IGF-I and IGFBP-2 were localized in GFAP-positive astrocytic processes, many of which were perineuronal. Peak staining intensity was found 4-7 days after transection and immunoreactivity still was present after 21-35 days. IGFR mRNA was found in some regenerating neurons; however, IGFR peptide was not detected in these neurons or in any other cells in the facial nucleus. Our findings suggest that astrocytic production of IGF-I and IGFBP-2 may accompany regeneration of neurons undergoing retrograde changes induced by axotomy.     

Meninges play a neurotrophic role in the regeneration of vasopressin nerves after hypophysectomy

Following hypophysectomy the regenerating fibers of magnocellular neurons are known to establish new neurohemal connections with reorganized vasculatures in the median eminence, which lead to establishment of a posterior pituitary-like structure. In order to examine the role of the meninges (the pia mater and the arachnoid) in this regeneration process, we implanted the meningeal tissues obtained from neonatal rat pups into the third ventricle of the adult rats, and then hypophysectomized the host animals. Ten days after hypophysectomy, the meningeal tissue grafts were found to be densely innervated by regenerating vasopressin-immunoreactive fibers. Such fibers had dots and frequently formed large punctuations. On the contrary, few vasopressin fibers were found within the cortical tissue grafts. Further, the exposure of primary hypothalamic cell cultures to the medium conditioned by meningeal cell cultures promoted not only the survival of vasopressin-immunoreactive neurons but also the outgrowth and arborization of the neurites. The survivals of cortical and cerebellum neurons in culture were also promoted by the conditioned medium. These findings raise the possibility that the meninges play an important role in the axonal regeneration process after hypophysectomy.     

Glycosaminoglycans co-administration enhance insulin-like growth factor-I neuroprotective and neuroregenerative activity in traumatic and genetic models of motor neuron disease: a review.

In this report it is shown how glycosaminoglycans and insulin-like growth factor-I (IGF-I) promote muscle reinnervation and prevent motor neuron death in experimental models of motor neuron disease. Such effect appears to be mediated by insulin-like growth factor-1. The glycosaminoglycan moiety of proteoglycans is a constituent of the basal lamina active on nerve regeneration by means of the interaction with laminin and with several growth factors. We have previously shown that supplementation by means of subcutaneous injections of glycosaminoglycans affects neuronal degeneration and regeneration. In this study we report that following neonatal lesion of the rat sciatic nerve, glycosaminoglycan treatment promoted extensor digitorum longus muscle reinnervation with consequent improvement of muscle morphology. In saline-treated rats, reinnervation was only partial and there was a marked muscle fibre atrophy, whereas, glycosaminoglycan treatment of lesioned rats increased IGF-I mRNA and protein in the reinnervated muscle, and IGF-I and insulin-like growth factor binding protein-3 plasma levels. Similarly, treatment of lesioned rats with IGF-I promoted muscle reinnervation, and prevented muscle fibre atrophy, higher levels of IGF-I in the reinnervated muscle, of IGF-I, and insulin-like growth factor binding proteins in plasma. In the wobbler mouse IGF-I and glycosaminoglycans alone promote only a partial motor neuron survival and the preservation of forelimb function decays after 3 weeks of treatment. However when glycosaminoglycans and insulin-like growth factor are administered together the motor neuron disease in the wobbler mouse is halted and there is no more loss of motor neurons.     

Effect of cycloheximide administered to rats in early postnatal life: Prolonged inhibition of DNA synthesis in the developing brain

Administration of cycloheximide in a single dose of 0.6 mg/kg to 7-day-old rats was used to induce short-term inhibition of protein synthesis at the period of brain ‘growth spurt’. Measurement of the rate of [¹⁴C]lysine incorporation indicated that the initial inhibition of protein synthesis in the brain (by 75%) was released within about 12 h. The normal rate of protein synthesis was attained by 48 h after cycloheximide administration; there was no sign of protein synthesis stimulation. The estimation of [¹⁴C]thymidine incorporation into brain DNA showed that inhibition of DNA synthesis was greater and longer lasting in the forebrain and olfactory bulbs (by about 80%) than in the cerebellum (by about 40%). Similar differential inhibition of thymidine kinase activity was observed in the olfactory bulbs (by 75%) and cerebellum (by 30%) at 24 h after cycloheximide, suggesting that the formation of [¹⁴C]thymidine nucleotides may have been impaired. However, a retardation of DNA accumulation was found in the forebrain and cerebellum at 72 h after cycloheximide. Thus, the short-term inhibition of protein synthesis produced prolonged inhibition of DNA synthesis and altered cell proliferation in the developing brain.     

Effects of human growth hormone on peripheral nerve regeneration

Systemic administration of human growth hormone (hGH) to intact or hypophysectomized rats was found to stimulate regeneration of the crush-lesioned sciatic nerve. The IGF-I levels of serum of the treated rats did not correlate with regeneration of the nerve but hGH treatment increased the IGF-I content of the nerve. The results suggest that a GH-mediated increase of IGF-I in the sciatic nerve could be involved in the stimulatory effect of GH on neurite outgrowth.     

Notexin causes greater myotoxic damage and slower functional repair in mouse skeletal muscles than bupivacaine

Although the myotoxins bupivacaine and notexin are employed for studying processes that regulate muscle regeneration after injury, no studies have compared their efficacy in causing muscle damage or assessing functional regeneration in mouse skeletal muscles. Bupivacaine causes extensive injury in rat muscles but its effects on mouse muscles are variable. We compared functional and morphological properties of regenerating mouse extensor digitorum longus (EDL) muscles after notexin or bupivacaine injection and tested the hypothesis that muscle damage would be more extensive and functional repair less complete after notexin injection. Bupivacaine caused degeneration of 45% of fibers and reduced maximum force (Po) to 42% of control after 3 days. In contrast, notexin caused complete fiber breakdown and loss of functional capacity after 3 days (P < 0.05). At 7 and 10 days after bupivacaine, Po was restored to 65% and 71% of control, respectively, whereas Po of notexin-injected muscles was only 10% and 39% of control at these time-points, respectively (P < 0.05). At 7 and 10 days after bupivacaine, approximately 30% of fibers were centrally nucleated (regenerating), whereas notexin-injected muscles were comprised entirely of regenerating fibers (P < 0.05). The results demonstrate that notexin causes a more extensive and complete injury than bupivacaine, and is a useful model for studying muscle regeneration in mice.     

Nerve regeneration and serum levels of insulin-like growth factor-I in rats with streptozotocin-induced insulin deficiency

Peripheral nerve regeneration was studied in female Sprague-Dawley rats with streptozotocin-induced insulin deficiency. Nerve regeneration was provoked by a crush lesion on the sciatic nerve 21 days after the streptozotocin injection. The regeneration was assessed by a pinch test at different time-points after injury. The rate of regeneration in insulin-deficient animals, 2.5 mm/day, was significantly lower than in control animals, 2.9 mm/day (P less than 0.05). There was no difference in the initial delay, i.e. the period before regeneration attains a constant velocity. One group of insulin-deficient rats was treated with insulin during the regeneration period by means of implanted osmotic mini-pumps. This treatment prevented the decrease in regeneration. After 6 days the sciatic nerves of insulin-deficient rats had regenerated 12.3 +/- 0.3 mm (mean +/- S.E.M.), while the corresponding value for insulin-treated rats was 15.7 +/- 0.6 mm. (P less than 0.01). The streptozotocin-treated rats were found to have a 39% reduction in the serum level of insulin-like growth factor-I (IGF-I) compared to control rats (0.33 +/- 0.02 micrograms/ml and 0.54 +/- 0.02 micrograms/ml respectively, P less than 0.001). Insulin treatment during the regeneration period completely restored the IGF-I level back to normal.     

Adult-onset deficiency in growth hormone and insulin-like growth factor-I decreases survival of dentate granule neurons: insights into the regulation of adult hippocampal neurogenesis

Insulin-like growth factor-I (IGF-I), long thought to provide critical trophic support during development, also has emerged as a candidate for regulating ongoing neuronal production in adulthood. Whether and how IGF-I influences each phase of neurogenesis, however, remains unclear. In the current study, we used a selective model of growth hormone (GH) and plasma IGF-I deficiency to evaluate the role of GH and IGF-I in regulating cell proliferation, survival, and neuronal differentiation in the adult dentate gyrus. GH/IGF-I-deficient dwarf rats of the Lewis strain were made GH/IGF-I replete throughout development via twice daily injections of GH, and then GH/IGF-I deficiency was initiated in adulthood by removing animals from GH treatment. Bromodeoxyuridine (BrdU) labeling revealed no effect of GH/IGF-I deficiency on cell proliferation, but adult-onset depletion of GH and plasma IGF-I significantly reduced the survival of newly generated cells in the dentate gyrus. Colabeling for BrdU and markers of immature and mature neurons revealed a selective effect of GH/IGF-I deficiency on the survival of more mature new neurons. The number of BrdU-labeled cells expressing the immature neuronal marker TUC-4 did not differ between GH/IGF-I-deficient and -replete animals, but the number expressing only the marker of maturity NeuN was lower in depleted animals. Taken together, results from the present study suggest that, under conditions of short-term GH/IGF-I deficiency during adulthood, dentate granule cells continue to be produced, to commit to a neuronal fate, and to begin the process of neuronal maturation, whereas survival of the new neurons is impaired.     

IGF-I gene delivery promotes corticospinal neuronal survival but not regeneration after adult CNS injury

An unmet challenge of spinal cord injury research is the identification of mechanisms that promote regeneration of corticospinal motor axons. Recently it was reported that IGF-I promotes corticospinal axon growth during nervous system development. We therefore investigated whether IGF-I also promotes regeneration or survival of adult lesioned corticospinal neurons. Adult Fischer 344 rats underwent C3 dorsal column transections followed by grafts of IGF-I-secreting marrow stromal cell grafts into the lesion cavity. IGF-I secreting cell grafts promoted growth of raphespinal and cerulospinal axons, but not corticospinal axons, into the lesion/graft site. We then examined whether IGF-I-secreting cell grafts promote corticospinal motor neuron survival or axon growth in a subcortical axotomy model. IGF-I expression coupled with infusion of the IGF binding protein inhibitor NBI-31772 significantly prevented corticospinal motor neuron death (93% cell survival compared to 49% in controls, P < 0.05), but did not promote corticospinal axon regeneration. Coincident with observed effects of IGF-I on corticospinal survival but not growth, expression of IGF-I receptors was restricted to the somal compartment and not the axon of adult corticospinal motor neurons. Thus, whereas IGF-I influences corticospinal axonal growth during development, its application to sites of adult spinal cord injury or subcortical axotomy fails to promote corticospinal axonal regeneration under conditions that are sufficient to prevent corticospinal cell death and promote the growth of other supraspinal axons. We conclude that developmental patterns of growth factor responsiveness are not simply recapitulated after adult injury, potentially due to post-natal shifts in patterns of IGF-I receptor expression.     

Perikaryal routing of newly synthesized proteins in regenerating neurons: Quantitative electron microscopic autoradiography

Intracellular transport of newly synthesized proteins through organelles in the perikarya of regenerating goldfish retinal ganglion cells was studied using electron microscopic autoradiography. Retinas were removed 14 or 30 days after optictract cut or sham operation, pulse-labeled in [³H]proline-containing medium for 5 min, and then chase-incubated in medium containing unlabeled proline for various times up to 55 min before fixation. Fourteen days after axotomy, during rapid growth of the regenerating axons, the time course of change of relative grain density (% grains/% area) in the rough endoplasmic reticulum in regenerating cells was almost identical to that in control cells. However, the grain distribution analysis revealed an increased delivery of newly synthesized proteins to the Golgi apparatus, perikaryal plasma membrane and nucleus in regenerating cells. Thirty days after axotomy, during synaptogenesis, Golgi apparatus labeling in the regenerating cells became significantly higher than control, but the increase was delayed compared to the increase seen 14 days after axotomy. Labeling of the plasma membrane and nucleus did not rise above control in 30-day regenerating cells chase-incubated for up to 55 min. Thus the pattern of intracellular transport of newly synthesized proteins varies with the stage of axonal regeneration.