新型温敏型肝素-泊洛沙姆水凝胶包载神经生长因子对糖尿病外周神经损伤修复的作用
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摘要
目的探讨神经生长因子-肝素泊洛沙姆水凝胶(NGF-HP)对糖尿病大鼠坐骨神经损伤的修复作用。方法成年雄性SD大鼠腹腔注射链脲佐菌素以制备糖尿病(DM)模型。成模后,暴露并分离术侧的坐骨神经,运用静脉夹压迫神经以造成外周神经损伤(PNI)。同时,将其随机分为模型组(PNI-diabetics),游离NGF组(free NGF)及NGF-HP水凝胶组(NGF-HP hydrogel)。每组给予对应的药物治疗,同时在神经损伤后的每周进行斜板试验和BBB评分。30 d后,处死各组大鼠并收集术侧的坐骨神经,运用免疫印迹检测各组神经内部结构和功能蛋白的表达,运用免疫荧光、Masson三色染色和透射电镜观察各组神经内轴突和髓鞘的再生。结果该水凝胶可装载一定剂量的神经生长因子(NGF)并缓控其释放。体内实验结果表明,单次注射一定体积的NGF-HP水凝胶后,PNI-diabetics大鼠运动功能恢复良好,神经内GAP-43、MAP-2、MBP及S-100的含量出现显著地上调,神经纤维的再生效果良好且脱髓鞘情况改善显著。结论 NGF-HP对PNI-diabetics疾病具有优越的疗效,为该药应进入临床提供理论依据。
OBJECTIVE To investigate the effects of NGF-HP thermosensitive hydrogel on facilitating structural and functional regeneration in diabetic rats with sciatic nerve crush injury. METHODS Eight-week-old male SD rats( 200-220 g) were intraperitoneally injected wither steptozocin( STZ) to induce diabetes. After success of model establishment,the sciatic nerve of the diabetes rats were made crushed through two vascular clips force. Muscle and skin were then closed with 5-0 stitches. Following surgery,the rats were randomly divided into three groups: PNI-diabetics group,free NGF group and NGF-HP hygrogel group. Each group received corresponding therapeutic drugs through a microsyringe. The motor recovery in all tested rats was assessed using Basso-Beattie-Bresnahan( BBB) locomotion scale and inclined plane test at indicated time points. After 30 d,rats were sacrificed,the crushed nerve and corresponding gastrocnemius muscle were harvested and the pathology index was assessed. The expressions of structural and functional proteins were detected through immunoblotting. The improvement of axon and myelination regeneration were evaluated via immunofluorescence,Masson's trichrom stain and transmission electron microscope. RESULTS NGF-HP not only had a good affinity for a certain amounts of nerve growth factor( NGF),but also controlled its release in a steady fashion in vitro. In vivo,compared with administration of direct free NGF,single injection of NGF-HP hydrogel was more effective at upregulating the expression of nerve associated structural and functional proteins,enhancing axonal regeneration and remyelination,as well as improving motor function recovery. CONCLUSION This new type of hydrogel loaded with NGF shows striking effects on functional and morphometric recovery on peripheral nerve injury( PNI) following diabetes,which may provide a theoretical basis strategies for remedying PNI-diabetes in clinical populations.
OBJECTIVE To investigate the effects of NGF-HP thermosensitive hydrogel on facilitating structural and functional regeneration in diabetic rats with sciatic nerve crush injury. METHODS Eight-week-old male SD rats( 200-220 g) were intraperitoneally injected wither steptozocin( STZ) to induce diabetes. After success of model establishment,the sciatic nerve of the diabetes rats were made crushed through two vascular clips force. Muscle and skin were then closed with 5-0 stitches. Following surgery,the rats were randomly divided into three groups: PNI-diabetics group,free NGF group and NGF-HP hygrogel group. Each group received corresponding therapeutic drugs through a microsyringe. The motor recovery in all tested rats was assessed using Basso-Beattie-Bresnahan( BBB) locomotion scale and inclined plane test at indicated time points. After 30 d,rats were sacrificed,the crushed nerve and corresponding gastrocnemius muscle were harvested and the pathology index was assessed. The expressions of structural and functional proteins were detected through immunoblotting. The improvement of axon and myelination regeneration were evaluated via immunofluorescence,Masson's trichrom stain and transmission electron microscope. RESULTS NGF-HP not only had a good affinity for a certain amounts of nerve growth factor( NGF),but also controlled its release in a steady fashion in vitro. In vivo,compared with administration of direct free NGF,single injection of NGF-HP hydrogel was more effective at upregulating the expression of nerve associated structural and functional proteins,enhancing axonal regeneration and remyelination,as well as improving motor function recovery. CONCLUSION This new type of hydrogel loaded with NGF shows striking effects on functional and morphometric recovery on peripheral nerve injury( PNI) following diabetes,which may provide a theoretical basis strategies for remedying PNI-diabetes in clinical populations.
引文
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[16] BASSO D M,BEATTIE M S,BRESNAHAN J C. A sensitive and reliable locomotor rating scale for open field testing in rats[J]. J Neurotrauma,1995,12(1):1-21.
[17] MOHAMMADI R,SANAEI N,AHSAN S,et al. Stromal vascular fraction combined with silicone rubber chamber improves sciatic nerve regeneration in diabetes[J]. Chin J Traumatol(中华创生杂志),2015,18(4):212-218.
[18] ZHAO Y Z,LV H F,LU C T,et al. Evaluation of a novel thermosensitive heparin-poloxamer hydrogel for improving vascular anastomosis quality and safety in a rabbit model[J]. PLoS One,2013,8(8):e73178.
[19] GRASSELLI G,STRATA P. Structural plasticity of climbing fibers and the growth-associated protein gap-43[J]. Front Neural Circuits,2013,7(3):25-34.
[20] MERCERON-MARTINEZ D,ALMAGUER-MELIAN W,ALBERTIAMADOR E,et al. Amygdala stimulation promotes recovery of behavioral performance in a spatial memory task and increases gap-43and map-2 in the hippocampus and prefrontal cortex of male rats[J]. Brain Res Bull,2018,142(3):8-17.
[21] ZIERATH D,KUNZE A,FECTEAU L,et al. Promiscuity of autoimmune responses to mbp after stroke[J]. J Neuroimmunol,2015,285(29):101-105.
[22] MOHANRAJ A,SRINIVASAN S. Role of s-100 immunostaining in demonstration of nerve changes and quantification of dendritic cells in leprosy[J]. J Clin Diagn Res,2014,8(3):38-40.
[23] MOORE A M,KASUKURTHI R,MAGILL C K,et al. Limitations of conduits in peripheral nerve repairs[J]. Hand,2009,4(2):180-186.
[24] FORBES S J,ROSENTHAL N. Preparing the ground for tissue regeneration:from mechanism to therapy[J]. Nat Med,2014,20(8):857-868.
[25] GIANNACCINI M,CALATAYUD M P,POGGETTI A,et al.Magnetic nanoparticles for efficient delivery of growth factors:stimulation of peripheral nerve regeneration[J]. Adv Healthc Mater,2017,6(5):57-71.
[26] GORDON T. Neurotrophic factor expression in denervated motor and sensory schwann cells:relevance to specificity of peripheral nerve regeneration[J]. Exp Neuro,2014,254(4):99-108.
[27] LIU D,JIANG T,CAI W,et al. An in situ gelling drug delivery system for improved recovery after spinal cord injury[J]. Adv Healthc Mater,2016,5(12):1513-1521.
[28] CHANG E I,GALVEZ M G,GLOTZBACH J P,et al. Vascular anastomosis using controlled phase transitions in poloxamer gels[J]. Nat Med,2011,17(9):1147-1158.
[29] WANG Q,HE Y,ZHAO Y,et al. A thermosensitive heparinpoloxamer hydrogel bridge afgf to treat spinal cord injury[J].ACS Appl Mater Interfaces,2017,9(8):6725-6747.
[30] ZHAO Y Z,JIANG X,LIN Q,et al. Thermosensitive heparinpoloxamer hydrogels enhance the effects of gdnf on neuronal circuit remodeling and neuroprotection after spinal cord injury[J].J Biomed Mater Res A,2017,105(10):2816-2829.
[31] WIJELATH E,NAMEKATA M,MURRAY J,et al. Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity[J]. J Cell Biochem,2010,111(2):461-468.
[32] KIM H A,MINDOS T,PARKINSON D B. Plastic fantastic:schwann cells and repair of the peripheral nervous system[J]. Stem Cells Transl Med,2013,2(8):553-557.
[2] GUARIGUATA L,WHITING D R,HAMBLETO I,et al. Global estimates of diabetes prevalence for 2013 and projections for 2035[J]. Diabetes Res Clin Pract,2014,103(2):137-149.
[3] ZENKER J,ZIEGLER D,CHRAST R. Novel pathogenic pathways in diabetic neuropathy[J]. Trends Neurosci,2013,36(8):439-449.
[4] KAPLAN S. Diabetic neuropathy-a review[J]. Nat Clin Pract Neurol,2007,3(6):331-340.
[5] TSUKAMOTO M,SANGO K,NIIMI N,et al. Upregulation of galectin-3 in immortalized schwann cells ifrs1 under diabetic conditions[J]. Neurosci Res,2015,92(69):80-85.
[6] DEY I,MIDHA N,SINGH G,et al. Diabetic schwann cells suffer from nerve growth factor and neurotrophin-3 underproduction and poor associability with axons[J]. Glia,2013,61(12):1990-1999.
[7] RAIVICH G,HELLWEG R,KREUTZBERG G W. Ngf receptor-mediated reduction in axonal ngf uptake and retrograde transport following sciatic nerve injury and during regeneration[J].Neuron,1991,7(1):151-164.
[8] SOFRONIEW M V,HOWE C L,MOBLEY W C. Nerve growth factor signaling,neuroprotection,and neural repair[J]. Annu Rev Neurosci,2001,24(3):1217-1281.
[9] KEMP S W,WEBB A A,DHALIWAI S,et al. Dose and duration of nerve growth factor(ngf)administration determine the extent of behavioral recovery following peripheral nerve injury in the rat[J]. Exp Neurol,2011,229(2):460-470.
[10] KARAMOYSOYLI E,BURNAND R C,TOMLINSON D R,et al. Neuritin mediates nerve growth factor-induced axonal regeneration and is deficient in experimental diabetic neuropathy[J].Diabetes,2008,57(1):181-189.
[11] GROSHEVA M,NOHROUDI K,SCHWARZ A,et al. Comparison of trophic factors'expression between paralyzed and recovering muscles after facial nerve injury. A quantitative analysis in time course[J]. Exp Neurol,2016,279(35):137-148.
[12] ZOCHODNE D W,CHENG C. Neurotrophins and other growth factors in the regenerative milieu of proximal nerve stump tips[J]. J Anat,2010,196(2):279-283.
[13] TSAI C C,LU M C,CHEN Y S,et al. Locally administered nerve growth factor suppresses ginsenoside rb1-enhanced peripheral nerve regeneration[J]. Am J Chin Med,2003,31(5):665-673.
[14] ZHAO Y Z,LV H F,LU C T,et al. Correction:evaluation of a novel thermosensitive heparin-poloxamer hydrogel for improving vascular anastomosis quality and safety in a rabbit mode[J].PLoS One,2013,8(8):e73178.
[15] TIAN J L,ZHAO Y Z,JIN Z,et al. Synthesis and characterization of poloxamer 188-grafted heparin copolymer[J]. Drug Dev Ind Pharm,2010,36(7):832-838.
[16] BASSO D M,BEATTIE M S,BRESNAHAN J C. A sensitive and reliable locomotor rating scale for open field testing in rats[J]. J Neurotrauma,1995,12(1):1-21.
[17] MOHAMMADI R,SANAEI N,AHSAN S,et al. Stromal vascular fraction combined with silicone rubber chamber improves sciatic nerve regeneration in diabetes[J]. Chin J Traumatol(中华创生杂志),2015,18(4):212-218.
[18] ZHAO Y Z,LV H F,LU C T,et al. Evaluation of a novel thermosensitive heparin-poloxamer hydrogel for improving vascular anastomosis quality and safety in a rabbit model[J]. PLoS One,2013,8(8):e73178.
[19] GRASSELLI G,STRATA P. Structural plasticity of climbing fibers and the growth-associated protein gap-43[J]. Front Neural Circuits,2013,7(3):25-34.
[20] MERCERON-MARTINEZ D,ALMAGUER-MELIAN W,ALBERTIAMADOR E,et al. Amygdala stimulation promotes recovery of behavioral performance in a spatial memory task and increases gap-43and map-2 in the hippocampus and prefrontal cortex of male rats[J]. Brain Res Bull,2018,142(3):8-17.
[21] ZIERATH D,KUNZE A,FECTEAU L,et al. Promiscuity of autoimmune responses to mbp after stroke[J]. J Neuroimmunol,2015,285(29):101-105.
[22] MOHANRAJ A,SRINIVASAN S. Role of s-100 immunostaining in demonstration of nerve changes and quantification of dendritic cells in leprosy[J]. J Clin Diagn Res,2014,8(3):38-40.
[23] MOORE A M,KASUKURTHI R,MAGILL C K,et al. Limitations of conduits in peripheral nerve repairs[J]. Hand,2009,4(2):180-186.
[24] FORBES S J,ROSENTHAL N. Preparing the ground for tissue regeneration:from mechanism to therapy[J]. Nat Med,2014,20(8):857-868.
[25] GIANNACCINI M,CALATAYUD M P,POGGETTI A,et al.Magnetic nanoparticles for efficient delivery of growth factors:stimulation of peripheral nerve regeneration[J]. Adv Healthc Mater,2017,6(5):57-71.
[26] GORDON T. Neurotrophic factor expression in denervated motor and sensory schwann cells:relevance to specificity of peripheral nerve regeneration[J]. Exp Neuro,2014,254(4):99-108.
[27] LIU D,JIANG T,CAI W,et al. An in situ gelling drug delivery system for improved recovery after spinal cord injury[J]. Adv Healthc Mater,2016,5(12):1513-1521.
[28] CHANG E I,GALVEZ M G,GLOTZBACH J P,et al. Vascular anastomosis using controlled phase transitions in poloxamer gels[J]. Nat Med,2011,17(9):1147-1158.
[29] WANG Q,HE Y,ZHAO Y,et al. A thermosensitive heparinpoloxamer hydrogel bridge afgf to treat spinal cord injury[J].ACS Appl Mater Interfaces,2017,9(8):6725-6747.
[30] ZHAO Y Z,JIANG X,LIN Q,et al. Thermosensitive heparinpoloxamer hydrogels enhance the effects of gdnf on neuronal circuit remodeling and neuroprotection after spinal cord injury[J].J Biomed Mater Res A,2017,105(10):2816-2829.
[31] WIJELATH E,NAMEKATA M,MURRAY J,et al. Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity[J]. J Cell Biochem,2010,111(2):461-468.
[32] KIM H A,MINDOS T,PARKINSON D B. Plastic fantastic:schwann cells and repair of the peripheral nervous system[J]. Stem Cells Transl Med,2013,2(8):553-557.