Neuroprotective Effects Of Insulin-like Growth Factor 1 On Cultured Dorsal Root Ganglion Neurons With Neurotoxicity Induced By Glutamate

Insulin-like growth factor 1(IGF-1),was reported as a multifunctional trophic factor for neurons which has extensive expression in vivo.IGF-1 has been shown to have neurotrophic actions on different types of central and peripheral nervous system neurons in vitro.IGF-1 could inhibit neurons apoptosis induced by various factors.Several lines of evidence converged to indicate that IGF-1 participates in structural and functional plasticity of the dorsal root ganglia(DRG) neurons.IGF-1 may induct the intracellular events,such as mitochondrial transport or accumulate at regions of focal IGF-1 stimulation,and intracellular Ca²⁺ homeostasis.However,it's not clear whether IGF-1 has neuroprotective effect of IGF-1 on cultured DRG neurons injured by Glu.Glu,one of excitatory neurotransmitters,could be found in the mammaliam central and peripheral nervous system.Excess release of Glu can lead to N-Methyl-D-aspartate(NMDA) receptor over-activation which is a key excitotoxic stimulus that leads to increases in intracellular calcium and activation of downstream signaling pathways and then induce cell apoptosis.Therefore,in this study we exert Glu(200μmol/L) on dissociated cultured DRG neurons to determine the neuroprotective effects of IGF-1 on cultured DRG neurons injured by Glu.The abstract of the full text is as follows:In this study,DRG neurons of Wistar rats embryoes were cultured in vitro for 48 h and then the cultures were divided into 3 groups.There was not any agents was added to the cultures of the normal control group.At the culture age of 48h,Glu was added to the media at a final concentration of 200μmol/L in the injured group.In the groups of IGF-1,after Glu was added,IGF-1 was added to the media at a final concentration of 10nmmol/L.All the above groups were incubated at 37℃5%CO₂ to continue.The living cells were observed by inverse contrast microscopy at different culture age.At the designed culture age all the samples were processed as follows:The identification of dissociated DRG neurons by using fluorescent double staining of microtubule associated protein 2(MAP2) and 4',6-Diamidino-2-phenylindole(DAPI);The apoptosis rate of DRG cells was determined by using flow cytometry;The fluorescent intensity of intracellular Ca²⁺ by using confocal laser scanning microscope(CLSM);The level of procaspase-3 protein by using Western blot.The neuroprotective effects of IGF-1 on cultured DRG neurons injured by Glu.were analyzed according to the above data.The results are as follows:(1)The results of MAP2 and DAPI double staining:Fluorescent photomicrographs of MAP2 and DAPI double staining of dissociated DRG neurons at 48 h of culture age showed that the majority of cells in our study were neurons,the minority of cells are were non-neurons.(2) The results of the morphology character of DRG neurons observed by inverted phase contrast microscope:The single layered DRG neurons sent neurites to form a network in normal control group.The DRG neuronal cell bodies swelled or shrunk and the neurites broke,became shorter or even disappeared after incubated at a concentration of 200μmmol/L of Glu.In the groups that glutamic acid with IGF-1,the shape of neuronal cell bodies or neurite networks was almost the same as the normal control group.(3) The apoptosis rate of cells increased after incubated at a concentration of 200μmmol/L of Glu for 24h in DRG cultures.IGF-1 can decrease the apoptosis rate of cells injured by Glu obviously.(4) The results of fluorescent intensity of intracellular Ca²⁺:The fluorescent intensity of Fluo-3 labeled Ca²⁺ increased in the Glu injured DRG and neurons.In the groups that Glu with IGf-1,the fluorescent intensity decreased in most of the DRG neurons,only some injured neurons had strong fluorescent signals.Quantitive analysis indicated that the fluorescent intensity in IGF-1 with Glu group was lower than that in glutamic acid group(P＜0.001).The fluorescent intensity in the normal group was lower than that in Glu group(P＜0.001).There was no significance of the fluorescent intensity between IGF-1 with Glu group and normal control group (P＞0.05).(5) The results of procaspase-3 expression:The levels of procaspase-3 protein expression decreased after Glu treatment.But the levels of procaspase-3 protein expression increased after IGF-with Glu treatment.These results indicated that IGF-1 might prevent the morphological functional alterations of cultured DRG neurons in vitro injured by Glu.The neuroprotective mechanisms may be:Exogenous IGF-1 may prevent cultured DRG and spinal cord neurons from intracellular Ca²⁺ overloaded and maintain intracellular Ca²⁺ homeostasis.IGF-1 may be inhibit neuronal apopatosis by influencing caspase-3 activity,then protects DRG neurons from neurotoxicity induced by Glu.