An Experimental Study Of The Effects Of IGF-1on Attenuation Of Paclitaxel-induced Neurotoxicity Of Dorsal Root Ganglion Neurons

Paclitaxel (PT) is one of the most commonly used anti-neoplastic agents and produces a dose-limiting side effect that is chronic sensory peripheral neuropathy and subsequent neuropathic pain that is resistant to standard analgesics. PT-induced neurotoxicity is a significant problem associated with successful treatment of cancers. The pathogenesis of neurotoxicity or neuropathy induced by PT is still unclear. The neuropathic pain symptoms caused by PT are relevant to the neurotoxicity of dorsal root ganglion (DRG) primary sensory neurons. Hence, to explore the alterations of neurochemical phenotypes of DRG neurons is important for clarifying the mechanisms of PT-neurotoxicity. To explore the attenuation of PT-neurotoxicity is particular critical fro improving PT-induced neuropathy. Primary cultured DRG neurons and PT-induced neuropathy rat animal model were used for determining the effects of insulin-like growth factor-1(IGF-1) on attenuation of paclitaxel-induced neurotoxicity of DRG neurons.Part I The protective effects of IGF-1on cultured dorsal root ganglion neurons with paclitaxel-induced neurotoxicity The neuropeptide-immunoreactive (IR) and neurofilament (NF)-IR neurons are two major neurochemical phenotypical classes in DRG. Calcitonin gene-related peptide (CGRP), a sensory neuron-associated neuropeptide expressed in DRG neurons, produced multi-biological effects. CGRP-IR neurons represent neuropeptide-IR neurons in DRG Neurofilaments (NFs) are neuron-specific intermediate filaments. Neurofilament-200plays an important role in healthy neurons. NF-200-IR neurons represent NF-IR neurons in DRG Whether PT influences neurochemical phenotypes of DRG neurons remains unknown.IGF-1is a growth-promoting peptide hormone that has been shown to have neurotrophic properties. IGF-1promoted neuronal survival by activating its tyrosine kinase receptor IGF-1R. IGF-1and its receptor IGF-1R are expressed in DRG neurons. IGF-1and its receptor IGF-1R signalling play an important role in promoting axonal growth and regulating sensory neuropeptide expression. Whether IGF-1through regulating extracellular signal-regulated protein kinase1/2(ERK1/2) and phosphatidylinositol3-kinase (PI3K)/serine-threonine kinase (Akt) signaling pathways regulates neurochemical phenotypes of DRG neurons with PT-induced neurotoxicity is still to be clarified.To determine the dose effect of PT on neurite outgrowth, The DRG neuronal cultures at48h post-culture were exposed to different doses of PT (0.01μmol/L,0.1μmol/L, and1μmol/L, respectively) for an additional24h. After that, the neuronal growth status was observed under phase contrast miroscope and the total neurite length of each neuron was measured by fluorescent labeling of microtubule-associated protein2(MAP2). The result showed that PT exposure caused a neurite retraction in a dose-dependent manner.To assess the neuroprotective effects of IGF-1on DRG neurons with PT-induced neurotoxicity, the primary cultured DRG neurons were exposed to PT (1μmol/L), PT (1μmol/L) plus IGF-1(20nmol/L), ERK1/2inhibitor PD9805930min before treatment with PT (1μmol/L) plus IGF-1(20nmol/L), PI3K inhibitor LY29400230min before treatment with PT (1μmol/L) plus IGF-1(20nmol/L) for an additional24 h, respectively. The DRG neurons were continuously exposed to culture media as a control. After that, the neuronal growth status was observed under phase contrast miroscope. The total neurite length of each neuron was measured by fluorescent labeling of MAP2. The cell viability was evaluated by a WST-1kit. The apoptotic neuronal cell death was observed by using Hoechst33342staining. The alterations of neuronal phenotypes were determined by double immunofluorescent labeling of MAP2and CGRP or NF-200. The mRNA levels of CGRP and NF-200were analyzed with real-time PCR. The protein levels of CGRP and NF-200were analyzed with Western blot assay. The results showed that PT exposure caused a decrease of cell viability and increase of ratio to apoptotic cells which could be reversed by IGF-1. The percentage of CGRP-IR neurons and NF-200-IR neurons, mRNA and protein levels of CGRP and NF-200decreased significantly after treatment with PT. IGF-1administration had protective effects on CGRP-IR neurons, but not NF-200-IR neurons. Either ERK1/2inhibitor PD98059or PI3K inhibitor LY294002blocked the effect of IGF-1. The results imply that IGF-1may through attenuate apoptosis to improve neuronal cell viability and promote neurite growth of DRG neurons with PT-induced neurotoxicity. Moreover, these results support an important neuroprotective role of exogenous IGF-1on distinct subpopulations of DRG neurons which is responsible for skin sensation. The effects of IGF-1might be through ERK1/2or PI3K/Akt signaling pathways. These findings provide experimental evidence for IGF-1administration to alleviate neurotoxicity of distinct subpopulations of DRG neurons induced by PT.Part II The attenuation effects of IGF-1on neuropathic pain symptoms induced by paclitaxel in ratsPT-induced neuropathic pain is resistant to standard analgesics. To improve neuronal functional status through neurotrophic actions is a potential research area for clinical therapy development. The improvement of different neuronal injury insults and the paticular neurotrophic actions of IGF-1might provide novel therapeutic targets for relieving neuropathic pain induced by PT. In the present study, the PT-induced neuropathic pain rat animal model was established by intraperitoneally injection (i.p.). Mechanical allodynia and thermal hyperalgesia were measured after intrathecal injection of IGF-1, ERK1/2inhibitor PD98059+IGF-1, or PI3K inhibitor LY294002+IGF-1. The expression of CGRP and NF-200their mRNAs in L4-6DRG was also determined. The results showed that intrathecal injection of IGF-1relieved mechanical allodynia and thermal hyperalgesia behaviors. The mRNA and protein levels of CGRP increased and the mRNA and protein levels of NF-200decreased significantly after PT treatment. The levels of CGRP and its mRNA, but not NF-200and its mRNA, recovered to normal after intrathecal administration of IGF-1. Either ERK1/2inhibitor PD98059or PI3K inhibitor LY294002blocked the effect of IGF-1on animal pain behavoirs and the levels of CGRP and its mRNA. These results imply that IGF-1, throuh activating ERK1/2and PI3K/Akt signaling pathways, to relieve neuropathic pain induced by PT. These data provide novel experimental evidence for improving PT-induced neuropathy.