Growth-associated protein-43 expression in cocultures of dorsal root ganglion neurons and skeletal muscle cells with different neurotrophins.

INTRODUCTION: Both target skeletal muscle (SKM) cells and neurotrophins (NTs) are essential for the maintenance of neuronal function and nerve-muscle communication. The effects of different NTs and SKM cells on growth-associated protein-43 (GAP-43) expression in dorsal root ganglion (DRG) neurons have not been clarified. METHODS: The morphological relationship between DRG neurons and SKM cells in neuromuscular cocultures was observed by scanning electron microscopy. The levels of GAP-43 and its mRNA were determined after administration of different NTs. RESULTS: DRG neurons demonstrated dense neurite outgrowth in the presence of NTs. Distinct NTs promoted GAP-43 and its mRNA expression in neuromuscular cocultures of DRG neurons and SKM cells. CONCLUSIONS: These results offer new clues for a better understanding of the effects of distinct NTs on GAP-43 expression in DRG sensory neurons in the presence of target SKM cells and implicate NTs and target SKM cells in DRG neuronal regeneration.

Insulin-like growth factor-1 regulates neurite outgrowth and neuronal migration from organotypic cultured dorsal root ganglion.

ABSTRACT Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor and plays an important role in promoting axonal growth from neurons. Whether IGF-1 could promote neurite outgrowth and neuronal migration of dorsal root ganglion (DRG) explants in vitro remains unknown. In the present study, organotypic rat DRG explant culture model was established. Using this unique culture system, outgrowth of neurites from the peripheral nerve attached to DRG explant and migration of neurons from DRG explant to the peripheral area were quantified in the presence (5 nmol/L, 10 nmol/L, 20 nmol/L) or absence of IGF-1. The number of nerve fiber bundles extended from DRG explant increased significantly in the presence of IGF-1 (5 nmol/L, 19.25 ± 3.11, p < .05; 10 nmol/L, 20.92 ± 2.31, p < .01; 20 nmol/L, 23.00 ± 4.09, p < .001) as compared with that in the absence of IGF-1 (16.58 ± 2.94). The number of neurons migrated from DRG explant increased significantly in the presence of IGF-1 (5 nmol/L, 104.08 ± 16.70, p < .05; 10 nmol/L, 115.25 ± 13.68, p < .001; 20 nmol/L, 138.75 ± 18.05, p < .001) as compared with that in the absence of IGF-1 (90.25 ± 8.53). These data implicated that IGF-1 could promote neurite outgrowth and neuronal migration from DRG explants in vitro.

Insulin-like growth factor-1 modulates Ca2+ homeostasis and apoptosis of cultured dorsal root ganglion neurons with excitotoxicity induced by glutamate.

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor and a potent anti-apoptotic factor. IGF-1 plays an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons and prevents apoptosis in DRG neurons. Whether IGF-1 could modulate Ca2+ homeostasis and apoptosis of sensory DRG neurons with excitotoxicity induced by glutamate (Glu) is still unknown. In the present study, primary cultured DRG neurons were used to determine the effects of IGF-1 on Ca2+ homeostasis and apoptosis of sensory DRG neurons with excitotoxicity induced by Glu. Intracellular Ca2+ concentration ([Ca2+]i) in isolated DRG neurons using the fluorescent Ca2+ indicator fura-3 was measured by confocal laser scanning microscope (CLSM). Procaspase-3 expression was detected by Western blot analysis. Application of 0.2 mmol/L Glu evoked an increase in [Ca2+]i, confirming the excitatory effect of Glu at this stage. The decrease of procaspase-3 expression levels after application of 0.2 mmol/L Glu suggested the apoptotic effects of Glu. These effects could be inhibited by the presence of IGF-1. In conclusion, we demonstrated that IGF-1 could modulate Ca2+ homeostasis and apoptosis of sensory DRG neurons with excitotoxicity induced by Glu. Both Ca2+ homeostasis and caspase-3 processing were implicated as the underlying neuroprotective mechanisms of IGF-1.

Pioglitazone inhibits hypertrophy induced by high glucose and insulin in cultured neonatal rat cardiomyocytes.

The aim of the present study was to determine whether the antidiabetic agent pioglitazone has a direct inhibiting effect on myocardial hypertrophy induced by high glucose and insulin in primary cultured neonatal rat cardiomyocytes. Culture preparations of ventricular muscle cells newborn rats were utilized. At 72 h of culture age, the cardiomyocytes were incubated for another 48 h with 25.5 mmol/L glucose plus 0.1 micromol/L insulin (group 2), 25.5 mmol/L glucose and 0.1 micromol/L insulin plus 10 micromol/L pioglitazone (group 3), 10 micromol norepinephrine (group 4), respectively. Cells cultured continuously in medium served as control (group 1). Cellular surface area, protein content, atrial natriuretic factor (ANF) mRNA, and cardiotrophin-1 (CT-1) mRNA were assessed after treatment with different agents. All those parameters increased significantly after treatment with high glucose and insulin as compared with control (P < 0.01). These effects were inhibited markedly by pioglitazone. The cellular surface area and ANF mRNA in group 3 were decreased as compared with group 2 (P < 0.01). The protein content and CT-1 mRNA in group 3 were also decreased as compared with group 2 (P < 0.05). We concluded that a the cellular level myocardial hypertrophy induced by high glucose and insulin was inhibited directly by pioglitazone in primary cultured cardiac myocytes. CT-1 may be involved in myocardial hypertrophy induced by high glucose andinsulin and inhibiting effects of pioglitazone on myocardial hypertrophy.

Expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 in cocultures of dissociated DRG neurons and skeletal muscle cells in administration of NGF or NT-3.

Both neurotrophins (NTs) and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons and nerve-muscle communication. However, much less is known about the association of target SKM cells with distinct NTs on the expression of mRNAs for preprotachykinin (PPT), calcitonin-gene related peptide (CGRP), neurofilament 200 (NF-200), and microtubule associated protein 2 (MAP-2) in dorsal root ganglion (DRG) sensory neurons. In the present study, a neuromuscular coculture model of dissociated dorsal root ganglion (DRG) neurons and SKM cells was established. The morphology of DRG neurons and SKM cells in coculture was observed with an inverted phase contrast microscope. The effects of nerve growth factor (NGF) or neurotrophin-3 (NT-3) on the expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 was analyzed by real time-PCR assay. The morphology of DRG neuronal cell bodies and SKM cells in neuromuscular coculture at different conditions was similar. The neurons presented evidence of dense neurite outgrowth in the presence of distinct NTs in neuromuscular cocultures. NGF and NT-3 increased mRNA levels of PPT, CGRP, and NF-200, but not MAP-2, in neuromuscular cocultures. These results offer new clues towards a better understanding of the association of target SKM cells with distinct NTs on the expression of mRNAs for PPT, CGRP, NF-200 and MAP-2, and implicate the association of target SKM cells and NTs with DRG sensory neuronal phenotypes.

Regulatory effects of insulin-like growth factor-1 on the expression of sensory neuropeptide mRNAs in cultured dorsal root ganglion neurons with excitotoxicity induced by glutamate.

OBJECTIVE: To determine the effects of insulin-like growth factor-1 (IGF-1) on the expression of preprotachykinin (PPT) mRNA encoding substance P (SP) and calcitonin gene-related peptide (CGRP) mRNA in cultured dorsal root ganglion (DRG) neurons with excitotoxicity induced by glutamate (Glu). METHODS: DRGs were dissected from embryonic day 15 Wistar rats. DRG neurons were dissociated and cultured for 48 h and then exposed to Glu (0.2 mmol/L) or Glu (0.2 mmol/L) plus IGF-1 (5 nmol/L, 10 nmol/L and 20 nmol/L) for 12 h. The DRG neurons in control group were exposed to only growth media throughout the experiment. After that, the living DRG neurons were observed under inverted phase contrast microscope and microphotographs were taken. The expression levels of PPT and CGRP mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: IGF-1 could inhibit Glu-induced shortening of neurite. Besides, IGF-1 could significantly increase the levels of PPT mRNA and CGRP mRNA in primary cultured DRG neurons with Glu-induced excitotoxicity, in a dose-dependent manner. CONCLUSION: IGF-1 may exert neuroprotective effects on DRG neurons against Glu-induced excitotoxicity, probably through regulating the expression levels of PPT and CGRP mRNAs.