大鼠面神经切断及修复后面神经核内鞘脂激活蛋白原的变化
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摘要
背景研究证实,面神经损伤后,面神经元自身将合成鞘脂激活蛋白原促进神经元修复。
目的观察模拟人体面神经损伤SD大鼠的面神经创伤模型面神经核鞘脂激活蛋白原阳性产物的时程变化及再生条件下的合成规律。设计、时间及地点神经病理学的随机对照动物实验,于2007-03/2008-09在重庆医科大学完成。
材料成年雄性SD大鼠48只,随机分为面神经单纯切断组和面神经切断后即刻端端吻合组,每组24只。兔抗鼠Prosaposin多克隆抗体、即用型SABC免疫组化试剂盒、兔抗鼠Prosaposin一抗工作液(1:500)由武汉中美科技技术有限公司提供。
方法面神经单纯切断组结扎左侧面神经耳后支远端主干,结扎点远端切除长约5 mm的神经干。面神经切断后即刻端端吻合组切断左侧面神经耳后支远端主干后,立即行神经外膜吻合。以两组动物右侧面神经作为正常对照。
主要观察指标损伤后1,3,7,14,21,35d,以免疫组织化学方法检测两组大鼠面神经核中鞘脂激活蛋白原阳性神经元数量和免疫反应强度的变化。
结果面神经单纯切断组:损伤侧面神经核鞘脂激活蛋白原阳性神经元的数量和免疫反应强度于术后第l天即开始增多,3d时升高明显,表达水平均高于对照侧,7d下降至最低。面神经切断后即刻端端吻合组:术后7d内损伤侧面神经核内鞘脂激活蛋白原阳性神经元数量和免疫反应强度变化规律与面神经单纯切断组相同,损伤侧面神经核内鞘脂激活蛋白原阳性神经元数量和免疫反应强度的回升最早出现在吻合后第14天,术后35d恢复接近至正常水平。术后14,21,35d面神经核鞘脂激活蛋白原阳性神经元的数量和免疫反应强度高于面神经单纯切断组(P<0.05),大鼠触须节律性拂动恢复,且大鼠触须节律性拂动恢复的时间与鞘脂激活蛋白原阳性神经元数量回升的时间基本一致。
结论面神经损伤后7d内,面神经核鞘脂激活蛋白原表达呈先升后降的趋势,实施面神经修复对其表达影响不大。面神经损伤14d后,实施神经吻合可的表达。面神经核鞘脂激活蛋白原的回升与神经再支配同步出现。
BACKGROUND Studies have demonstrated that damaged facial nerves synthesize prosaposin to promote repair of facial neurons.
OBJECTIVE To observe time-course changes of prosaposin expression in the facial nerve nucleus of Sprague Dawley rats following facial nerve transection and repair.
DESIGN, TIME AND SETTING A randomized control neuropathological animal experiment was performed in Chongqing Medical University between March 2007 and September 2008.
MATERIALS A total of 48 adult, male, Sprague Dawley rats were selected and randomly divided into transection and transection + end-to-end anastomosis groups (n=24). Rabbit anti-rat prosaposin antibody, instant SABC immunohistochemical kit, and antibody dilution solution were purchased from Wuhan Uscn Sciences Co.,Ltd,China.
METHODS In the transection group, the nerve trunk of the distal retroauricular branch of the left facial nerves was ligated in Sprague Dawley rats, and a 5-mm nerve trunk at the distal end of the ligation site was removed. In the transection + end-to-end anastomosis group, epineurial anastomosis was performed immediately following transection of the left facial nerves. The right facial nerves in the two groups served as the normal control group.
MAIN OUTCOME MEASURES The number of prosaposin-positive neurons, as well as intensity of immunostaining in facial nerve nucleus, following transection and end-to-end anastomosis were determined by immunohistochemistry at 1, 3, 7, 14, 21, and 35 days after injury.
RESULTS Transection group: transection of facial nerves resulted in increased number of prosaposin-positive neurons and immunoreactivity intensity in the facial nucleus on day 1. These values significantly increased by day 3. Expression was greater than in the control side. The peak of the reduction was reached at 7 days post-surgery. Transection + end-to-end anastomosis group: the number of prosaposin-positive neurons and immunoreactivity intensity was reduced in the facial nerve nucleus following immediate end-to-end anastomosis on day 7 post-surgery. These values began to gradually increase by day 14 post-anastomosis. By day 35 post-anastomosis, the number of prosaposin-positive neurons in the operated side recovered to normal levels. The number of prosaposin-positive neurons, as well as immunoreactivity intensity, was significantly greater in the facial nerve nucleus, compared with the transection group on days 14, 21, and 35 post-surgery (P < 0.05). The rhythmic whisking of vibrissa recovered, and recovery time was consistent with increased numbers of prosaposin-positive neurons.
CONCLUSION Within 7 days after injury, prosaposin expression in the facial nerve nucleus exhibited an initial increase, followed by a decrease, and was not affected by facial nerve repair. Following facial nerve damage, neural anastomosis was shown to increase prosaposin expression in the facial nerve nucleus after 14 days. Recovery of prosaposin occurred simultaneously with reinnervation.
目的观察模拟人体面神经损伤SD大鼠的面神经创伤模型面神经核鞘脂激活蛋白原阳性产物的时程变化及再生条件下的合成规律。设计、时间及地点神经病理学的随机对照动物实验,于2007-03/2008-09在重庆医科大学完成。
材料成年雄性SD大鼠48只,随机分为面神经单纯切断组和面神经切断后即刻端端吻合组,每组24只。兔抗鼠Prosaposin多克隆抗体、即用型SABC免疫组化试剂盒、兔抗鼠Prosaposin一抗工作液(1:500)由武汉中美科技技术有限公司提供。
方法面神经单纯切断组结扎左侧面神经耳后支远端主干,结扎点远端切除长约5 mm的神经干。面神经切断后即刻端端吻合组切断左侧面神经耳后支远端主干后,立即行神经外膜吻合。以两组动物右侧面神经作为正常对照。
主要观察指标损伤后1,3,7,14,21,35d,以免疫组织化学方法检测两组大鼠面神经核中鞘脂激活蛋白原阳性神经元数量和免疫反应强度的变化。
结果面神经单纯切断组:损伤侧面神经核鞘脂激活蛋白原阳性神经元的数量和免疫反应强度于术后第l天即开始增多,3d时升高明显,表达水平均高于对照侧,7d下降至最低。面神经切断后即刻端端吻合组:术后7d内损伤侧面神经核内鞘脂激活蛋白原阳性神经元数量和免疫反应强度变化规律与面神经单纯切断组相同,损伤侧面神经核内鞘脂激活蛋白原阳性神经元数量和免疫反应强度的回升最早出现在吻合后第14天,术后35d恢复接近至正常水平。术后14,21,35d面神经核鞘脂激活蛋白原阳性神经元的数量和免疫反应强度高于面神经单纯切断组(P<0.05),大鼠触须节律性拂动恢复,且大鼠触须节律性拂动恢复的时间与鞘脂激活蛋白原阳性神经元数量回升的时间基本一致。
结论面神经损伤后7d内,面神经核鞘脂激活蛋白原表达呈先升后降的趋势,实施面神经修复对其表达影响不大。面神经损伤14d后,实施神经吻合可的表达。面神经核鞘脂激活蛋白原的回升与神经再支配同步出现。
BACKGROUND Studies have demonstrated that damaged facial nerves synthesize prosaposin to promote repair of facial neurons.
OBJECTIVE To observe time-course changes of prosaposin expression in the facial nerve nucleus of Sprague Dawley rats following facial nerve transection and repair.
DESIGN, TIME AND SETTING A randomized control neuropathological animal experiment was performed in Chongqing Medical University between March 2007 and September 2008.
MATERIALS A total of 48 adult, male, Sprague Dawley rats were selected and randomly divided into transection and transection + end-to-end anastomosis groups (n=24). Rabbit anti-rat prosaposin antibody, instant SABC immunohistochemical kit, and antibody dilution solution were purchased from Wuhan Uscn Sciences Co.,Ltd,China.
METHODS In the transection group, the nerve trunk of the distal retroauricular branch of the left facial nerves was ligated in Sprague Dawley rats, and a 5-mm nerve trunk at the distal end of the ligation site was removed. In the transection + end-to-end anastomosis group, epineurial anastomosis was performed immediately following transection of the left facial nerves. The right facial nerves in the two groups served as the normal control group.
MAIN OUTCOME MEASURES The number of prosaposin-positive neurons, as well as intensity of immunostaining in facial nerve nucleus, following transection and end-to-end anastomosis were determined by immunohistochemistry at 1, 3, 7, 14, 21, and 35 days after injury.
RESULTS Transection group: transection of facial nerves resulted in increased number of prosaposin-positive neurons and immunoreactivity intensity in the facial nucleus on day 1. These values significantly increased by day 3. Expression was greater than in the control side. The peak of the reduction was reached at 7 days post-surgery. Transection + end-to-end anastomosis group: the number of prosaposin-positive neurons and immunoreactivity intensity was reduced in the facial nerve nucleus following immediate end-to-end anastomosis on day 7 post-surgery. These values began to gradually increase by day 14 post-anastomosis. By day 35 post-anastomosis, the number of prosaposin-positive neurons in the operated side recovered to normal levels. The number of prosaposin-positive neurons, as well as immunoreactivity intensity, was significantly greater in the facial nerve nucleus, compared with the transection group on days 14, 21, and 35 post-surgery (P < 0.05). The rhythmic whisking of vibrissa recovered, and recovery time was consistent with increased numbers of prosaposin-positive neurons.
CONCLUSION Within 7 days after injury, prosaposin expression in the facial nerve nucleus exhibited an initial increase, followed by a decrease, and was not affected by facial nerve repair. Following facial nerve damage, neural anastomosis was shown to increase prosaposin expression in the facial nerve nucleus after 14 days. Recovery of prosaposin occurred simultaneously with reinnervation.
引文
[1] O'Brien JS, Kishimoto Y. Saposin proteins: structure, function, and role in human lysosomal storage disorders[J]. FASEB J.1991;5(3):301-308.
[2] Kotani Y, Matsuda S, Sakanaka M, et al. Prosaposin facilitates sciatic nerve regeneration in vivo[J]. J Neurochem. 1996;66(5):2019-2025.
[3] Sano A, Matsuda S, Wen TC, et al. Protection by prosaposin against ischemia-induced learning disability and neuronal loss[J]. Biochem Biophys Res Commun.1994;204(2):994-1000.
[4] Kotani Y, Matsuda S, Wen TC, et al. A hydrophilic peptide comprising 18 amino acid residues of the prosaposin sequence has neurotrophic activity in vitro and in vivo[J]. J Neurochem.1996;66(5):2197-2200.
[5] Taylor EM, Otero DA, Banks WA, et al. Designing stable blood-brain barrier-permeable prosaptide peptides for treatment of central nervous system neurodegeneration[J]. J Pharmacol Exp Ther. 2000;293(2):403-409.
[6] Calcutt NA, Campana WM, Eskeland NL, et al. Prosaposin gene expression and the efficacy of a prosaposin-derived peptide in preventing structural and functional disorders of peripheral nerve in diabetic rats[J]. J Neuropathol Exp Neurol.1999;58(6):628-636.
[7] Jolivalt CG, Vu Y, Mizisin LM, et al. Impaired prosaposin secretion during nerve regeneration in diabetic rats and protection of nerve regeneration by a prosaposin-derived peptide[J]. J Neuropathol Exp Neurol. 2008;67(7):702-710.
[8] O'Brien JS, Carson GS, Seo HC, et al. Identification of prosaposin as a neurotrophic factor[J]. Proc Natl Acad Sci U S A.1994;91(20):9593-9596.
[9] O'Brien JS, Carson GS, Seo HC, et al. Identification of the neurotrophic factor sequence of prosaposin[J]. FASEB J.1995;9(8):681-685.
[10] Campana WM, Mohiuddin L, Misasi R, et al. Prosaposin-derived peptides enhanced sprouting of sensory neurons in vitro and induced sprouting at motor endplates in vivo[J]. J Peripher Nerv Syst. 2000;5(3):126-130.
[11] Unuma K, Chen J, Saito S, et al. Changes in expression of prosaposin in the rat facial nerve nucleus after facial nerve transection[J]. Neurosci Res. 2005 ;52(3):220-227.
[12] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.
[13]骆文龙,林代诚.面神经外膜与束膜缝合法的实验研究[J].华西医科大学学报, 1994,25(1):112-116.
[14] Li F, Luo WL.Agmatine promotes expression of brain-derived neurotrophic factor in brainstem facial nucleus in the rat facial nerve injury model[J].Neural Regen Res.2008,3(6):618-620.
[15]季兴,熊绍虎.面神经损伤与修复对大鼠面神经核内信号转导子和转录激活子3活性的影响[J].中国临床康复,2005,9(25):44-46.
[16]包新民,苏斯云.大鼠脑立体定位图谱[M].人民卫生出版社,1991: 76-84.
[17] Chu Z, Sun Y, Kuan CY, et al. Saposin C: neuronal effect and CNS delivery by liposomes. Ann N Y Acad Sci.2005;1053:237-246.
[18] Misasi R, Sorice M, Di Marzio L, et al. Prosaposin treatment induces PC12 entry in the S phase of the cell cycle and prevents apoptosis: activation of ERKs and sphingosine kinase[J]. FASEB J.2001;15(2):467-474.
[19] Sorice M, Molinari S, Di Marzio L, et al. Neurotrophic signalling pathway triggered by prosaposin in PC12 cells occurs through lipid rafts[J]. FEBS J.2008;275(19):4903-4912.
[20] Ochiai T, Takenaka Y, Kuramoto Y, et al. Molecular mechanism for neuro-protective effect of prosaposin against oxidative stress: Its regulation of dimeric transcription factor formation[J]. Biochim Biophys Acta. 2008;1780(12):1441-1447.
[21] Che YH, Tamatani M, Tohyama M. Changes in mRNA for post-synaptic density-95 (PSD-95)and carboxy-terminal PDZ ligand of neuronal nitric oxide synthase following facial nerve transection[J]. Brain Res Mol Brain Res. 2000;76(2):325-335.
[22] Hiraiwa M, Taylor EM, Campana WM, et al. Cell death prevention, mitogen-activated protein kinase stimulation, and increased sulfatide concentrations in Schwann cells and oligodendrocytes by prosaposin and prosaptides.Proc Natl Acad Sci USA. 1997;94(9):4778-478.
[2] Kotani Y, Matsuda S, Sakanaka M, et al. Prosaposin facilitates sciatic nerve regeneration in vivo[J]. J Neurochem. 1996;66(5):2019-2025.
[3] Sano A, Matsuda S, Wen TC, et al. Protection by prosaposin against ischemia-induced learning disability and neuronal loss[J]. Biochem Biophys Res Commun.1994;204(2):994-1000.
[4] Kotani Y, Matsuda S, Wen TC, et al. A hydrophilic peptide comprising 18 amino acid residues of the prosaposin sequence has neurotrophic activity in vitro and in vivo[J]. J Neurochem.1996;66(5):2197-2200.
[5] Taylor EM, Otero DA, Banks WA, et al. Designing stable blood-brain barrier-permeable prosaptide peptides for treatment of central nervous system neurodegeneration[J]. J Pharmacol Exp Ther. 2000;293(2):403-409.
[6] Calcutt NA, Campana WM, Eskeland NL, et al. Prosaposin gene expression and the efficacy of a prosaposin-derived peptide in preventing structural and functional disorders of peripheral nerve in diabetic rats[J]. J Neuropathol Exp Neurol.1999;58(6):628-636.
[7] Jolivalt CG, Vu Y, Mizisin LM, et al. Impaired prosaposin secretion during nerve regeneration in diabetic rats and protection of nerve regeneration by a prosaposin-derived peptide[J]. J Neuropathol Exp Neurol. 2008;67(7):702-710.
[8] O'Brien JS, Carson GS, Seo HC, et al. Identification of prosaposin as a neurotrophic factor[J]. Proc Natl Acad Sci U S A.1994;91(20):9593-9596.
[9] O'Brien JS, Carson GS, Seo HC, et al. Identification of the neurotrophic factor sequence of prosaposin[J]. FASEB J.1995;9(8):681-685.
[10] Campana WM, Mohiuddin L, Misasi R, et al. Prosaposin-derived peptides enhanced sprouting of sensory neurons in vitro and induced sprouting at motor endplates in vivo[J]. J Peripher Nerv Syst. 2000;5(3):126-130.
[11] Unuma K, Chen J, Saito S, et al. Changes in expression of prosaposin in the rat facial nerve nucleus after facial nerve transection[J]. Neurosci Res. 2005 ;52(3):220-227.
[12] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.
[13]骆文龙,林代诚.面神经外膜与束膜缝合法的实验研究[J].华西医科大学学报, 1994,25(1):112-116.
[14] Li F, Luo WL.Agmatine promotes expression of brain-derived neurotrophic factor in brainstem facial nucleus in the rat facial nerve injury model[J].Neural Regen Res.2008,3(6):618-620.
[15]季兴,熊绍虎.面神经损伤与修复对大鼠面神经核内信号转导子和转录激活子3活性的影响[J].中国临床康复,2005,9(25):44-46.
[16]包新民,苏斯云.大鼠脑立体定位图谱[M].人民卫生出版社,1991: 76-84.
[17] Chu Z, Sun Y, Kuan CY, et al. Saposin C: neuronal effect and CNS delivery by liposomes. Ann N Y Acad Sci.2005;1053:237-246.
[18] Misasi R, Sorice M, Di Marzio L, et al. Prosaposin treatment induces PC12 entry in the S phase of the cell cycle and prevents apoptosis: activation of ERKs and sphingosine kinase[J]. FASEB J.2001;15(2):467-474.
[19] Sorice M, Molinari S, Di Marzio L, et al. Neurotrophic signalling pathway triggered by prosaposin in PC12 cells occurs through lipid rafts[J]. FEBS J.2008;275(19):4903-4912.
[20] Ochiai T, Takenaka Y, Kuramoto Y, et al. Molecular mechanism for neuro-protective effect of prosaposin against oxidative stress: Its regulation of dimeric transcription factor formation[J]. Biochim Biophys Acta. 2008;1780(12):1441-1447.
[21] Che YH, Tamatani M, Tohyama M. Changes in mRNA for post-synaptic density-95 (PSD-95)and carboxy-terminal PDZ ligand of neuronal nitric oxide synthase following facial nerve transection[J]. Brain Res Mol Brain Res. 2000;76(2):325-335.
[22] Hiraiwa M, Taylor EM, Campana WM, et al. Cell death prevention, mitogen-activated protein kinase stimulation, and increased sulfatide concentrations in Schwann cells and oligodendrocytes by prosaposin and prosaptides.Proc Natl Acad Sci USA. 1997;94(9):4778-478.