Article

Insulin-like growth factor (IGF-I) as a stimulator of regeneration in the freeze-injured rat sciatic nerve

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Abstract

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.

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... A role for IGF in peripheral nerve regeneration has been proposed by several authors (Hansson et al., 1986(Hansson et al., , 1987a(Hansson et al., , 1988aSjoberg and Kanje, 1989;Kanje et al., 1990). The latter authors showed that local administration of IGF-I to a crush lesion of the rat sciatic nerve stimulated nerve regeneration. ...
... Aside from embryogenesis, Schwann cells also proliferate during nerve regeneration and tumourgenesis. Evidence for a role of IGF-I in regeneration has been provided by several studies (Hansson et al., 1986(Hansson et al., , 1987aKanje et al., 1990;Sjoberg and Kanje, 1989). Cut or crush of the sciatic nerve resulted in increased Schwann cell IGF-I immunoreactivity within 1 day of injury, the levels remaining high for several weeks (Hansson et al., 1986(Hansson et al., , 1987a. ...
... In this regard IGF-I has been shown to play a role in neurite outgrowth, and neuronal survival in vitro (Caroni and Grandes, 1990;Torres-Aleman et al., 1990;Svrzic and Schubert, 1990). Further evidence for an in vivo role of IGF-I in stimulating Schwann cell proliferation is provided by Sjoberg and Kanje, (1989) and Kanje et al., (1990). In these studies IGF-I perfusion was shown to enhance the regeneration rates of freeze injured sciatic nerves. ...
Thesis
The factors required to stimulate short-term Schwann cell DNA synthesis in serum-free medium (containing insulin) are thoroughly analysed and compared with the growth factor requirements of long-term ''autocrine" Schwann cells. Evidence is presented to demonstrate the importance of cAMP in Schwann cell DNA synthesis. Measurements of intracellular cAMP levels by radioimmunoassay in both Schwann cell types is related to the differing mitogen requirements of these cells. Further tissue culture studies show that insulin-like growth factor-I (IGF-I) plays a role in Schwann cell DNA synthesis, and that cultured Schwann cells are IGF immunoreactive. Binding studies and Scatchard analysis using 125I-IGF-I provide evidence for a type 1 IGF receptor on cultured Schwann cells. An immunohistochemical analysis on the distribution of IGF in vivo in the rat sciatic nerve is reported. Dried cell preparations and and 2hr cultures are used to document the presence of IGF in the Schwann cells from embryonic to adult rats. Teased nerve preparations from postnatal rats are used to study the distribution of IGF in neurones. Results obtained suggest that IGF-I may be acting as an autocrine/paracrine Schwann cell mitogen in vivo. An immunohistochemical investigation reveals that the growth associated protein GAP-43, hitherto considered to be associated with neurones and certain CNS glia, is present in non-myelin-forming Schwann cells. Nerve sections and dried cell preparations are used to document the developmental distribution of GAP-43 in Schwann cells in vivo. Further analyses on the developmental regulation of GAP-43 expression in Schwann cells has been carried out in tissue culture. Gel electrophoresis and western blotting techniques are used to confirm the identity of Schwann cell GAP-43. Evidence that GAP-43 is widely distributed in the neurones of the adult peripheral nervous system (PNS) is provided. Immunohistochemical studies and western blotting demonstrate that GAP-43 is present in high amounts in all three sub divisions of the autonomic nervous system (ANS) of the adult rat. Taken together with known distribution of GAP-43 in areas of the CNS associated with plasticity, these findings suggest a role for GAP-43 in the plasticity of the ANS.
... IGFs enhance tissue regeneration, including pancreas (Smith et al., 1991), muscle (Jennische and Matejka, 1992), and kidney (Matejka and Jennische, 1992; Verstrepen et al., 1993). In the nervous system, IGF infusion enhances motor nerve regeneration (Sjoberg and Kanje, 1989; Near et al., 1992) and, in vitro, stimulates regeneration of adult sensory neurons (Fernyhough et a!., 1993). ...
... The lOP family of ligands, receptors, and binding proteins is instrumental in normal nervous system development (Daughaday and Rotwein, 1989; Liu et al., 1991) and in the repair of nervous tissues (Sjoberg and Kanje, 1989; Jennische and Matejka, 1992; Near et al., 1992; Fernyhough et al., 1993). In this study, we examined the pattem of lOP-I and lOP-I receptor gene expression after sciatic nerve transection. ...
Article
The insulin-like growth factors (IGFs) are trophic factors whose growth-promoting actions are mediated via the IGF-I receptor and modulated by six IGF binding proteins (IGFBPs). In this study, we observed increased transcripts of both IGF-I and IGF-I receptor after rat sciatic nerve transection. Schwann cells (SCs) were the main source of IGF-I and IGFBP-5 immunoreactivity until 7 days after nerve transection, when invading macrophages in the distal nerve stumps were strongly IGF-I positive. In vitro, IGF-I promoted SC mitogenesis. Northern analysis revealed that SCs expressed IGF-I receptor and IGFBP-5. IGF-I treatment increased the intensity of IGFBP-5 without affecting gene expression. Des(1–3)IGF-I, an IGF-I analogue with low affinity for IGFBP, had no such effect. Incubation of recombinant human IGFBP-5 with SC conditioned media revealed IGF-I protection of IGFBP-5 from proteolysis, implying the presence of an IGFBP-5 protease in SC conditioned media. Collectively, these data support the concept that, in response to nerve injury, invading macrophages produce IGF-I and SC express the IGF-I receptor, to facilitate regeneration. This regenerative process may be augmented further by the ability of SC to secrete IGFBPs, which in turn may increase local IGF-I bioavailability.
... Oppenheim and coworkers also demonstrated that IGF-I prevented the programmed death of motoneurons during development . In vivo, the IGFs promote the regeneration of both motor and sensory neurons following sciatic nerve injury Sjoberg and Kanje, 1989;Near et al., 1992;Vergara et al., 1993;Contreras et al., 1995). However while demonstrating that IGFs can function as neurotrophic factors, these studies do not address whether the IGFs play a role during the normal development and functioning of the adult nervous system. ...
... IGF-I might act directly on DRG sensory neurons and spinal cord motor neurons to exert its function as a neurotrophic factor to promote axonal outgrowth and axonal thickening. Supporting evidence for this first model come from previous observations that IGF-I facilitates axonal regeneration following nerve injury in vivo Sjoberg and Kanje, 1989;Near et al., 1992;Lewis et al., 1993;Vergara et al., 1993;Contreras et al., 1995). Alternatively, IGF-I might act on other cells within the peripheral nerves-in particular, the Schwann glial cells, which may indirectly influence peripheral nerve fiber growth. ...
Article
Although insulin-like growth factor-I (IGF-I) can act as a neurotrophic factor for peripheral neurons in vitro and in vivo following injury, the role IGF-I plays during normal development and functioning of the peripheral nervous system is unclear. Here, we report that transgenic mice with reduced levels (two genotypes: heterozygous Igf1+/− or homozygous insertional mutant Igf1m/m) or totally lacking IGF-I (homozygous Igf1−/−) show a decrease in motor and sensory nerve conduction velocities in vivo. In addition, A-fiber responses in isolated peroneal nerves from Igf1+/− and Igf1−/− mice are impaired. The nerve function impairment is most profound in Igf1−/− mice. Histopathology of the peroneal nerves in Igf1−/− mice demonstrates a shift to smaller axonal diameters but maintains the same total number of myelinated fibers as Igf1+/+ mice. Comparisons of myelin thickness with axonal diameter indicate that there is no significant reduction in peripheral nerve myelination in IGF-I–deficient mice. In addition, in Igf1m/m mice with very low serum levels of IGF-I, replacement therapy with exogenous recombinant hIGF-I restores both motor and sensory nerve conduction velocities. These findings demonstrate not only that IGF-I serves an important role in the growth and development of the peripheral nervous system, but also that systemic IGF-I treatment can enhance nerve function in IGF-I–deficient adult mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 142–152, 1999
... In contrast, local delivery of IGF-1 can avoid systemic distribution and daily parenteral injections while allowing for controlled dose titration to achieve maximal efficacy. Local infusion of IGF-1 at the site of nerve injury enhances axonal regeneration, muscle reinnervation, and return of motor function in animal models for nerve injury-and-repair [10,[26][27][28][29][30]. However, these studies relied on implanted infusion pumps to achieve sustained, local delivery of IGF-1, which is impractical and unlikely to be adopted for human clinical use. ...
... Consistent with prior translational studies assessing local IGF-1 delivery via mini-pump [10,[26][27][28][29][30], our findings of greater reinnervation of motor endplates and improved functional recovery suggest improved axonal regeneration with IGF-1 NP treatment. However, retrograde labelling would be helpful in future studies to delineate the number of motor neurons contributing to end-organ reinnervation, as IGF-1 is known to promote sprouting into muscle [49] and motor unit expansion may contribute to these observations. ...
Article
Functional recovery following peripheral nerve injury is limited by progressive atrophy of denervated muscle and Schwann cells (SCs) that occurs during the long regenerative period prior to end-organ reinnervation. Insulin-like growth factor 1 (IGF-1) is a potent mitogen with well-described trophic and anti-apoptotic effects on neurons, myocytes, and SCs. Achieving sustained, targeted delivery of small protein therapeutics remains a challenge. We hypothesized that a novel nanoparticle (NP) delivery system can provide controlled release of bioactive IGF-1 targeted to denervated muscle and nerve tissue to achieve improved motor recovery through amelioration of denervation-induced muscle atrophy and SC senescence and enhanced axonal regeneration. Biodegradable NPs with encapsulated IGF-1/dextran sulfate polyelectrolyte complexes were formulated using a flash nanoprecipitation method to preserve IGF-1 bioactivity and maximize encapsulation efficiencies. Under optimized conditions, uniform PEG-b-PCL NPs were generated with an encapsulation efficiency of 88.4%, loading level of 14.2%, and a near-zero-order release of bioactive IGF-1 for more than 20 days in vitro. The effects of locally delivered IGF-1 NPs on denervated muscle and SCs were assessed in a rat median nerve transection-without- repair model. The effects of IGF-1 NPs on axonal regeneration, muscle atrophy, reinnervation, and recovery of motor function were assessed in a model in which chronic denervation is induced prior to nerve repair. IGF-1 NP treatment resulted in significantly greater recovery of forepaw grip strength, decreased denervation-induced muscle atrophy, decreased SC senescence, and improved neuromuscular reinnervation.
... Multiple studies have found that following PNI, IGF-1 increases axon number and maintains SC proliferation at near-normal levels while also enhancing NMJ recovery to promote end-organ reinnervation (Caroni and Grandes, 1990;Kanje et al., 1991;Apel et al., 2010;Emel et al., 2011;Bayrak et al., 2017). Studies administering anti-IGF-1 antibodies to a sciatic nerve crush model further validated the role of IGF-1 in PNI, finding a diminished capacity for regeneration Sjoberg and Kanje, 1989). ...
... Mini-osmotic pumps provide a sustained, local delivery of exogenous IGF-1 ( Table 5; Kanje et al., 1989;Sjoberg and Kanje, 1989;Ishii and Lupien, 1995;Tiangco et al., 2001;Fansa et al., 2002;Apel et al., 2010;Luo et al., 2016). This technique involves subcutaneous implantation of an osmotic pump in the abdomen with extension of a catheter from the pump to the transected nerve site. ...
Article
Full-text available
Patients who sustain peripheral nerve injuries (PNIs) are often left with debilitating sensory and motor loss. Presently, there is a lack of clinically available therapeutics that can be given as an adjunct to surgical repair to enhance the regenerative process. Insulin-like growth factor-1 (IGF-1) represents a promising therapeutic target to meet this need, given its well-described trophic and anti-apoptotic effects on neurons, Schwann cells (SCs), and myocytes. Here, we review the literature regarding the therapeutic potential of IGF-1 in PNI. We appraised the literature for the various approaches of IGF-1 administration with the aim of identifying which are the most promising in offering a pathway toward clinical application. We also sought to determine the optimal reported dosage ranges for the various delivery approaches that have been investigated.
... However, Pio is not specific to the PPAR-γ pathway, and more recent studies have demonstrated that some of Pio's beneficial effects come from activation of the insulin-like growth factor-1 (IGF-1) pro-proliferative pathway of Ras/Raf/ MEK (MAPK/ERK kinases)/ERK (extracellular signalregulated kinases), with Ras being a small G protein and Raf a protein kinase [14,15]. Furthermore, other laboratories have revealed beneficial effects of IGF-1 administration on peripheral nerve regeneration, including the CN in the rat after cryoablation [16][17][18]. Therefore, the purpose of this experiment was to determine if the IGF-1 pathway had a significant role in the erectile function recovery of rats post-BCNI. ...
... This suggests that Pio improves erectile function recovery by stimulating the IGF-1 pathway shown in Figure 4. This is consistent with other literature that has documented that IGF-1 is beneficial for peripheral nerve regeneration in rat models of nerve injury [16,17]. The IGF-1R is known to have two major downstream pathways, one for metabolism and one for proliferation. ...
Article
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To determine if the insulin-like growth factor-1 (IGF-1) pathway is involved in the improvement in erectile function recovery in rats after nerve crush injury treated with pioglitazone (Pio). Sprague-Dawley rats were divided into four groups. The first group received sham operation (n = 5). The second group underwent bilateral cavernous nerve injury (BCNI, n = 7). The third group received BCNI and Pio treatment (BCNI + Pio, n = 7), whereas the fourth group underwent BCNI with Pio treatment and IGF-1 inhibition (BCNI + Pio + JB-1, n = 7). The IGF-1 receptor (IGF-1R) was inhibited by JB-1, a small molecular antagonist of the receptor. After 14 days of treatment, erectile function was measured via intracorporal pressure normalized to mean arterial pressure (ICP/MAP) and the major pelvic ganglion and cavernous nerve harvested for western blot and immunohistochemistry (IHC) of phosphorylated-IGF-1Rβ (p-IGF-1Rβ), phosphorylated-ERK1/2 (p-ERK1/2), and neuronal NOS (nNOS). BCNI + Pio animals exhibited improvements in ICP/MAP, similar to Sham animals, and BCNI + Pio + JB-1 rats demonstrated a reduced ICP/MAP similar to BCNI-only rats at all measured voltages. Western blot results showed upregulation of p-IGF-1Rβ was observed in the BCNI + Pio group. Low levels of p-ERK1/2 were seen in the JB-1-treated animals. The immunoblot results were supported by IHC findings. Intense IHC staining of nNOS was detected in the BCNI + Pio group. The group treated with JB-1 showed minimal protein expression of p-ERK1/2, nNOS, and p-IGF-1Rβ. Pio improves erectile function in rats undergoing BCNI via an IGF-1-mediated pathway.
... VEGF is a potent angiogenic factor or neurotrophic factor, that can not only stimulate angiogenesis and provide directions for the migration of SCs, but also promote neurite outgrowth and SC proliferation and activate the FLK-1 pathway to enhance nerve survival [40,47] (Fig. 18). IGF stimulates nerve regeneration by promoting the synthesis of proteins and lipids necessary for nerve regeneration [48]. TGF-b, PDGF, and FGF act as mitogens of SCs [49]. ...
Article
Full-text available
Background: Developing biocompatible nerve conduits that accelerate peripheral nerve regeneration, lengthening and functional recovery remains a challenge. The combined application of nerve microtissues and platelet-rich plasma (PRP) provides abundant Schwann cells (SCs) and various natural growth factors and can compensate for the deficiency of SCs in the nerve bridge, as well as the limitations of applying a single type of growth factor. Multimodal ultrasound evaluation can provide additional information on the stiffness and microvascular flow perfusion of the tissue. This study was designed to investigate the effectiveness of a novel tissue-engineered nerve graft composed of an autogenous vein, nerve microtissues and PRP in reconstructing a 12-mm tibial nerve defect and to explore the value of multimodal ultrasound techniques in evaluating the prognosis of nerve repair. Methods: In vitro, nerve microtissue activity was first investigated, and the effects on SC proliferation, migration, factor secretion, and axonal regeneration of dorsal root ganglia (DRG) were evaluated by coculture with nerve microtissues and PRP. In vivo, seventy-five rabbits were equally and randomly divided into Hollow, PRP, Micro-T (Microtissues), Micro-T + PRP and Autograft groups. By analysing the neurological function, electrophysiological recovery, and the comparative results of multimodal ultrasound and histological evaluation, we investigated the effect of these new nerve grafts in repairing tibial nerve defects. Results: Our results showed that the combined application of nerve microtissues and PRP could significantly promote the proliferation, secretion and migration of SCs and the regeneration of axons in the early stage. The Micro-T + PRP group and Autograft groups exhibited the best nerve repair 12 weeks postoperatively. In addition, the changes in target tissue stiffness and microvascular perfusion on multimodal ultrasound (shear wave elastography; contrast-enhanced ultrasonography; Angio PlaneWave UltrasenSitive, AngioPLUS) were significantly correlated with the histological results, such as collagen area percentage and VEGF expression, respectively. Conclusion: Our novel tissue-engineered nerve graft shows excellent efficacy in repairing 12-mm defects of the tibial nerve in rabbits. Moreover, multimodal ultrasound may provide a clinical reference for prognosis by quantitatively evaluating the stiffness and microvescular flow of nerve grafts and targeted muscles.
... Following peripheral nerve injury, the local or systemic delivery of IGF-1 improves the rate of sciatic nerve regeneration in age matched control or streptozotocin (STZ)-induced type 1 diabetic rats [5,17,18]. Hyperalgesia was also prevented/reversed in STZ-induced diabetic rats treated with IGF-1 [19]. Lumbar intrathecal injection of IGF-1 reversed indices of neuropathy including the deficit in intraepidermal nerve fiber (IENF) density, sural nerve axonal degeneration, and reduced sensory and motor nerve conduction velocities in STZ-induced type 1 diabetic rats [20,21]. ...
Article
Full-text available
Objective Diabetic sensorimotor polyneuropathy (DSPN) affects approximately half of diabetic patients leading to significant morbidity. There is impaired neurotrophic growth factor signaling, AMP-activated protein kinase (AMPK) activity and mitochondrial function in dorsal root ganglia (DRG) of animal models of type 1 and type 2 diabetes. We hypothesized that sub-optimal insulin-like growth factor 1 (IGF-1) signaling in diabetes drives loss of AMPK activity and mitochondrial function, both contributing to development of DSPN. Methods Age-matched control Sprague-Dawley rats and streptozotocin (STZ)-induced type 1 diabetic rats with/without IGF-1 therapy were used for in vivo studies. For in vitro studies, DRG neurons from control and STZ-diabetic rats were cultured and treated with/without IGF-1 in the presence or absence of inhibitors or siRNAs. Results Dysregulation of mRNAs for IGF-1, AMPKα2, ATP5a1 (subunit of ATPase), and PGC-1β occurred in DRG of diabetic vs. control rats. IGF-1 up-regulated mRNA levels of these genes in cultured DRGs from control or diabetic rats. IGF-1 treatment of DRG cultures significantly (P < 0.05) increased phosphorylation of Akt, P70S6K, AMPK and acetyl-CoA carboxylase (ACC). Mitochondrial gene expression and oxygen consumption rate (spare respiratory capacity), ATP production, mtDNA/nDNA ratio and neurite outgrowth were augmented (P < 0.05). AMPK inhibitor, Compound C, or AMPKα1-specific siRNA suppressed IGF-1 elevation of mitochondrial function, mtDNA and neurite outgrowth. Diabetic rats treated with IGF-1 exhibited reversal of thermal hypoalgesia and, in a separate study, reversed the deficit in corneal nerve profiles. In diabetic rats, IGF-1 elevated the levels of AMPK and P70S6K phosphorylation, raised Complex IV-MTCO1 and Complex V-ATP5a protein expression, and restored the enzyme activities of Complex IV and I in the DRG. IGF-1 prevented TCA metabolite build-up in nerve. Conclusions In DRG neuron cultures IGF-1 signals via AMPK to elevate mitochondrial function and drive axonal outgrowth. We propose that this signaling axis mediates IGF-1-dependent protection from distal dying-back of fibers in diabetic neuropathy.
... The historical scientific development has granted a previously undiscovered role to insulin-like growth factors in peripheral nerve regeneration, to detect the presence of receptors for IGF, located in the growing points and along the axis of the axons [13]. Local infusion of IGF-I has been shown to increase the distance of regeneration of sensory axons injured rat sciatic nerves [14,15]. ...
Article
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This project aims to study the regeneration of non-repairable lesions of peripheral nerve by muscle grafts enhanced with growth factors. The experiment was carried out in two phases. The first one compared direct suture of a critical defect in the sciatic nerve of ten rats, with the interposition of autologous muscle graft, denatured by heat, in another ten. The second phase compared ten rats with nerve repair using an acellular muscle graft, with injection of 2cc of IGF-1 (10mg/ml mecasermin, Injectable solution) into the acellular graft of another ten. A clinical and functional follow-up was carried out including, ambulation, footprint measurement, and «Grasping Test». The animals were sacrificed at 90-100 days, and samples obtained for macro- and microscopic studies with toluidine blue, haematoxylin-eosin and Masson's trichrome staining. The first experiment showed the characteristic findings of nerve tissue in muscle graft level sections. The second was an enhancement of the results: post-surgical clinical improvement, early ambulation, decrease in the rate of pressure ulcers in toes, recovery of the footprint, and increasing the percentage of nerve endings in distal sciatic regeneration (47-62%). In this study the experimental and clinical possibilities of nerve defect repair by denatured muscle are demonstrated, confirming the suitability of the technique. Furthermore, it confirms our hypothesis with clinical and cellular determinations enriched by the addition of growth factors that promote nerve regeneration.
... The historical scientific development has granted a previously undiscovered role to insulin-like growth factors in peripheral nerve regeneration, to detect the presence of receptors for IGF, located in the growing points and along the axis of the axons [13]. Local infusion of IGF-I has been shown to increase the distance of regeneration of sensory axons injured rat sciatic nerves [14,15]. ...
... IGF1 and IGF2 are elevated in denervated Schwann cells prior to the invasion of strongly IGF1 positive macrophages (Aperghis et al., 2004;Cheng et al., 1996;English, 2003;Glazner et al., 1993;Glazner et al., 1994;Ishii et al., 1994;Streppel et al., 2002). This elevated expression has positive effects on nerve regeneration that include increasing Schwann cell viability (Kanje et al., 1991;Meier et al., 1999;Near et al., 1992;Syroid et al., 1999) and myelination (Chattopadhyay and Shubayev, 2009;Liang et al., 2007), promoting outgrowth of motor nerves (Lewis et al., 1993;Vergara et al., 1993), increasing regeneration rate (Hansson et al., 1986;Kanje et al., 1991;Pu et al., 1999;Sjoberg and Kanje, 1989;Xu et al., 2004) and enhancing axonal regeneration in chronically injured neurons (Houle et al., 1996). 4.8. ...
Article
After peripheral nerve injury, recovery of motor performance negatively correlates with the poly-innervation of neuromuscular junctions (NMJ) due to excessive sprouting of the terminal Schwann cells. Denervated muscles produce short-range diffusible sprouting stimuli, of which some are neurotrophic factors. Based on recent data that vibrissal whisking is restored perfectly during facial nerve regeneration in blind rats from the Sprague Dawley (SD)/RCS strain, we compared the expression of brain derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), insulin growth factors 1 and 2 (IGF1, IGF2) and nerve growth factor (NGF) between SD/RCS and SD-rats with normal vision but poor recovery of whisking function after facial nerve injury. To establish which trophic factors might be responsible for proper NMJ-reinnervation, the transected facial nerve was surgically repaired (facial-facial anastomosis, FFA) for subsequent analysis of mRNA and proteins expressed in the levator labii superioris muscle. A complicated time course of expression included (1) a late rise in BDNF protein that followed earlier elevated gene expression, (2) an early increase in FGF2 and IGF2 protein after 2 days with sustained gene expression, (3) reduced IGF1 protein at 28 days coincident with decline of raised mRNA levels to baseline, and (4) reduced NGF protein between 2 and 14 days with maintained gene expression found in blind rats but not the rats with normal vision. These findings suggest that recovery of motor function after peripheral nerve injury is due, at least in part, to a complex regulation of lesion-associated neurotrophic factors and cytokines in denervated muscles. The increase of FGF-2 protein and concomittant decrease of NGF (with no significant changes in BDNF or IGF levels) during the first week following FFA in SD/RCS blind rats possibly prevents the distal branching of regenerating axons resulting in reduced poly-innervation of motor endplates.
... IGF-1 therapy also augments peripheral nerve regeneration and neuroprotection. For example, IGF-1 peptide improved nerve regeneration in both normal and streptozotocin (STZ)-induced type 1 diabetic rats after sciatic nerve crush [13][14][15]. Intrathecal delivery of IGF-1 reversed sensory and motor nerve conduction velocity deficits and prevented intra-epidermal nerve fiber (IENF) loss and axonal degeneration in the sural nerve of STZ-induced diabetic rats [16,17]. Furthermore, overexpression of IGF-binding protein 5 (an intrinsic IGF-1 inhibitor) or depletion of IGF1R promoted a neurodegeneration phenotype in mice that resembled the nerve damage observed in humans with diabetic neuropathy [18]. ...
Article
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Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer’s disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly ( P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPβ, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPβ overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPβ can be a promising therapeutic approach.
... Following sciatic nerve crush injury, infusion of IGF-1 locally and at dorsal root ganglia improved axonal regeneration, while regeneration was diminished by administration of anti-IGF-1 antibodies. [40,41] Local administration of IGF-1 has also been shown to enhance axonal regeneration, neuromuscular junction reinnervation, and return of motor function following nerve transection and repair. [42][43][44] The efficacy of IGF-1 in enhancing peripheral nerve regeneration and muscle reinnervation was found to be equivalent in young and aging rats. ...
Article
Introduction: Peripheral nerve injuries often result in debilitating motor and sensory deficits. There are currently no therapeutic agents that are clinically available to enhance the regenerative process. Following surgical repair, axons often must regenerate long distances to reach and reinnervate distal targets. Progressive atrophy of denervated muscle and Schwann cells (SCs) prior to reinnervation contributes to poor outcomes. Growth hormone (GH)-based therapies have the potential to accelerate axonal regeneration while at the same time limiting atrophy of muscle and the distal regenerative pathway prior to reinnervation. Areas covered: In this review, we discuss the potential mechanisms by which GH-based therapies act on the multiple tissue types involved in peripheral nerve regeneration to ultimately enhance outcomes, and review the pertinent mechanistic and translational studies that have been performed. We also address potential secondary benefits of GH-based therapies pertaining to improved bone, tendon and wound healing in the setting of peripheral nerve injury. Expert opinion: GH-based therapies carry great promise for the treatment of peripheral nerve injuries, given the multi-modal mechanism of action not seen with other experimental therapies. A number of FDA-approved drugs that augment the GH axis are currently available, which may facilitate clinical translation.
... In human aortic cells, PGZ has been demonstrated to upregulate IGF-1 independently of PPAR-gamma [20] . IGF-1 has been demonstrated to stimulate nerve regeneration and survival in models of sciatic nerve injury and CNS neuronal injury after hypoxemic injury [21] . Therefore, it is possible that an IGF-1 dependent mechanism may play a role in the neuroprotective and regenerative properties of PGZ. ...
Article
Full-text available
Cavernosal nerve injury is a common complication after radical prostatectomy and causes erectile dysfunction (ED). Our recent publication established that pioglitazone (PGZ) improves cavernosal nerve function after crush injury in the rat model by both neural protection and neuroregeneration. This result is clinically significant for the many men who undergo treatment for localized prostate cancer. A better understanding of the effects of PGZ on pelvic ganglion neurons after cavernosal nerve injury is warranted. In this Research Highlight, we discuss the implications of our investigation from a molecular and clinical perspective.
... IGF1 and IGF2 expression increases in denervated Schwann cells prior to the invasion of strongly IGF1 -positive macrophages (Aperghis et al., 2004;Cheng et al., 1996b;English, 2003;Glazner et al., 1993Glazner et al., , 1994Ishii et al., 1994;Streppel et al., 2002). This elevated IGF expression has positive effects on nerve regeneration that include increasing Schwann cell viability (Kanje et al., 1991;Meier et al., 1999;Near et al., 1992;Syroid et al., 1999) and myelination of regenerated axons (Chattopadhyay and Shubayev, 2009;Liang et al., 2007), promoting outgrowth of motor nerves (Lewis et al., 1993;Vergara et al., 1993), increasing rate of regeneration (Hansson et al., 1986;Kanje et al., 1991;Pu et al., 1999;Sjoberg and Kanje, 1989;Xu et al., 2004), and enhancing axonal regeneration in chronically injured neurons (Houle et al., 1996). ...
Article
It is well-known that, after nerve transection and surgical repair, misdirected regrowth of regenerating motor axons may occur in three ways. The first way is that the axons enter into endoneurial tubes that they did not previously occupy, regenerate through incorrect fascicles and reinnervate muscles that they did not formerly supply. Consequently the activation of these muscles results in inappropriate movements. The second way is that, in contrast with the precise target-directed pathfinding by elongating motor nerves during embryonic development, several axons rather than a single axon grow out from each transected nerve fiber. The third way of misdirection occurs by the intramuscular terminal branching (sprouting) of each regenerating axon to culminate in some polyinnervation of neuromuscular junctions, i.e. reinnervation of junctions by more than a single axon. Presently, "fascicular" or "topographic specificity" cannot be achieved and hence target-directed nerve regeneration is, as yet, unattainable. Nonetheless, motor and sensory reinnervation of appropriate endoneurial tubes does occur and can be promoted by brief nerve electrical stimulation. This review considers the expression of neurotrophic factors in the neuromuscular system and how this expression can promote functional recovery, with emphasis on the whisking of vibrissae on the rat face in relationship to the expression of the factors. Evidence is reviewed for a role of neurotrophic factors as short-range diffusible sprouting stimuli in promoting complete functional recovery of vibrissal whisking in blind Sprague Dawley (SD)/RCS rats but not in SD rats with normal vision, after facial nerve transection and surgical repair. Briefly, a complicated time course of growth factor expression in the nerves and denervated muscles include (1) an early increase in FGF2 and IGF2, (2) reduced NGF between 2 and 14days after nerve transection and surgical repair, (3) a late rise in BDNF and (4) reduced IGF1 protein in the denervated muscles at 28days. These findings suggest that recovery of motor function after peripheral nerve injury is due, at least in part, to a complex regulation of nerve injury-associated neurotrophic factors and cytokines at the neuromuscular junctions of denervated muscles. In particular, the increase of FGF2 and concomittant decrease of NGF during the first week after facial nerve-nerve anastomosis in SD/RCS blind rats may prevent intramuscular axon sprouting and, in turn, reduce poly-innervation of the neuromuscular junction.
... Insulin pathway affects regeneration DAF-2 (insulin receptor) dependent activation of DAF-16 regulates age-dependent inhibition of GABA motor neuron axon regeneration in parallel or upstream of DLK-1 pathway IGF-1 (insulin growth factor) stimulates injured segment of rat sciatic nerve regeneration Byrne et al., 2014;Sjoberg and Kanje, 1989 EFA-6 ...
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Regeneration and wound healing are complex processes that allow organs and tissues to regain their integrity and functionality after injury. Wound healing, a key property of epithelia, involves tissue closure that in some cases leads to scar formation. Regeneration, a process rather limited in mammals, is the capacity to regrow (parts of) an organ or a tissue, after damage or amputation. What are the properties of organs and the features of tissue permitting functional regrowth and repair? What are the cellular and molecular mechanisms underlying these processes? These questions are crucial both in fundamental and applied contexts, with important medical implications. The mechanisms and cells underlying tissue repair have thus been the focus of intense investigation. The last decades have seen rapid progress in the domain and new models emerging. Here, we review the fundamental advances and the perspectives that the use of C. elegans as a model have brought to the mechanisms of wound healing and cellular plasticity, axon regeneration and transdifferentiation in vivo.
... While the present review focuses mainly on the physiological role of IGF-1 in the CNS, and lesioninduced plasticity and IGF1 was discussed above, a specific note on its neuroprotective and regenerative roles post-injury is prudent. In accordance with the role of IGF-1 as a molecular multi-tasker, it has crucial roles to play in neural and axonal regeneration in the injured CNS, effects teased out in the literature over the past quarter of a century Sjoberg and Kanje, 1989;Nachemson et al., 1990). In models of diffuse injury to the CNS such as Traumatic Brain Injury (TBI) and local injury such as direct nerve injuries such as transection and crush injuries, IGF-1 has emerged as a crucial neuroprotective and regenerative factor in the nervous system and as an essential mediator in neural and axonal regeneration. ...
Article
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Insulin-Like Growth Factor 1 (IGF-1) is a phylogenetically ancient neurotrophic hormone with crucial roles to play in CNS development and maturation. Recently, IGF-1 has been shown to have potent effects on cellular neuroplasticity. Neuroplasticty refers to the adaptive changes made by the CNS in the face of changing functional demands and is crucial in processes such as learning and memory. IGF-1, signaling through its glycoprotein receptor (IGF-1R), and canonical signaling pathways such as the PI3K-Akt and Ras-Raf-MAP pathways, has potent effects on cellular neuroplasticity in the CNS. In the present review, the role of IGF-1 in brain development is reviewed, followed by a detailed discussion of the role played by IGF in cellular neuroplasticity in the CNS. Findings from models of perturbed and reparative plasticity detailing the role played by IGF-1 are discussed, followed by the electrophysiological, structural and functional evidence supporting this role. Finally, the post-lesion and post-injury roles played by IGF-1 are briefly evaluated. We discuss the putative neurobiology underlying these changes, reviewing recent evidence and highlighting areas for further research.
... [15] The combined use of PDGF and IGF has a synergistic effect and support wound healing. [16] Studies have shown that VEGF accelerated the accumulation of granulation tissue in dermal wounds in rabbit ears and rat skin. [15] Animal studies had shown that transforming growth factor beta (TGF-b) had positive effects on partial and full-thickness wound in lower extremities of horses, on eardrum perforations of rats, on chronic ulcers, hypertrophic scars and intestinal mucosa ulcers of rats. ...
... Thus, protecting neurons or reducing infarct size is essential for the restoration of hearing after cerebral infarction. No previous studies have investigated the effects of IGF1 on hearing recovery after cerebral infarction, but IGF1 is shown to be a factor in post-injury neural and axonal regeneration over the past quarter of a century Sjöberg and Kanje, 1989;Nachemson et al., 1990). Serhan et al. reported that post-stroke systemic injections of IGF1 exert neuroprotective effects in rats (Fig. 1G) . ...
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Insulin-like growth factor 1 (IGF1) exerts an influence on almost every organ system in the body and plays an important role in growth, development, and metabolism. In the nervous system, IGF1 acts by promoting the development and growth of neurons and glial cells, differentiation of Schwann cells and their migration to axons, neurite outgrowth, and neuronal survival. The lack of IGF1 is associated with several pathological conditions, including severe prenatal growth retardation, postnatal growth failure, microcephaly, mental retardation, and bilateral sensorineural hearing loss. In addition to its physiological effects, based on the findings of in vivo and in vitro experiments and clinical trials, IGF1 is considered to play a potential role in the treatment of various types of neuronal damage. In this review, we discuss the potential use of IGF1 as a therapeutic molecule in the nervous system: (1) auditory system, including hair cells, cochlear ribbon synapses, auditory nerve, and central nervous systems, and (2) other peripheral nervous systems, especially the olfactory system and facial nerve. The role of IGF1 in the progression of age-related sensory deficits, especially hearing loss and olfactory dysfunction, is also discussed. Recent studies on IGF1 demonstrated that exogenous IGF1 can be applied in many fields, thus supporting the continued evaluation of IGF1 as a potential therapeutic molecule. Additional scientific investigations should be conducted to further supplement recent findings.
... Τhere is a growing body of evidence showing that IGF-I serves an important role in the regeneration of several different tissues following injury (8)(9)(10)(11)(12). Interestingly, despite this growing body of literature showing the wound healing effects of IGF-I in animals (13)(14)(15)(16), there are only a few studies investigating its effects in humans. ...
Article
The induction of wound healing by insulin-like growth factor-I (IGF-I) has been demonstrated in several animal studies; however, there are disproportionately fewer studies assessing its value in humans. The aim of the present review is to provide a comprehensive summary of all the available evidence pertaining to the effects of IGF-I administration on the process of wound anaplasias, both in human tissues in vivo and in cells in vitro. A systematic search of Medline, Scopus and Google Scholar was performed for relevant studies published until May 2020. Overall, 11 studies were included. Of these, 2 studies were conducted in human subjects, whereas the rest of them were performed using in vitro models of human cell lines. All studies demonstrated a positive association between IGF-I and wound anaplasias; IGF-I promoted the migration of keratinocytes, thus playing an important role in wound epithelialization as well as enabling wound bed contraction, and it also stimulated hyaluronan synthesis. The wound healing-promoting effect of IGF-I may be a great asset in dealing with the healing of challenging wounds; thus, this type of treatment could be extremely useful in addressing patients with large burn wounds, chronic diabetic ulcers and patients with impaired wound healing. Nevertheless, the route of recombinant IGF-I administration, the recommended dosage, as well as the indications for clinical use of this growth factor remain to be determined and thus, additional clinical trials are required, with a focus on the medical use of recombinant IGF-I in wound anaplasias.
... IGF-I has been shown to stimulate: fibroblast migration (Khaw et al. 1994b); ECM contraction (Assouline et al. 1992;Khaw et al. 1994b); fibroblast proliferation (Khaw et al. 1994b) and ECM synthesis (Bird and Tyler, 1994;Khaw et al. 1994b). Roles for IGF-I in healing in vivo have also been shown, with this factor stimulating bone formation (Hock et al. 1988), regeneration o f neural tissue following crush injury Sjoberg and Kanje, 1989) and in enhanced healing in vivo in combination with PDGF (Lynch et al. 1987). ...
Thesis
The wound healing response, in which the fibroblast is a key player, is a major cause of clinical morbidity throughout the human body. This response is of particular importance in the eye, as it plays a role in the pathogenesis or failure of treatment of many visually disabling or blinding conditions in the world today. An example of this is the failure of the surgical treatment of glaucoma due to excessive scarring. Previous studies have shown that single exposures to antimetabolites result in the long term growth arrest of ocular fibroblasts in vitro, and reduced scarring in vivo following glaucoma surgery. The overall effects of these treatments on fibroblast wound healing behaviour are not known. Additionally, growth arrested fibroblast feeder layers have been routinely used to serially cultivate keratinocytes for over twenty years, the mechanisms underlying this support of keratinocyte growth being unclear. This thesis investigated the effects of single exposures to antimetabolites on molecular and cellular aspects of ocular fibroblast wound healing behaviour. Growth arrested ocular fibroblasts were found to produce a number of regulatory molecules both at the message and protein levels using a quantitative reverse transcriptase polymerase chain reaction technique and enzyme linked immunosorbent assays. The molecules produced included: growth factors (transforming growth factor beta-1 and basic fibroblast growth factor); growth factor receptors (transforming growth factor beta type II receptor, fibroblast growth factor receptor and epidermal growth factor receptor); and extracellular matrix molecules (collagen type I, collagen type III and fibronectin) up to 48 days post-growth arrest. Fibroblasts were also found to migrate and contract collagen following growth arrest. Additionally, matrix metalloproteinases were identified as a novel, essential and possibly ubiquitous component of fibroblast-mediated collagen contraction. The experiments in this thesis have led to an increased understanding of the biology of growth arrest in vitro, and may explain how growth arrested fibroblasts support keratinocyte growth in vitro. Additionally, these findings may have clinical implications with respect to the modulation of scarring in vivo. Finally, the identification of a novel and potentially ubiquitous mechanism of collagen contraction may lead to the development of new anti-scarring strategies.
... VEGF was shown to support and enhance the growth of regenerating nerve fibers (Sondell et al., 2000;Lopes et al., 2011). IGF-1 was shown to exert important growth supporting effects on regenerating peripheral nerves (Hansson et al., 1986;Kanje et al., 1989;Sjöberg and Kanje, 1989). Unfortunately, these findings have not yet led to a clinical treatment improving peripheral nerve repair. ...
Article
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The peripheral nervous system has an intrinsic ability to regenerate after injury. However, this process is slow, incomplete, and often accompanied by disturbing motor and sensory consequences. Sciatic nerve injury (SNI), which is the most common model for studying peripheral nerve injury, is characterized by damage to both motor and sensory fibers. The main goal of this study is to examine the feasibility of administration of human muscle progenitor cells (hMPCs) overexpressing neurotrophic factor (NTF) genes, known to protect peripheral neurons and enhance axon regeneration and functional recovery, to ameliorate motoric and sensory deficits in SNI mouse model. To this end, hMPCs were isolated from a human muscle biopsy, and manipulated to ectopically express brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF-1). These hMPC-NTF were transplanted into the gastrocnemius muscle of mice after SNI, and motor and sensory functions of the mice were assessed using the CatWalk XT system and the hot plate test. ELISA analysis showed that genetically manipulated hMPC-NTF express significant amounts of BDNF, GDNF, VEGF, or IGF-1. Transplantation of 3 × 106 hMPC-NTF was shown to improve motor function and gait pattern in mice following SNI surgery, as indicated by the CatWalk XT system 7 days post-surgery. Moreover, using the hot-plate test, performed 6 days after surgery, the treated mice showed less sensory deficits, indicating a palliative effect of the treatment. ELISA analysis following transplantation demonstrated increased NTF expression levels in the gastrocnemius muscle of the treated mice, reinforcing the hypothesis that the observed positive effect was due to the transplantation of the genetically manipulated hMPC-NTF. These results show that genetically modified hMPC can alleviate both motoric and sensory deficits of SNI. The use of hMPC-NTF demonstrates the feasibility of a treatment paradigm, which may lead to rapid, high-quality healing of damaged peripheral nerves due to administration of hMPC. Our approach suggests a possible clinical application for the treatment of peripheral nerve injury.
... Insulin receptors have been found on peripheral axons, sensory neurons, Schwann cells, endothelial cells and pericytes in peripheral nervous system (PNS) (Sugimoto et al., 2000;Zochodne, 2007). Insulin is considered a potent neuronal growth factor and plays an important role in regeneration of peripheral nerve, i.e. beyond its role in glucose metabolism (Sjöberg and Kanje, 1989;Edbladh et al., 1994). Application of low doses of insulin near peripheral axons in rodents may improve electrophysiological alterations associated with diabetes (Singhal et al., 1997). ...
Article
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Peripheral diabetic neuropathy (PDN) is one of the most common complications of diabetes mellitus. Previous studies showed an association between dietary iron load and inflammation in the development of PDN in a rat model of type 1 diabetes (T1D). Here we investigated the role of iron and neural inflammation in development of PDN in a animal model of obesity and type 2 diabetes (T2D). 3-month-old db/db mice were fed with a high, standard or low iron diet for 4 months. High iron chow lead to a significant increase in motor nerve conduction velocities compared to mice on standard and low iron chow. Direct beneficiary effects on lowering blood glucose and HbA1c concentrations were shown in the high iron treated diabetic mice. Numbers of pro-inflammatory M1 macrophages were reduced in nerve sections, and anti-inflammatory M2 macrophages were increased in db/db mice on high iron diet compared to other groups. These results confirm and extend our previous findings in STZ-diabetic rats by showing that dietary non-hem iron supplementation may partly prevent the development of PDN in opposition to iron restriction. The identification of these dietary iron effects on the metabolic and inflammatory mechanisms of PDN supports a role of dietary iron and leads us to suggest testing for iron levels in human diabetic patients.
... There is increasing evidence to indicate that IGF-1 plays a main role in the regeneration of different tissues following injury (11,(14)(15)(16)(17). Of note however, despite the escalating number of animal studies (18)(19)(20), there are fewer studies investigating the role of IGF-1 in the wound healing process in humans. ...
Article
The ability of neurotrophic factors to regulate developmental neuronal survival and adult nervous system planticity suggests the use of these molecuales to treat neurodegeneration associated with human diseases. Solid rationales exist for the use of NGF and neurotrophin-3 in the treatment of neuropathies of the peripheral sensory system, insulin-like growth factor and ciliary neurotrophic factor in motor neuron atrophy, and NGF in Alzheimer's disease. Growth factors have been identified for neurons affected in Parkinson's disease, Huntington's disease, and acute brain and spinal cord injury. Various strategies are actively pursued to deliver neurotrophic factors to the brain, and develop therapeutically useful molecules that mimic neurotrophic factor actions or stimulate their production or receptor mechanisms. 1994 John Wiley & Sons, Inc.
Article
Neurotrophic factors are molecules that specifically promote neuronal survival, axonal growth, and synaptic plasticity. Different populations of neurons respond to specific neurotrophic factors, thereby providing potential tools for targeting various neurological disorders. In animal models of human diseases—including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), stroke, spinal cord injury, and peripheral neuropathy—neurotrophic factors have convincingly exhibited the ability to promote neuronal rescue, elicit axonal growth, and generate functional recovery. Yet clinical trials of neurotrophic factors have yielded little success to date, demonstrating preliminary efficacy for the treatment of peripheral neuropathy but failing to show benefit in several other neurological diseases. An emerging concept in the application of this extremely powerful and promising class of molecules for the treatment of nervous system disease is that their mode of delivery to the nervous system is critical: neurotrophic factors must be delivered specifically and accurately to neuronal targets, a difficult feat when delivering large molecules across the blood–brain barrier. Several novel approaches for delivering neurotrophic factors to the nervous system are under development, including gene therapy, focal intraparenchymal brain infusion, carrier-mediated transport, and small molecular analogs to neurotrophic factors that can cross the blood–brain barrier. These approaches are likely to establish neurotrophic factors as a mainstay of neurological therapy in the future, providing not only a means of compensating for nervous system disease once it has occurred, but for the first time, preventing or reducing the rate of disease progression. MRDD Research Reviews 1998;4:212–222. © 1998 Wiley-Liss, Inc.
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IntroductionThis project aims to study the regeneration of non-repairable lesions of peripheral nerve by muscle grafts enhanced with growth factors.Material and methodsThe experiment was carried out in two phases. The first one compared direct suture of a critical defect in the sciatic nerve of ten rats, with the interposition of autologous muscle graft, denatured by heat, in another ten. The second phase compared ten rats with nerve repair using an acellular muscle graft, with injection of 2 cc of IGF-1 (10 mg/ml mecasermin, Injectable solution) into the acellular graft of another ten.A clinical and functional follow-up was carried out including, ambulation, footprint measurement, and «Grasping Test». The animals were sacrificed at 90-100 days, and samples obtained for macro- and microscopic studies with toluidine blue, haematoxylin-eosin and Masson's trichrome staining.ResultsThe first experiment showed the characteristic findings of nerve tissue in muscle graft level sections. The second was an enhancement of the results: post-surgical clinical improvement, early ambulation, decrease in the rate of pressure ulcers in toes, recovery of the footprint, and increasing the percentage of nerve endings in distal sciatic regeneration (47-62%).Conclusions In this study the experimental and clinical possibilities of nerve defect repair by denatured muscle are demonstrated, confirming the suitability of the technique. Furthermore, it confirms our hypothesis with clinical and cellular determinations enriched by the addition of growth factors that promote nerve regeneration.
Article
Preclinical studies involving intramuscular injection of plasmid into animals have revealed at least four significant variables that effect levels of gene expression (i.e., >fivefold effect over controls), including the formulation, injection technique, species and pretreatment of the muscle with myotoxic agents to induce muscle damage. The uptake of plasmid formulated in saline has been shown to be a saturable process, most likely via a receptor-mediated event involving the T tubules and caveolae. Pharmacokinetic studies have demonstrated that the bioavailability of injected plasmid to muscle cells is very low, due to rapid and extensive plasmid degradation by extracellular nucleases. We have developed protective, interactive, non-condensing (PINC) delivery systems designed to complex plasmids and to (i) protect plasmids from rapid nuclease degradation, (ii) disperse and retain intact plasmid in the muscle and (iii) facilitate the uptake of plasmid by muscle cells. PINC systems result in up to at least a one log increase in both the extent and levels of gene expression over plasmid formulated in saline. We have combined the PINC delivery systems with two different muscle-specific expression plasmids. After direct intramuscular injection of these gene medicines, we have shown both local myotrophic and neurotrophic effects of expressed human insulin-like growth factor (hIGF-I) and the secretion of biologically active human growth hormone (hGH) into the systemic circulation.
Article
Trauma to a peripheral nerve trunk is a complex injury because it involves not only repair processes locally at the peripheral level, but it also engages repair and compensation mechanisms at central levels. The main actor is the fascinating and unique neuron with its supporting cells, which consist mainly of Schwann cells. In the neuron and in the Schwann cells, intracellular signaling mechanisms are initiated by the peripheral nerve injury and aim to turn the intracellular processes into a regenerative and proliferative state. The intracellular signaling mechanism is called signal transduction and works along the entire neuron, including the intracellular axonal transport system. A very delicate interaction occurs between the growth cones formed by the distal tip of the outgrowing axons and the environment into which the axons grow. A large number of changes occur in this environment due to the process of Wallerian degeneration caused by the injury. A thorough knowledge of the cellular and molecular repair mechanisms after peripheral nerve injury is the basis on which we can build new research with the aim to improve results after this devastating injury, because there are limitations in the pure surgical treatment of peripheral injury.
Article
Preclinical studies involving intramuscular injection of plasmid into animals have revealed at least four significant variables that effect levels of gene expression (i.e., >fivefold effect over controls), including the formulation, injection technique, species and pretreatment of the muscle with myotoxic agents to induce muscle damage. The uptake of plasmid formulated in saline has been shown to be a saturable process, most likely via a receptor-mediated event involving the T tubules and caveolae. Pharmacokinetic studies have demonstrated that the bioavailability of injected plasmid to muscle cells is very low, due to rapid and extensive plasmid degradation by extracellular nucleases. We have developed protective, interactive, non-condensing (PINC) delivery systems designed to complex plasmids and to (i) protect plasmids from rapid nuclease degradation, (ii) disperse and retain intact plasmid in the muscle and (iii) facilitate the uptake of plasmid by muscle cells. PINC systems result in up to at least a one log increase in both the extent and levels of gene expression over plasmid formulated in saline. We have combined the PINC delivery systems with two different muscle-specific expression plasmids. After direct intramuscular injection of these gene medicines, we have shown both local myotrophic and neurotrophic effects of expressed human insulin-like growth factor (hIGF-I) and the secretion of biologically active human growth hormone (hGH) into the systemic circulation.
Article
Abstract Glial cell line-derived neurotrophic factor (GDNF), first characterized for its effect on dopamine uptake in central dopaminergic neurons, appears to be a powerful neurotrophic factor for motor neurons. GDNF has recently been shown to signal through a multisubunit receptor. This receptor is composed of a ligand-binding subunit, called GDNF receptor α (GDNFRα), and a signalling tyrosine kinase subunit, Ret. To gain further insight into GDNF function, we investigated the expression of GDNF and its receptors after nerve lesion in adult mice. Analysis of expression in muscle, nerve and spinal cord by RNase protection assay and in situ hydridization revealed that, in adult non-lesioned mice, GDNF mRNA was expressed in the nerve and GDNFRα mRNA in the nerve and the spinal cord, while the expression of Ret was restricted to spinal cord motor neurons. After a sciatic nerve crush a rapid increase in GDNF mRNA was observed in the distal part of the nerve and a delayed elevation in the muscle, while GDNFRα mRNA was up-regulated in the distal part of the sciatic nerve but not in proximal nerve or spinal cord. The lesion also induced a rapid increase in Ret mRNA expression, but the increase was observed only in spinal cord motor neurons and in dorsal root ganglion neurons. A pattern of expression of GDNF and its receptors similar to that seen after lesion in the adult was detected during embryonic development. Administration of GDNF enhanced sciatic nerve regeneration measured by the nerve pinch test. Taken together, these results suggest that GDNF has an important role during regeneration after nerve damage in the adult.
Article
FK506 is a frequently used immunosuppressant with neuroregenerative effects. The neuroregenerative and immunosuppressive mechanisms of FK506, however, are distinct, suggesting that FK506 may stimulate nerve regeneration at lower doses than are needed to induce immunosuppression. The effects of cold preservation, a technique known to improve axonal regeneration through nerve allografts, are not well studied in nerve isografts and are also reported here. To determine the effects of subimmunosuppressive doses of FK506 and cold preservation on nerve regeneration in isografts. The neuroregenerative properties of immunosuppressive and subimmunosuppressive doses of FK506 were compared in a murine model receiving either fresh or cold preserved nerve isografts. Sixty female BALB/cJ mice were randomized into six groups. Animals in groups I, III and V received fresh nerve isografts. Animals in groups II, IV and VI received cold-preserved nerve isografts. Mice in groups I and II received no medical therapy, while those in groups III and IV received subimmunosuppressive doses of FK506, and those in groups V and VI received immunosuppressive doses as confirmed by mixed lymphocyte reactivity assays. Nerve regeneration was evaluated with histomorphometry and functional recovery was evaluated with walking track analysis. Pretreatment with cold preservation did not significantly affect neural regeneration. The potent neuroregenerative effect of immunosuppressive doses of FK506 was confirmed, and the ability of subimmunosuppressive doses of FK506 to stimulate axonal regeneration in murine nerve isografts is reported. Less toxic subimmunosuppressive doses of FK506 retaining some neuroregenerative properties may have a clinical role in treating extensive nerve injuries.
Article
Insulin-like growth factor-1 (IGF-1) is a naturally occurring neurotrophic factor that plays an important role in promoting cell proliferation and differentiation during normal brain development and maturation. The present review examines recent evidence that endogenous IGF-1 also plays a significant role in recovery from insults such as hypoxia-ischemia and that giving additional exogenous IGF-1 can actively ameliorate damage. It is now well established that neurons and other cell types die many hours or even days after initial injury due to activation of programmed cell death pathways. IGF-1 and its binding proteins and receptors are intensely induced within damaged brain regions following brain injury, suggesting a possible a role for IGF-1 in brain recovery. Exogenous administration of IGF-1 within a few hours after brain injury is now known to be protective in both gray and white matter and leads to improved somatic function. In contrast, pre-treatment is ineffective, likely reflecting limited intracerebral penetration of IGF-1 into the uninjured brain. The neuroprotective effects of IGF-1 are mediated by IGF-1 receptors and its binding proteins and are specific to particular cellular phenotypes and brain regions. The window of opportunity for treatment with IGF-1 is limited to a few hours after normothermic brain injury, reflecting its specific actions on early, intracellular events in the apoptotic cascade. However, injury-associated mild post-hypoxic hypothermia, which delays the development of cell death, can shift and dramatically extend the window of opportunity for delayed treatment with IGF-1. Such a combined approach is likely to be essential for any clinical treatment.
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Conference Paper
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Background: White adipose tissue (WAT) and insulin-like growth factor-1 (IGF-1) have shown potential to enhance peripheral nerve regeneration. We hypothesized that white adipose tissue flap (WATF) enriched with IGF-1 as an in vivo biologic scaffold would provide functional and histological benefits in a sciatic nerve crush injury model. Methods: Forty male Sprague-Dawley rats were divided into four arms. All rats underwent a crush injury to one sciatic nerve and received a pedicled WATF, controlled local release of IGF-1, both, or no treatment at the lesion area for 4 weeks. The WATF was composed of adipose tissue derived from the inguinal region. IGF-1 was delivered from polylactic-co-glycolic acid (PLGA) microspheres embedded in fibrin gel adjacent to the crush injury. Tibialis anterior (TA) muscle weights and maximum isometric tetanic force (ITF) of the TA muscle normalized to the contralateral side were recorded. Histological sections 1mm distal to the lesion were histomorphometrically analyzed. Data were analyzed utilizing Student’s t-test and two-way ANOVA. Results: At 4 weeks, the mean normalized ITFs were as follows: WATF 51.9±12.3%, IGF-1 33.5±5.7%, WATF/IGF-1 46.7±22.8%, and control 44.8±12.8%. The presence of the WATF as a main effect resulted in a statistically significant 26.1% increase in normalized ITF (p<0.05). There was a statistically significant 11.3% increase in myelin thickness and 13.1% increase in total axon count in the WATF-only group versus control (p<0.01). The TA muscle weights were similar across all groups. Both functional and histomorphometric data suggest that the presence of IGF-1 suppressed the effect of the WATF rather than enhanced it. Conclusions: We induced a sciatic nerve axonotmesis experimental model in rats, which yielded promising functional and histomorphometrical results utilizing a pedicled WATF. IGF-1 does not appear to enhance the regenerative effect of the WATF.
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Background: Combination treatments of botulinum toxin type-A and other rejuvenation agents or instruments are gradually becoming more popular. After observing a high incidence of therapy failure following simultaneous applications of botulinum toxin type-A and platelet-rich plasma mesotherapy, we aimed to investigate whether PRP has an inhibitory effect on botulinum toxin type-A. Methods: Twenty-four New Zealand white rabbits were divided into 4 groups, and the anterior auricular muscle and overlying skin were used for injections. Groups I and II both received onabotulinumtoxinA intramuscular injections. In addition, autologous platelet-rich plasma mesotherapy was performed in Group I while Group II received saline mesotherapy. Group III was designed as the in vitro mixture group in which onabotulinumtoxinA and platelet-rich plasma were mixed and then administered intramuscularly. Group IV received saline within the mixture instead of platelet-rich plasma. The contralateral ears of all the rabbits served as control and were only treated with onabotulinumtoxinA. Visual evaluation of ear positions and electroneuromyographic studies were done prior to all procedures and at day 14. Anterior auricular muscles were harvested at day 14 and were evaluated with quantitative real-time PCR. Results: Visual and electroneuromyographic studies revealed less onabotulinumtoxinA activity in Groups I and III. When platelet-rich plasma was administered through skin mesotherapy, onabotulinumtoxinA activity failure was more severe in comparison with direct contact. No significant difference in SNAP-25 mRNA expression through quantitative real-time PCR was observed between groups. Conclusion: Although we could not explain the exact mechanism underlying this interaction, platelet-rich plasma applications result in less onabotulinumtoxinA muscle paralysis activity.
Article
Growth factors play a central role in the regulation of normal and injury‐induced regenerative cell growth. The purpose of this article is to summarize the available data on the expression of different growth factors and their receptors in the injured peripheral nervous system and to discuss their possible role in promoting peripheral nerve regeneration.
Chapter
Recent researches have shown that hormone plays an impotent role in the process of aging. The secretion of growth hormones is related to the nueroendocrine regulation. Decrease in the rate of growth hormone secretion in response to stimuli in an aging individual can be observed. This establishes the fact that growth hormones have a relation with aging as well. Aging has a direct impact on the memory of the individual. The inter relation between cerebrovasculature and aging also forms an integral part of the discussion in this chapter. The crux of the discussion in the chapter is the eminent role of Insulin like growth factor in the development of brain, and its relation with aging.
Chapter
This chapter discusses roles of insulin-like growth factors (IGF) in peripheral nerve regeneration and motor neuron survival. The human IGF-II gene is located downstream of the insulin gene, on chromosome 11. There is a regulatory mechanism to modulate levels of insulin and IGFs. Infusion of supraphysiological concentrations of rhIGF-I into test subjects produces a substantial decline in IGF-II and insulin content in serum. The distribution of IGFs in brain and presence of variant IGF receptors suggest that IGFs are likely to play important roles in the nervous system. Adult neurons might lose responsiveness or may respond to different growth factors, but adult sensory neurons continue to extend neurites in response to IGFs. In addition to increasing neurite outgrowth, low concentrations of IGFs are found to enhance the survival of cultured embryonic sensory and sympathetic cells, as well as fetal cortical neurons. The data indicate that endogenous IGF-I and IGF-II may both contribute to IGF-dependent sciatic nerve regeneration. The axon regeneration and down-regulation of IGF mRNAs are also elaborated.
Article
In the experimental model of the rat sciatic nerve, three different strategies to improve peripheral nerve regeneration in order to obtain better outcomes have been studied. Firstly, brain-derived neurotrophic factor (BDNF) delivered through osmotic pumps has demonstrated a significant capacity for improving the presence of motoneurons in the ventral spinal horn and thus promoting nerve regeneration thorough nerve autografts without the problems inherent in multiple anaesthesia or device exchange. Secondly, therapeutic doses of FK506 administered through osmotic pumps was effective for preventing nerve allograft rejection across a major histocompatibility difference in rats without any serious toxic response at the dosage of 3.5 mg/kg/day. Moreover, diminishing the antigenicity of nerves in a UWCSS solution during 3 weeks at 4°C reaches the same results when compared to allografts treated with FK506. Finally, applying scar inhibitors at the level of the anastomosis such as the inhibitor peptide of TGF-ß1 (p144®) has demonstrated a significant capacity to accelerate the functional activity of the lower limb in rats, and thereby encourage nerve regeneration through nerve repair. The sciatic nerve of the rat has been used as the experimental model due to its accessibility and the possibility of performing functional studies with proven reliability.
Article
Insulin/insulin-like growth factor signalling (IIS) is a critical regulator of an organism's most important biological decisions from growth, development, and metabolism to reproduction and longevity. It primarily does so through the activity of the DAF-16 transcription factor (forkhead box O (FOXO) homologue), whose global targets were identified in Caenorhabditis elegans using whole-worm transcriptional analyses more than a decade ago. IIS and FOXO also regulate important neuronal and adult behavioural phenotypes, such as the maintenance of memory and axon regeneration with age, in both mammals and C. elegans, but the neuron-specific IIS/FOXO targets that regulate these functions are still unknown. By isolating adult C. elegans neurons for transcriptional profiling, we identified both the wild-type and IIS/FOXO mutant adult neuronal transcriptomes for the first time. IIS/FOXO neuron-specific targets are distinct from canonical IIS/FOXO-regulated longevity and metabolism targets, and are required for extended memory in IIS daf-2 mutants. The activity of the forkhead transcription factor FKH-9 in neurons is required for the ability of daf-2 mutants to regenerate axons with age, and its activity in non-neuronal tissues is required for the long lifespan of daf-2 mutants. Together, neuron-specific and canonical IIS/FOXO-regulated targets enable the coordinated extension of neuronal activities, metabolism, and longevity under low-insulin signalling conditions.
Article
Growth factors are signal proteins that regulate the cellular processes in wound healing. By manipulating the actions of growth factors, it may be possible to accelerate or modify wound healing. Currently, intense research is being conducted throughout the world to investigate this possibility. In otolaryngology, the impact of improved wound healing could be tremendous. This article discusses the involvement of growth factors in soft-tissue healing that is relevant to otolaryngology.
Thesis
This study has investigated the mechanism of growth hormone (GH) action in the skin and its innervation as well as in skeletal muscle. Two main issues have been addressed: (1) To what extent are GH effects on extrahepatic targets the result of direct actions of this hormone, or, indirect effects mediated by local production of insulin-like growth factor I (IGF-I)? (2) Can GH, acting either directly or indirectly, be regarded as a key mediator of neurotrophic functions? These issues have been examined using three approaches: i) Muscle biopsies were taken from adult growth hormone-deficient patients before and after six to twelve months of recombinant human growth hormone (r-hGH) treatment, as part of a double-blind placebo controlled clinical trial. These patients exhibit reductions in muscle bulk and strength which can be reversed with r-hGH treatment. Muscle fibre types and fibre size were examined by light microscopy for an influence of r-hGH therapy. IGF-I message was localised to muscle fibre cytoplasm and fibre-type specific mRNA was quantified by combined non-radioactive in situ hybridisation with computerised image analysis. Circulating IGF-I levels were also assayed, making it possible to compare morphological changes in muscle fibre size with changes in circulating and muscle-derived IGF-I. The results obtained are consistent with a direct effect of GH on skeletal muscle resulting in generation of IGF-I by the muscle fibres. However, the influence of circulating IGF-I could not be dismissed from this scheme. ii) The innervation, morphology and function of eccrine sweat glands was examined in GH-deficient subjects who display reduced sweating, in patients with acromegaly (a GH hypersecretory state with excessive sweating) and in control subjects. Pilocarpine iontophoresis sweat tests were used to assess changes in sweating. An increase in the cholinergic innervation accompanied restoration of sweat rates in r-hGH treated subjects, whilst acromegalics displayed hypertrophy of sweat glands and elevated sweat gland innervation. These data imply an effect of GH on the sweat glands with repercussions for their innervation. A GH-mediated influence on neurotrophic support to the sweat gland nerves can thus be envisaged. iii) Aged rats were utilised to investigate direct effects of r-hGH administration in the vicinity of the footpad eccrine sweat glands, with the aim of minimising the confounding influence of serum IGF-I. Ageing is a state of relative GH deficiency and sweat glands are believed to be targets for GH action. Further aged rats display reductions in sweat gland size and innervation. An immunohistochemical method was utilised to stain for nerves and neurotrophin receptor sites around sweat glands. Due to injection-induced inflammatory effects, the results from this study were not clear, aside from an increase in sweat gland acinar size on r-hGH administration and the demonstration of parallel alterations in neurotrophin receptor expression and periacinar nerve density. These results allow the hypothesis of a direct effect of GH on target tissues to be advanced, resulting in local generation of IGF-I and, perhaps, of other target-derived neurotrophic factors.
Thesis
There is a clinical need to improve functional outcome after peripheral nerve reconstruction, and also to find a replacement for autologous nerve grafts currently used for this purpose. Schwann cells (SC) are essential for adequate axonal regeneration and the aim of this work was to develop a methodology to enable investigation and characterisation of the effects of SC transplantation and genetic manipulation on peripheral nerve regeneration. Initially an identifiable and pure population of cultured SC was obtained. In vitro evaluation showed that chemical labelling adversely affected SC properties. Transduction of lacZ genetic label was carried out and a stable population of genetically modified SC was obtained. Transduced SC properties and lacZ expression were preserved in vitro for 6 months of continuous culture. Suspension matrix is required for SC transplantation and the suitability of alginate hydrogel was confirmed by in vitro tests to support SC proliferation and neurite sprouting in a neuron-glial co-culture. Defects in the rat sciatic nerve were bridged using resorbable poly-3-hydroxybutyrate (PHB) conduits containing SC. The results showed that the optimal number of SC required to enhance axonal regeneration was 80×l0⁶/ml. Following transplantation of transduced SC, syngeneic SC could be identified for up to 6 weeks and allogeneic SC for up to 3 weeks, as identified by X-gal staining. Immunohistochemistry was used to characterise SC, immune response, and axonal regeneration and the staining quantified by image analysis. Transplantation of both syngeneic and allogeneic SC improved axonal regeneration distance, the quantity of regeneration was better and more sustained with syngeneic SC. Furthermore, addition of liquid fibronectin to alginate improved regeneration which was further enhanced when SC were present. Finally a new technique for nonviral gene delivery of insulin-like growth factor I and mechano growth factor gene with alginate hydrogel matrix showed promising results in improving peripheral nerve regeneration.
Article
Methods: Twenty-eight Japanese white rabbits were used. A 15-mm sciatic nerve defect was created on the left limb. The resected nerve was used as a reverse autologous nerve. The rabbits were randomly divided into two groups. In group A (n = 10), only nerve grafting was performed. In group B (n = 18), nerve grafting was performed with local PRP administration. Right limbs were used as control (group C, n = 28). The rabbits in each group were equally divided into two subgroups based on the evaluation period of 4 and 12 weeks after grafting. Electrophysiological evaluation, muscle wet-weight, histological evaluation, and multiple immunofluorescence staining were performed to investigate the regenerative effect of PRP. Results: The mean regenerative axon diameter of the graft portion in group B (2.02 ± 0.22-μm) was significantly larger than that in group A (1.89 ± 0.16-μm) at 4 weeks. The regenerative axon number at the distal portion showed a greater increase in group B (9017 ± 2224/mm(2) ) than in group A (4955 ± 3117/mm(2) ) at 12 weeks. Electrophysiological evaluation and muscle wet-weight revealed no significant differences. On immunohistological evaluation, the number of activated SCs increased to a larger extent in group B (188 ± 90/mm(2) ) than in group A (117 ± 51/mm(2) ). Conclusions: Local PRP administration increases the regenerative axon diameter and the regenerative axon number at the distal portion. PRP accelerates SC proliferation in vivo.
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Objective In this study, it was aimed to evaluate and compare biochemical and histopathological effects of platelet-rich plasma (PRP), ozone and hyperbaric oxygen (HBO) on wound healing which was formed experimentally in oral cavity of rats.Materials and methodsIn this study, thirty-six Wistar Hannover rats with weight of 250–350 g, fed with standard feeds, were anesthetized to create intraoral wound on the hard palate. Rats were divided into four groups as the following: control group, PRP, HBO and Ozone groups. 0.1 ml of PRP was injected on the wound edges of each rat in the PRP group on days 1, 3 and 7. Each rat in the ozone group was systemically injected with 2.3–3.0 ml ozone gas on the 1st, 3rd and 7th days. The wounds of the rats in the control group were not performed anything. The rats in the HBO group were placed in the HBO pressure chamber and were treated with one session (2 h for each session) of 100% oxygen each day for 7 days. Rats in all groups were killed on the 15th day and the hard palates were excised for histopathological examination. Inflammation severity, neovascularization, fibroblast proliferation, collagen density and epithelization were evaluated.ResultsThe rate of intense wound closure (epithelialization intensity) was significantly higher in all the three treatment groups, the PRP group was the highest, than in the control group (p < 0.05). Fibroblast proliferation level was higher in PRP group (p < 0.05), followed by ozone, HBO and control groups, respectively. Histologically, inflammation was significantly higher in the control group than the treatment groups (p < 0.05). For the treatment groups; it was lowest in the PRP group, followed by the ozone and HBO groups, respectively. The rates of angiogenesis and collagenization were significantly higher in all the three treatment groups, the PRP group was the highest, than in the control group (p < 0.05). In this study, no significance difference was found between the groups in terms of blood glucose levels (p = 0.21).Conclusion In this study, all three treatment modalities were found to be effective in wound healing, and PRP was found to be more effective than the others.
Article
Background: Previous studies have documented improvement in erectile function after bilateral cavernous nerve injury (BCNI) in rats with the use of pioglitazone. Our group determined this improvement to be mediated by the insulin-like growth factor-1 (IGF-1) pathway. Aim: To eliminate the systemic effects of pioglitazone and evaluate the local delivery of IGF-1 by polymeric microspheres after BCNI in the rat. Methods: Male Sprague-Dawley rats aged 10-12 weeks were assigned at random to 3 groups: sham operation with phosphate buffered saline (PBS)-loaded microspheres (sham group), crush injury with PBS-loaded microspheres (crush group), and crush injury with IGF-1-loaded microspheres (IGF-1 group). Poly(lactic-co-glycolic) acid microspheres were injected underneath the major pelvic ganglion (MPG). IGF-1 was released at approximately 30 ng/mL/day per MPG per rat. Outcomes: Functional results were demonstrated by maximal intracavernosal pressure (ICP) normalized to mean arterial pressure (MAP). Protein-level analysis data of IGF-1 receptor (IGF-1R), extracellular signal-regulated kinase (ERK)-1/2, and neuronal nitric oxide synthase (nNOS) were obtained using Western blot analysis and immunohistochemistry for both the cavernosal tissue and the MPG and cavernous nerve (CN). Results: At 2 weeks after nerve injury, animals treated with IGF-1 demonstrated improved erectile functional recovery (ICP/MAP) at all voltages compared with BCNI (2.5V, P = .001; 5V, P < .001; 7.5V, P < .001). Western blot results revealed that up-regulation of the IGF-1R and ERK-1/2 in both the nervous and erectile tissue was associated with improved erectile function recovery. There were no significant between-group differences in nNOS protein levels in cavernosal tissue, but there was an up-regulation of nNOS in the MPG and CN. Immunohistochemistry confirmed these trends. Clinical translation: Local up-regulation of the IGF-1R in the neurovascular bed at the time of nerve injury may help men preserve erectile function after pelvic surgery, such as radical prostatectomy, eliminating the need for systemic therapy. Strengths & limitations: This study demonstrates that local drug delivery to the MPG and CN can affect the CN tissue downstream, but did not investigate the potential effects of up-regulation of the growth factor receptors on prostate cancer tissue. Conclusion: Stimulating the IGF-1R at the level of the CN has the potential to mitigate erectile dysfunction in men after radical prostatectomy, but further research is needed to evaluate the safety of this growth factor in the setting of prostate cancer. Haney NM, Talwar S, Akula PK, et al. Insulin-Like Growth Factor-1-Loaded Polymeric Poly(Lactic-Co-Glycolic) Acid Microspheres Improved Erectile Function in a Rat Model of Bilateral Cavernous Nerve Injury. J Sex Med 2019;16:383-393.
Article
The ability of mature oxytocinergic (OT) and vasopressinergic (VP) neurons of the magnocellular neurosecretory system (MNS) to undergo axonal growth implies that one or more growth factors may be active in the adult MNS, yet little is known regarding their possible identity. One such potential factor is insulin-like growth factor I (IGF-I). We have examined the expression of IGF-I mRNA and IGF-I-immunoreactivity (IGF-I-ir) in the mature MNS and have also determined the in vivo response of OT and VP neurons to hypothalamic implants of IGF-I. In situ hybridization revealed moderate labeling of IGF-I mRNA in both the supraoptic (SON) and the paraventricular (PVN) nuclei of adult male rats. RT-PCR analysis confirmed the presence of authentic IGF-I mRNA in extracts of the basal hypothalamus. Faint IGF-I-ir was detected in scattered magnocellular neurons within both the PVN and the SON of normal rats, but IGF-I-ir was much more intense and the majority of MNS neurons including those in the accessory nuclei were immunoreactive in sections from rats given colchicine, as were some parvocellular neurons in the PVN. Confocal microscopy revealed that IGF-I-ir was present in both OT and VP neurons, but VP neurons contained the most intense IGF-I-ir. Finally, a dramatic growth response of OT but not of VP fibers was observed following implantation of polymer rods containing IGF-I into the hypothalamus. A dense OT fiber plexus grew along the cannula track and OT fibers invaded the leptomeninges ventral to the SON and encircled the rostral cerebral artery. To our knowledge this is the first demonstration of axonal sprouting by mature OT neurons in response to an identified growth factor and the first direct demonstration of sprouting in response to exogenous IGF-I in the adult CNS. These findings suggest that IGF-I is synthesized and transported by adult MNS neurons where it may act as an autocrine and/or paracrine growth factor.
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When prepared by methods utilized in our laboratory, pure populations of Schwann cells in culture do not divide, but, after recombination with peripheral sensory neurons or their processes, proliferate rapidly (Wood and Bunge, 1975, Nature (Lond.) 256:661--664). In this paper, we demonstrate that a membrane fraction prepared from sensory ganglion neurites is also mitogenic for Schwann cells and increases the labeling index (assessed by autoradiography after incubation of cells with tritiated thymidine) from less than 0.2 to 10% for primary cells, and from 0.4 to 18--19% for replated cells. The increased responsiveness of replated cells may reflect their greater access to the neurite membranes which is a consequence of the elimination of multiple cell layers after replating and the removal of the basal lamina. This stimulation was specific; addition of membrane preparations from other cell types (3T3, C1300, etc.) was not mitogenic. Ultrastructural analysis demonstrated apparent binding of neurite membranes to Schwann cells as well as significant phagocytosis of the membranes by the cells. The uptake of nonmitogenic membranes suggests that phagocytosis per se is not the stimulus of proliferation.
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Cultured rat Schwann cells were treated for 72 h with axolemma- and myelin-enriched fractions prepared from rat brainstem. [3H]Thymidine was added to the cultures 48 h before the termination of the experiment. Although, both fractions produced a dose-dependent uptake of label into Schwann cells, the shape of the dose response curves and rates at which [3H]thymidine was incorporated were different. The axolemma-enriched fraction produced a sigmoid dose response curve with a Hill coefficient of 2.05. The dose response curve for myelin rose sharply and saturated at a level that was approximately 50% of the maximal response observed with axolemma. Schwann cells that had been treated with axolemma exhibited little change in the rate of [3H]thymidine incorporation from 36-72 h after the addition of the membranes. In contrast, Schwann cells accumulated label three times faster during the 48-72-h period following the addition of myelin to the cultures when compared with the rate during the preceding 12-h interval. Furthermore, the mitogenic activity of the myelin-enriched fraction was decreased by the addition of ammonium chloride, a lysosomal inhibitor, whereas the activity of the axolemmal fraction was not impaired.
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We have examined the consequences of surface interactions with glial and nonglial cells on the in vitro growth of CNS neurons. When cerebellar or spinal cord cells were plated onto monolayers highly enriched in cortical astrocytes or sciatic nerve Schwann cells, neurons generally grew as single cells and showed relatively little tendency to aggregate. Similarly, neurites showed little tendency to fasciculate. In contrast, when plated onto fibroblast, heart muscle-fibroblast, or astrocyte-free meningeal monolayers, neurons rapidly aggregated, and neurite outgrowth was primarily in large fascicles. There were no glia detectable in the majority of aggregates or fascicles, suggesting that aggregation and fasciculation were due to interactions between neurons. Neurite outgrowth over 24 hr was also greater on astrocytes than on nonglia. Whether or not aggregation and fasciculation occurred was due to surface properties of the glial and nonglial cells. When neurons were added to astrocyte and nonglial monolayers growing in medium conditioned by a large excess of co-cultured nonglia or astrocytes, respectively, the pattern of neuronal growth was determined by the type of monolayer with which the neurons were in contact. Moreover, the initial growth of neurons on heat-killed astrocytes was indistinguishable from growth on living astrocytes. The pattern of neuronal growth on these different monolayers suggests that neurons are more adherent to glia than to other neurons but are more adherent to other neurons than to nonglia. Such an adherence hierarchy could explain the consistent finding of an apposition of neurons to glial surfaces during neuronal migration and axon outgrowth. Our findings also suggest that the interaction of axons with the non-neuronal milieu through which they grow may play an important role in regulating fasciculation, a process which has generally been treated as due primarily to axon-axon interactions.
Article
Full-text available
When prepared by methods utilized in our laboratory, pure populations of Schwann cells in culture do not divide, but, after recombination with peripheral sensory neurons or their processes, proliferate rapidly (Wood and Bunge, 1975, Nature (Lond.) 256:661--664). In this paper, we demonstrate that a membrane fraction prepared from sensory ganglion neurites is also mitogenic for Schwann cells and increases the labeling index (assessed by autoradiography after incubation of cells with tritiated thymidine) from less than 0.2 to 10% for primary cells, and from 0.4 to 18--19% for replated cells. The increased responsiveness of replated cells may reflect their greater access to the neurite membranes which is a consequence of the elimination of multiple cell layers after replating and the removal of the basal lamina. This stimulation was specific; addition of membrane preparations from other cell types (3T3, C1300, etc.) was not mitogenic. Ultrastructural analysis demonstrated apparent binding of neurite membranes to Schwann cells as well as significant phagocytosis of the membranes by the cells. The uptake of nonmitogenic membranes suggests that phagocytosis per se is not the stimulus of proliferation.
Article
Cultured rat Schwann cells were treated for 72 h with axolemma- and myelin-enriched fractions prepared from rat brainstem. [3H]Thymidine was added to the cultures 48 h before the termination of the experiment. Although, both fractions produced a dose-dependent uptake of label into Schwann cells, the shape of the dose response curves and rates at which [3H]thymidine was incorporated were different. The axolemma-enriched fraction produced a sigmoid dose response curve with a Hill coefficient of 2.05. The dose response curve for myelin rose sharply and saturated at a level that was approximately 50% of the maximal response observed with axolemma. Schwann cells that had been treated with axolemma exhibited little change in the rate of [3H]thymidine incorporation from 36-72 h after the addition of the membranes. In contrast, Schwann cells accumulated label three times faster during the 48-72-h period following the addition of myelin to the cultures when compared with the rate during the preceding 12-h interval. Furthermore, the mitogenic activity of the myelin-enriched fraction was decreased by the addition of ammonium chloride, a lysosomal inhibitor, whereas the activity of the axolemmal fraction was not impaired.
Article
An immunohistochemical investigation regarding the presence of S-100 protein in benign and malignant, primary and metastatic melanocytic tumors is reported. The studied series consisted of 15 benign nevi, 3 blue nevi, 4 juvenile melanomas, 1 balloon cell nevus, 30 primary malignant melanomas of skin, mucous membranes and conjunctiva and 30 metastatic malignant melanomas. The immunohistochemical analysis showed positive staining for S-100 protein within the majority of the tumor cells in all benign tumors examined, except the balloon cell nevus, as well as in all the primary and metastatic malignant melanomas, including low-differentiated epitheloid or spindle-cell types without demonstrable melanin pigment. The results indicate that S-100 protein is a valuable marker for melanocytic tumors, especially in the fairly frequent dilemma of malignant melanoma presenting as a solitary non-pigmented metastasis of uncharacteristic light-microscopic appearance, and without known primary tumor. A characterization and quantification of the S-100 protein immunoreactivity in 5 metastatic malignant melanomas is presented. Using immuno-electrophoresis, the presence of 3 antigenic S-100 determinants was demonstrated within homogenates from the malignant melanomas, including the previously characterized S-100 A (alpha/beta) and S-100 B (beta/beta), and suggesting the presence of a hitherto undescribed variant of S-100 protein, possibly consisting of 2 alpha-subunits. Using rocket immuno-electrophoresis, the amount of S-100 protein was estimated.
Article
The effect of inhibiting Schwann cell mitosis on the re-innervation of acellular autografts in the peripheral nervous system of the mouseReactive gliosis in the zone immediately proximal to transection of the sciatic nerve has been inhibited by intraneural injection of mitomycin C, an anti-mitotic agent known to arrest Schwann cell division after transection, crush or demyelination. Mitomycin C-pretreated proximal stumps were subsequently sutured to cellular or acellular autografts (0–5 cm long) and neurite growth into and within the grafts was examined during a 5-week post-operative period. Neurites grew into cellular autografts and became associated with the resident population of Schwann cells within the grafts, to the extent that remyelination was well established in the majority of Schwann cell basal lamina tubes by week 5 post-suture. In marked contrast, very few neurites grew into acellular grafts during this time, and where axons and Schwann cells were seen they tended to be grouped in ‘minifascicles’. The results suggest that neurite outgrowth from proximal stumps is dependent upon active Schwann cell participation.
Article
The spatial-temporal progress of nerve regeneration was examined in silicone chambers of three different volume capacities: 11, 25, and 75 μl. In all chambers, the stumps of a transected rat sciatic nerve were sutured into the ends of the chamber leaving a 10 mm gap between the stumps. Chambers were implanted empty (E chambers) or prefilled with saline (PF chambers). A coaxial and continuous fibrin matrix had formed in all chambers by 1 week. In E chambers, the matrices had a proximal-distal taper that was more pronounced in E25 and E75 chambers due to significantly larger matrix diameters in the proximal region. At 3 weeks, vascular and Schwann cell migration and axonal regeneration were less advanced in the E25 and E75 than in the control E11 chambers. The retardation correlated with the presence of an avascular organization of circumferential cells. Saline prefill-ing affected the caliber and density of fibrin fibers in the 1 week matrices of PF25 and PF75 chambers. The matrices did not have a prominent taper and diameters were progressively larger with increasing chamber volume. Saline prefilling did not affect regeneration progress in 3 week PF11 chambers but did enhance regeneration in the PF25 chambers; a 1.5-fold larger diameter nerve formed at 3 weeks that contained 2,6-fold more axons. Progress in the PF75 chamber was retarded. We conclude that the volume, timing, and nature of the fluid filling a silicone chamber have significant influence on the formation of fibrin matrices. Alterations in matrix formation correlate with substantial changes in the subsequent progress of intrachamber regeneration events.
Article
We have investigated mouse and rat ganglionic Schwann cells as possible sources of neurite outgrowth-promoting factors by co-culturing Schwann cells with nerve growth factor (NGF)-responsive PC12 pheochromocytoma cells primed by pretreatment with NGF. NGF-primed PC12 cells are capable of neurite regeneration when provided with an appropriate neurite promoting factor such as NGF. When primed PC12 cells were co-cultured with Schwann cells in the absence of exogenous NGF, PC12 cells that directly contacted Schwann cells became enlarged and flattened, attaining a neuron-like morphology within one day. When contact with Schwann cells was established, PC12 cells regenerated neurites by the first day of co-culture and these were maintained throughout the experiments (7 weeks). Most PC12 cells cultured in the same collagen-coated dishes with Schwann cells, but not directly in contact with them, failed to regenerate neurites. Instead, they began to proliferate, forming cell clusters. Neurite regeneration by PC12 cells in contact with Schwann cells was not blocked by antibody to NGF. These results demonstrate the presence of a neurite-promoting activity localized to the vicinity of the Schwann cell surface which is capable of eliciting regeneration and long-term maintenance of PC12 neurites in the absence of exogenous NGF. This activity does not appear to be due to NGF.
Article
Schwann cell mitosis has been demonstrated in chronically denervated cat tibial nerves re-innervated by axons regenerating from the proximal stump of a coapted peroneal nerve. Thymidine incorporation rose above baseline levels at the axon front, with no detectable increase in more distal regions occupied by denervated Schwann cells. Schwann cells therefore enter S phase upon the arrival of a regenerating axon in vivo as previously described in tissue culture. Intraneural treatment of the denervated distal stump with Mitomycin C prior to re-innervation delayed the subsequent appearance of myelin formation. This supports the notion that axonally stimulated division of Schwann cells is a prerequisite for myelination during nerve regeneration. Axonal advancement was also retarded by drug treatment, possibly because of a reduced level of trophic support provided by the compromised Schwann cells. A comparable absence of myelin and poor re-innervation was found in chemically untreated distal stumps that had been maintained in the denervated state for prolonged periods when Schwann cell columns are known to undergo progressive atrophy. These observations suggest that nerve repair should be delayed for limited periods if efficacious regeneration is desired.
Article
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 ofregeneration in insulin-deficient animals, 2.5 mm/day, was significantly lower than in control animals, 2.9 mm/day(P < 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 regenerationsw. After 6 days the sciatic nerves of insulin-deficient rats had regenerated 12.3 ±0.3mm(mean±S.E.M.), while the corresponding value for insulin-treated rats was 15.7 ±0.6 mm (P > 0.01). The streptozotocin-treated rats were found to have a 39% reduction in the serum level of insulin-like 1 growth factor-I (IGF-I)_compared to control rats (0.33 ± 0.22 μg/ml and 0.54 ± ml respectively, (P < 0.001). Insulin treatment 1830 1732 during the regeneration period completely restored the IGF-I level back to normal.
Article
Rat sciatic nerves can be transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers. Anatomical regeneration has been demonstrated across 10 mm long chambers containing both stumps, although little or no axonal outgrowth occurs in chambers omitting the distal stump or exceeding the 10 mm length. We have previously shown that chambers containing both proximal and distal stumps accumulate within days of implantation a clear fluid containing neuronotrophic factors (NTFs) directed to neurons from neonatal mouse dorsal root ganglia. We report here that these chamber fluids also have considerable neuronotrophic activity for chick embryo neurons from embryologic day 4 (E4) lumbar spinal cord, E12 sympathetic ganglia, E12 (but not E8) dorsal root ganglia and E8 ciliary ganglia. Thus, the neuronal types supported by trophic factors of these fluids include all those which contribute axons to the sciatic nerve, namely sensory, spinal motor, and sympathetic. In fluid collected 1 week after implantation, NTF levels directed to different neurons varied independently from one another in chambers with different nerve insertions, suggesting that these activities reside in separate factors. Fluid collected from chamber arrangements allowing little proximal fiber regrowth did not always contain correspondingly lower titers of NTFs. However, generally higher titers of all NTFs were found in chambers containing either or both nerve stumps that in nerve-free chambers. Fluids collected from nerve-containing chambers were subjected to heat, dialysis or trypsin treatments. The behavior of their neuronotrophic activities suggests their association with proteins.
Article
Immunohistochemistry has been used to demonstrate the presence of nerve growth factor (NGF)-like immunoreactivity in normal and sectioned mouse sciatic nerves. In normal nerves, immunoreactive material was not visible unless a silk ligature had previously been applied to constrict the nerves, and only then in the segment of nerve immediately distal to the ligation. Immunoreactivity was visible as early as 2 h after application of the ligature. When nerves were sectioned prior to ligation to prevent the transport of material from nerve terminals within innervated tissues, the NGF-like immunoreactivity continued to accumulate. This accumulation also occurred when a portion of the proximal stump from sectioned nerves was removed from the animal and placed in culture. Quantitative estimate of NGF concentrations with a sensitive immunoassay showed that the amount of NGF present within a segment of the proximal stump of sectioned nerves more than doubled in a 24 h period. The findings indicate that NGF is produced by cells within sectioned nerves, and further suggest that in the normal intact nerve at least a proportion of the NGF being transported derives from these cells.
Article
An in vivo technique which allows local application of drugs to the regenerating rat sciatic nerve for several days is presented. The sciatic nerve was transected at the knee level and a crush lesion was made proximal to the transection. The crush lesion and the nerve segment distal to it was enclosed in a chamber made of silicone tubing (STC). The STC was perfused via a catheter connected to a miniosmotic pump. Regeneration was evaluated with the 'pinch-test' at 3, 4 and 6 days after the enclosure of the nerve. The rate of regeneration in the STC-surrounded nerve segment was 3.5 mm/day after an initial delay of 1.6 days which are values similar to those in uncovered nerves with crush lesions. Perfusion of the STC with either vinblastine, cycloheximide, actinomycin D or mitomycin C inhibited regeneration. The effects were confined to the STC-covered region. Leakage of drugs was too small to affect the nerve outside the chamber. The results suggest that regeneration requires proliferation and protein synthesis in the cells surrounding the growing axons. This technique could be useful for studies of the local effects of various drugs, specific antibodies, potential growth factors etc. on regeneration of peripheral nerves.
Article
Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [3H]thymidine into DNA in these cells, the identity of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody alpha IR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125I-labeled IGF-I but not 125I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. alpha IR-3 competitively inhibits IGF-I-mediated stimulation of [3H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of alpha IR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of less than 1 microgram/ml, the effect of insulin to stimulate [3H]thymidine incorporation is not inhibited by alpha IR-3. However, the incremental effects of higher concentrations (greater than 1 microgram/ml) of insulin on [3H]thymidine incorporation are inhibited by alpha IR-3. alpha IR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself.
Article
The effect of human growth hormone (hGH) on regeneration of neuronal tissue have been studied in rats. A crush lesion was made on the sciatic nerve at the thigh level in intact or hypophysectomized rats. The hGH was administered systemically via subcutaneously implanted miniosmotic pumps. Regeneration was evaluated by the 'pinch-test' after 3, 4 and 6 days. Regeneration was significantly (P less than 0.05) impaired in hypophysectomized rats, but restored to normal after treatment with hGH (200 mIU/day). In intact rats treatment with 400 mIU/day hGH significantly (P less than 0.05) stimulated regeneration, whereas no effect was observed at 200 mIU/day. Immunoreactive insulin-like growth factor 1 (IGF-1) decreased in hypophysectomized rats, but rose again after hGH treatment. However, no consistent correlation between circulating IGF-1 and the rate of regeneration was found. Our results show that hGH can increase the rate of regeneration in peripheral nerves after injury. This can be due either to direct effects of hGH or to indirect effects via locally produced growth factors (e.g. somatomedins).
Article
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.
Article
Frog motor axons regenerate and grow back to reinnervate their targets, the original motor end plates, after a lesion. When the cutaneous pectoris muscle is cut away and a segment of peripheral nerve is placed in the vicinity of regenerating axons they turn and grow toward it. This is in marked contrast to the random pattern of axonal outgrowth seen in the absence of a target. The influence on the direction of axonal growth of motor neurons can be produced by a 1-mm segment of nerve satellite cells over a distance of more than 8 mm. The nerve satellite cells have no influence on the direction of growth of the regenerating axons after all the cells in the nerve segment have been killed, leaving only the Schwann cell basal lamina tubes intact. These results show that the cells in the segment of the nerve trunk contain cues that actively direct the growth of motor neurons. Two possible explanations for this effect might be (1) that the cells act indirectly by influencing the organization of the substructure over which axons regenerate or (2) that the nerve satellite cells release a diffusible substance that acts directly on the regenerating axons.
Article
The mechanisms influencing regeneration of peripheral nerves are incompletely known, but growth factors are supposed to play a key role. In the present study, we demonstrate, with the aid of immunohistochemical methods, that somatomedin C (Sm-C/insulin-like growth factor I/IGF-I) rapidly increased from low to high concentrations, reaching peak values in 2 weeks, in regenerating sciatic nerves of adult rats. In addition, IGF-I was demonstrated extracellularly, never observed in the control nerves. Reactive Schwann cells appeared to be the major source for IGF-synthesis. Higher concentrations were seen in tubulated nerves as compared to sutured ones. It is proposed that IGF-I exerts important growth supporting effects on regenerating peripheral nerves.
Article
The expression of insulin-like growth factor I (IGF-I, somatomedin C) was studied in regenerating skeletal muscle. Irreversible damage to skeletal muscle cells was induced in the extensor digitorum longus muscle (EDL) of adult rats by ischaemia, preceded by glycogen depletion, and the regeneration process was studied for periods up to 14 days after injury. The IGF-I was demonstrated by indirect immunofluorescence. Immunoreactivity against ribonucleotide reductase (RR) was used as a marker for DNA synthesis, that is, cell proliferation. Increased IGF-I immunoreactivity could be demonstrated within 24 h after injury in satellite cells, intramuscular nerves and in blood vessels. The IGF-I immunoreactivity remained virtually unchanged in the contralateral, undamaged EDL. An increasing number of satellite cells, expressing high IGF-I immunoreactivity, could be demonstrated in the injured EDL, and within 72 h myoblasts, expressing high IGF-I and RR immunoreactivity, were formed. Small immature muscle cells, displaying high IGF-I immunoreactivity, were observed 4 days after injury. Increased IGF-I immunoreactivity was still obvious in the regenerated muscle cells 14 days after injury while RR immunoreactivity was seen only in scattered satellite cells. It is concluded that IGF-I may act as a trophic factor during regeneration of skeletal muscle after injury.
Article
The scarcity of purified somatomedin/insulin-like growth factor (SM/IGF) has prevented investigation of the mechanisms of SM/IGF action. Recently insulin-like growth factor I (IGF-I) has been synthesized by recombinant DNA technology. The availability of large quantities of the biosynthetic IGF-I made it possible to study its effects by administering 120 micrograms/day via s.c. implanted minipump to rats for 7 days. After this 7 day administration of IGF-I, the body weight increased to 197.6 +/- 3.5% of initial values; the value was significantly greater than that of the control (179.4 +/- 3.7% of initial values, P less than 0.01). The body length and tibial epiphyseal width in IGF-I-treated rats were also significantly increased over those of control rats. The weights of kidneys, liver, testes and pituitary in IGF-I-treated rats were greater than those in control rats as well. These results provide a first demonstration that IGF-I stimulates growth in normal rats in vivo, and suggest that IGF-I might be useful in the treatment of growth retardation.
Article
Dorsal root ganglion neurons from embryonic rats, co-cultured with endoneurial explants from transected, adult rat sciatic nerve, extended neurites in the absence of exogenous nerve growth factor (NGF). The effect was seen with endoneurial explants from normal adult sciatic nerves or from nerves which had been permanently transected up to 51 days prior to explantation. The rate of outgrowth decreased at 5 and 7 days and reached a minimum at 14 days after transection. A second phase of increased neurite-promoting activity appeared in 28-, 35-, 41- and 51-day posttransection tissue. The early phase, but not the late phase, was partially inhibited by antisera to NGF.
Article
Pure human IGF I (43 and 103 micrograms/day) and IGF II (131 micrograms/day) were infused into hypophysectomized rats during 6 days by means of sc implanted minipumps. Their effects on several growth indices were compared with those of various doses of sc infused human growth hormone. Growth hormone infusion produced a dose-dependent rise of endogenous rat IGF from 39 (without growth hormone) to 86 microU equivalents/ml (with 400 mU hGH/day) as determined by a competitive protein binding assay with a human IGF standard. In rats receiving the two doses of IGF I, total serum IGF levels rose to 83 and 99 microU equivalents/ml, respectively, in those receiving the IGF II dose the total serum IGF level rose to 146 microU equivalents/ml. These increases corresponded to steady state levels of 168 and 286 ng/ml of immunoreactive insulin-like growth factor (IR-IGF) I and 320 ng/ml of IR-IGF II. IGF I, but not IGF II led to an increase in body weight similar to that induced by the low doses of hGH (12.5 and 25 mU, respectively). The rise of endogenous rat IGF as well as the infused human IGF I and II caused a widening of the tibial epiphysis and an increase of the [3H]thymidine incorporation into costal cartilage. With respect to these two indices IGF II was clearly less potent that IGF I. When expressed in microU equivalents of the protein binding assay, endogenous rat IGF induced by hGH appeared to be relatively more effective than infused human IGF I or II.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Explants of adult or 10-day-old rat sciatic and optic nerves were implanted as "bridges" through a silicon grease seal in a three-compartment chamber culture system, leading from a narrow center chamber to two adjacent side chambers. Dissociated newborn rat sympathetic or sensory neurons were plated into the center chamber and grown in the presence of optimal concentrations of nerve growth factor (NGF). By light microscopy, nerve fibers were seen to grow out of the sciatic nerve explants in the side chambers after 2 to 3 weeks. Electron microscopy showed large numbers of axons present inside the sciatic nerves, irrespective of the presence and number of living Schwann cells. Besides their tendency to fasciculate, axons grew with high preference on Schwann cell membranes and the Schwann cell side of the basal lamina, a situation identical to in vivo regeneration. In contrast to the sciatic nerves, no axons could be found under any condition in the optic nerves. This result points to the existence of extremely poor, non-permissive substrate conditions in the differentiated optic nerves which cannot be overcome by the strong fiber outgrowth-promoting effects of NGF.
Article
Axons of a cut peripheral nerve will grow across a gap (less than or equal to 10 mm in adult rodents) formed when the proximal and distal stumps are placed at opposite ends of an impermeable, inert tube, but will not grow to the end of a blind-ended tube in the absence of the distal stump [Williams et al, 1984]. Work reported here demonstrates that cultured peripheral nervous system (PNS) cells suspended in a collagen matrix will provide an effective milieu that directs and supports axonal regeneration from a severed nerve into a blind-ended tube in the absence of a distal stump. Adult mouse sciatic nerves were cut and the proximal stumps were inserted into close-ended tubes that contained either a collagen matrix containing dissociated cells from embryonic mouse dorsal root ganglia (DRG), a collagen matrix saturated with medium conditioned by cultured DRG cells, or a collagen matrix saturated with fresh medium. In all three cases cellular cables formed that ran the full length of the tubes, but myelinated and unmyelinated axons regenerated the length of the tubes only when cultured cells had been added. The critical factor in influencing axonal regeneration through the length of the tubes was the presence of cultured cells, since collagen alone or collagen saturated with conditioned medium did not support axonal regrowth even though cells had migrated into the chambers from the proximal stumps in all cases. Ordered structure was not a requisite for axonal growth, since the cultures consisted of random arrays of dissociated cells.
Article
We re-examined the hypothesis of Cajal³, later refuted by Weiss and Taylor²⁰, that cells in distal stumps of transected peripheral nerves exert an attractive (tropic) effect on regenerating axons. This question was re-assessed in vivo using surgical materials and assay procedures not available to those workers. Proximal stumps of transected rat sciatic or cat peroneal nerves were inserted into the single inlet end of a hollow, Y-shaped Silastic implant. Regenerating axons were provided with alternative targets consisting of a vacant arm vs one occupied by a sciatic nerve graft (rat), or a tibial (Tout) vs peroneal (Pout) distal nerve stump (cat). In some cases Pout was rendered metabolically compromised relative to Tout by exposing the former to dry ice and inhibitors of DNA and RNA synthesis. At 4.5 or 6 weeks postoperatively, the number of regenerating axons in each fork of the implant was assessed by morphometric analysis (total number of non-myelinated and myelinated axons greater than 1 μm in diameter at 4.5 weeks, and total number of myelinated axons at 6 weeks postoperatively), or by quantification of an axonally transported label.
Article
Cells prepared from the body walls of chicken embryos were plated in the absence of serum. Insulin-like growth factors (IGFs) barely stimulated cell replication, but preferentially enhanced the differentiation of muscle cells. Myoblast fusion was favoured in the presence of IGF (or insulin). Concomitantly, acetylcholinesterase activity increased. IGF I and IGF II were equipotent and active in low physiological concentrations, in contrast to insulin, which was known for a long time to exert such effects at pharmacological concentrations.
Article
Adult mice were divided into groups, all of which had the right sciatic nerve cut. In some groups, both the proximal and distal stumps were put into a plastic tube after various manipulations, while in other groups one or other stump was left out. Observations following survival periods of between 15 and 60 days showed that successful regeneration took place only in animals in which both proximal and distal stumps were present in the plastic tube and providing the cut ends of both stumps were facing each other. If the distal stumps were excluded from the tube or bent through 180°, regeneration occurred initially, but was eventually ineffective. If the distal stump alone was inserted in the tube, a proliferation of Schwann cells, fibroblasts and regularly aligned collagen occurred. These findings support the hypothesis that the distal stump is essential for successful regeneration, but that in order to enhance it, the peripheral segment must be perfectly aligned with the proximal stump within the tube. Reasons for the necessity of such an alignment are discussed.
Article
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 micrograms of protein per culture microwell or more was added. Half maximal stimulation was obtained with addition of axolemmal fraction equivalent to 4 micrograms 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, and of 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.
Article
Nerve segments approximately 7 mm long were excised from the predegenerated sciatic nerves of mice, and treated 5 times by repetitive freezing and thawing to kill the Schwann cells. Such treated nerve segments were grafted into the original places so as to be in contact with the proximal stumps. The animals were sacrificed 1, 2, 3, 5, 7 and 10 days after the grafting. The grafts were examined by electron microscopy in the middle part of the graft, i.e. 3-4 mm distal to the proximal end and/or near the proximal and distal ends of the graft. In other instances, the predegenerated nerve segments were minced with a razor blade after repetitive freezing and thawing. Such minced nerves were placed in contact with the proximal stumps of the same nerves. The animals were sacrificed 10 days after the grafting. Within 1-2 days after grafting, the dead Schwann cells had disintegrated into fragments. They were then gradually phagocytosed by macrophages. The basal laminae of Schwann cells, which were not attacked by macrophages, remained as empty tubes (basal lamina scaffolds). In the grafts we examined, no Schwann cells survived the freezing and thawing process. The regenerating axons always grew out through such basal lamina scaffolds, being in contact with the inner surface of the basal lamina (i.e. the side originally facing the Schwann cell plasma membrane). No axons were found outside of the scaffolds. One to two days after grafting, the regenerating axons were not associated with Schwann cells, but after 5-7 days they were accompanied by Schwann cells which were presumed to be migrating along axons from the proximal stumps. Ten days after grafting, proliferating Schwann cells observed in the middle part of the grafts had begun to sort out axons. In the grafts of minced nerves, the fragmented basal laminae of the Schwann cells re-arranged themselves into thicker strands or small aggregations of basal laminae. The regenerating axons, without exception, attached to one side of such modified basal laminae. Collagen fibrils were in contact with the other side, indicating that these modified basal laminae had the same polarity in terms of cell attachment as seen in the ordinary basal laminae of the scaffolds.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
A new peripheral nerve forms across a 10 mm gap within a silicone chamber regeneration model when the distal segment of a transected sciatic nerve, connected to its end organs, is sutured into the distal end of the chamber. We have tested the ability of other tissue inserts to support axonal regeneration in the chamber. When an isolated 2 mm piece of sciatic nerve was sutured into the distal end, fibrin matrix formation, cell immigration and axonal regeneration were identical to those occurring in the control. When the distal nerve insert was replaced with a 2 mm piece of skin or a ligation, a matrix did not form and subsequent cell immigration and axonal regeneration did not occur. When a 2 mm piece of tendon was inserted, a matrix did form at 1 week, but a structure across the gap was observed at later time periods in only 2 out of 7 chambers. The matrix either dissolved before cells could enter the chamber or did not promote cellular immigration and subsequent axonal regeneration. When the distal end was left open, a matrix formed and cells from the reactive tissue outside the chamber entered the matrix and formed a granulation tissue bridge across the gap. This tissue failed to support axonal regeneration; at 3 weeks, axons stopped 1 mm beyond the proximal stump at the interface with the granulation tissue. Thus, matrix formation and a cellular bridge are necessary but not sufficient to ensure regeneration. Successful regeneration across the silicone chamber gap requires humoral and/or cellular contributions available from peripheral nervous tissue and not from the other tested tissues.
Article
This chapter discusses the chemical and biological characterization, and properties of the insulin-like growth factors (IGF). The chapter also discusses their possible physiological role. Blood is a large reservoir of insulin-like activity. Insulin represents only a small portion of this activity. IGF I and II are polypeptides with molecular weights of 7649 and 7471. They contain 70 and 67 amino acid residues, respectively, each in a single polypeptide chain with three intra-chain disulfide bridges. Each of the two polypeptides contains two regions—domains—in which parts of the sequences can be aligned to identical parts in human pro-insulin: 38% of the sequence of residues 42 to 70 in IGF I and 44% of the sequence of residues 41 to 67 in IGF II (termed A-domains), are identical to the insulin A-chain. As in pro-insulin, the A- and B-domains are connected by a C-domain. However, in contrast to the C-peptide of pro-insulin, which consists of 35 amino acids, the C-regions of IGF I and II are only 12 and 8 residues long, respectively.
Article
Although growth hormone stimulates the growth of hypophysectomized rats, it has long been proposed1,2 that the effects are not direct but instead are mediated by the somatomedin peptides. Two of these are insulin-like growth factor I (IGF I) and insulin-like growth factor II (IGF II)3, so called because they are closely related to insulin in structure4,5. IGF I and IGF II have somatomedin activity in vitro6,7 but until now insufficient amounts of the peptides have been available to test their in vivo activity. We now report that pure IGF I stimulates the growth of hypophysectomized rats in a dose-dependent manner. This strongly supports the notion that the action of growth hormone is mediated by peptides of the somatomedin family.
Article
Axolemma-enriched fractions isolated from myelinated axons of rat and bovine CNS and PNS will stimulate a quiescent population of cultured rat Schwann cells to proliferate. The mitogen in the axolemma-enriched fraction is not lost in the extensive washing used to isolate the membrane fraction and is sensitive to heat and trypsin, suggesting that it is an integral membrane protein or glycoprotein. The axolemma-enriched preparations should provide an adequate source of Schwann cell mitogen for solubilization and purification of this membrane bound mitogen.
Article
The range of growth-promoting influences from a distal nerve stump on a regenerating proximal stump was determined using an experimental system in which a gap between cross-anastomosed rat sciatic nerves was encased by a cylindrical silicone chamber. Two arrangements were examined after 1 month in situ: A proximal-distal (PD) system in which both proximal and distal stumps were introduced into the ends of the chamber, and a proximal-open (PO) system in which the distal stump was omitted. When the gap was 6 mm long, a regenerated nerve extended all the way through the chamber in both the PD and PO systems. When the gap was increased to 10 mm, a similar regrowth occurred in the PD chamber, whereas in the PO chamber proximal regrowth was partial or nonexistent. When the gap was increased to 15 mm, no regeneration occurred, even in the presence of the distal stump. These observations confirm that the distal stump influences proximal regeneration and indicate that this influence can act only over a limited distance or volume. Such an influence could consist of humoral agents which support nerve growth and/or outgrowth from the distal stump.
Article
This paper describes a surgical method of manipulating feline peripheral nerve regeneration to separate spatially the temporal sequence of events of axon-Schwann cell interaction during nerve fibre formation. The method allows regenerating axons from the peroneal nerve to reinnervate the distal stump of an axon- and myelin-free, Schwann cell-enriched, chronically denervated tibial nerve distal stump. Three zones can be morphologically delineated in the tibial nerve stump after three weeks of reinnervation: (1) a proximalmyelinated zone, (2) a more distal, non-myelinated, axon-Schwann cellcontact zone, and (3) a distal axon-free Schwann cellnon-contact zone. The distal limit of the second zone can be determined accurately by the front of an axonally transported label. The large volume of available tissue makes this method suitable for interdisciplinary studies to elucidate the control of axon elongation, axon growth, and axon-Schwann cell inter-relationships.