光遗传技术调控新生神经元电活动对颅脑损伤小鼠认知功能的影响
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摘要
目的探讨光调控去极化对颅脑损伤(TBI)小鼠海马齿状回(DG)新生神经元的存活、成熟及认知功能的影响。方法将66只雄性C57BL/6小鼠随机分为假手术组(n=10)、TBI组(n=12)、TBI+增强型绿色荧光蛋白(EGFP)组(n=12)和TBI+视紫红质通道蛋白-2(ChR2)组(n=32);假手术组小鼠行单纯开颅手术,未行液压冲击;TBI组小鼠给予液压冲击制备TBI模型;TBI+EGFP组小鼠给予液压冲击制备TBI模型后感染慢病毒DCX-EGFP;TBI+ChR2组小鼠给予液压冲击制备TBI模型后感染慢病毒DCX-ChR2-EGFP。各组小鼠均于造模后连续7 d行腹腔内注射Brd U(100 mg·kg~(-1))。免疫荧光染色检测造模后7、28 d TBI组小鼠海马DG区新生神经元存活和成熟情况;倒置荧光显微镜观察假手术组和造模后不同时间点(3、5、7、9、12、18、28 d)TBI+ChR2组海马DG区DCX-EGFP阳性新生神经元变化;造模后21~26 d进行水迷宫实验检测各组小鼠认知能力;造模后28 d采用免疫荧光染色检测TBI组、TBI+EGFP组和TBI+ChR2组小鼠海马DG区表达DCX神经元的存活和成熟情况。结果造模后28 d,TBI组小鼠海马DG区新生成熟神经元数量显著少于造模后7 d(P<0.01)。造模后3、18、28 d,TBI+ChR2组与假手术组小鼠海马DG区DCX-EGFP阳性新生神经元数量比较差异无统计学意义(P>0.05);造模后5、7、9、12 d,TBI+ChR2组小鼠海马DG区DCX-EGFP阳性新生神经元数量均显著高于假手术组(P<0.05,P<0.01)。造模后9 d,TBI+ChR2组小鼠海马DG区DCX-EGFP阳性新生神经元数量均显著高于其他各时间点(P<0.05,P<0.01)。造模后21 d,4组小鼠寻找隐匿平台的潜伏期比较差异均无统计学意义(P>0.05)。造模后22~26 d,TBI组和TBI+EGFP组小鼠寻找隐匿平台的潜伏期均显著长于假手术组(P<0.01);TBI+ChR2组与假手术组小鼠寻找隐匿平台的潜伏期比较差异无统计学意义(P>0.05);TBI+EGFP组与TBI组小鼠寻找隐匿平台的潜伏期比较差异无统计学意义(P>0.05);TBI+ChR2组小鼠寻找隐匿平台的潜伏期均显著短于TBI组和TBI+EGFP组(P<0.01)。TBI组、TBI+EGFP组小鼠在目标象限停留时间和穿越平台次数均显著低于假手术组(P<0.01);TBI+ChR2组与假手术组小鼠在目标象限停留时间和穿越平台次数比较差异无统计学意义(P>0.05)。TBI+ChR2组小鼠在目标象限停留时间和穿越平台次数均高于TBI组和TBI+EGFP组(P<0.05);TBI+EGFP组与TBI组小鼠在目标象限停留时间和穿越平台次数比较差异无统计学意义(P>0.05)。造模后28 d,TBI+ChR2组小鼠海马DG区新生成熟神经元数量明显高于TBI组和TBI+EGFP组(P<0.01);TBI组与TBI+EGFP组小鼠海马DG区新生成熟神经元数量比较差异无统计学意义(P>0.05)。结论 TBI后光调控表达DCX-EGFP细胞的去极化可增强DG区新生神经元的存活和成熟,并显著改善TBI小鼠的认知功能。
Objective To investigate the effect of optical regulation depolarization on the survival,maturation of neonatal cells in dentate gyrus of hippocampus in mice with traumatic brain injury( TBI) and the cognitive function of mice with TBI. Methods Sixty-six male C57 BL/6 mice were randomly divided into sham operation group( n = 10),TBI group( n = 12),TBI + enhanced green fluorescent protein( EGFP) group( n = 12) and TBI + channelrhodopsin-2( ChR2) group( n = 32). The mice in sham operation group underwent simple craniotomy without hydraulic shock; the mice in TBI group were given hydraulic shock to prepare TBI model; the mice in TBI + EGFP group were given hydraulic shock to prepare TBI model and then infected with lentivirus DCX-EGFP; the mice in TBI + ChR2 group were given hydraulic shock to prepare TBI model and then infected with lentivirus DCX-ChR2-EGFP. All mice were injected Brd U( 100 mg·kg~(-1)) intraperitoneally for 7 days after modeling. The survival and maturation of the neonatal neurons in the DG area of the hippocampus of mice in the TBI group was detected by immunofluorescence staining on the 7 thand 28 thday after modeling; the changes of DCX-EGFP positive neurons in the DG area of the hippocampus of mice in the sham operation group and the TBI + ChR2 group at different time points( 3,5,7,9,12,18,28 days after modeling) were observed by inverted fluorescence microscopy; the cognitive function of mice in each group was detected by Morris water maze test at 21-26 days after modeling; the survival and maturation of cells expressing DCX in DG region of hippocampus in TBI group,TBI + EGFP group and TBI + ChR2 group were detected by immunofluorescence staining at 28 days after modeling. Results The number of neonatal mature neurons in the DG area of the hippocampus in TBI group at 28 days after modeling was significantly less than that at 7 days after modeling( P < 0. 01). There was no significant difference in the number of DCX-EGFP positive neonatal neurons in the DG area of the hippocampus of mice between TBI + ChR2 group and sham operation group at 3,18,28 days after modeling( P > 0. 05); the number of DCX-EGFP positive neonatal neurons in the DG area of the hippocampus of mice in the TBI + ChR2 group was significantly higher than that in the sham operation group at 5,7,9,12 days after modeling( P < 0. 05,P < 0. 01). The number of DCX-EGFP positive neonatal neurons in the DG area of the hippocampus of mice in TBI + ChR2 group at 9 days after modeling was significantly higher than other time points( P < 0. 05,P < 0. 01). There was no significant difference in the latency for searching hidden platform of mice among the four groups at 21 days after modeling( P > 0. 05). At 22-26 days after modeling,the latency for searching hidden platform of mice in TBI groupand TBI + EGFP group was significantly longer than that in the sham operation group( P < 0. 01);there was no significant difference in the latency for searching hidden platform of mice between TBI + ChR2 group and sham operation group( P > 0. 05); there was no significant difference in the latency for searching hidden platform of mice between TBI + EGFP group and TBI group( P > 0. 05); the latency for searching hidden platform of mice in TBI + ChR2 group was significantly shorter than that in the TBI group and TBI + EGFP group( P < 0. 01). The target quadrant staying time and the number of crossing platforms of mice in TBI group and TBI + EGFP group were significantly lower than those in the sham operation group( P < 0. 01); there was no significant difference in the target quadrant staying time and the number of crossing platforms of mice between TBI + ChR2 group and sham operation group( P > 0. 05); the target quadrant staying time and the number of crossing platforms of mice in TBI + ChR2 group were significantly higher than those in the TBI group and TBI + EGFP group( P < 0. 05); there was no significant difference in the target quadrant staying time and the number of crossing platforms of mice between TBI + EGFP group and TBI group( P > 0. 05). At 28 days after modeling,the number of neonatal mature neurons in the DG area of the hippocampus of mice in TBI + ChR2 group was significantly higher than that in the TBI group and TBI +EGFP group( P < 0. 01); there was no significant difference in the number of neonatal mature neurons in the DG area of the hippocampus of mice between TBI group and TBI + EGFP group( P > 0. 05). Conclusion Optical regulation depolarization of cells expressing DCX-EGFP can enhance the survival and maturation of newborn neurons in hippocampus dentate gyrus and significantly improve the cognitive deficits of mice after TBI.
Objective To investigate the effect of optical regulation depolarization on the survival,maturation of neonatal cells in dentate gyrus of hippocampus in mice with traumatic brain injury( TBI) and the cognitive function of mice with TBI. Methods Sixty-six male C57 BL/6 mice were randomly divided into sham operation group( n = 10),TBI group( n = 12),TBI + enhanced green fluorescent protein( EGFP) group( n = 12) and TBI + channelrhodopsin-2( ChR2) group( n = 32). The mice in sham operation group underwent simple craniotomy without hydraulic shock; the mice in TBI group were given hydraulic shock to prepare TBI model; the mice in TBI + EGFP group were given hydraulic shock to prepare TBI model and then infected with lentivirus DCX-EGFP; the mice in TBI + ChR2 group were given hydraulic shock to prepare TBI model and then infected with lentivirus DCX-ChR2-EGFP. All mice were injected Brd U( 100 mg·kg~(-1)) intraperitoneally for 7 days after modeling. The survival and maturation of the neonatal neurons in the DG area of the hippocampus of mice in the TBI group was detected by immunofluorescence staining on the 7 thand 28 thday after modeling; the changes of DCX-EGFP positive neurons in the DG area of the hippocampus of mice in the sham operation group and the TBI + ChR2 group at different time points( 3,5,7,9,12,18,28 days after modeling) were observed by inverted fluorescence microscopy; the cognitive function of mice in each group was detected by Morris water maze test at 21-26 days after modeling; the survival and maturation of cells expressing DCX in DG region of hippocampus in TBI group,TBI + EGFP group and TBI + ChR2 group were detected by immunofluorescence staining at 28 days after modeling. Results The number of neonatal mature neurons in the DG area of the hippocampus in TBI group at 28 days after modeling was significantly less than that at 7 days after modeling( P < 0. 01). There was no significant difference in the number of DCX-EGFP positive neonatal neurons in the DG area of the hippocampus of mice between TBI + ChR2 group and sham operation group at 3,18,28 days after modeling( P > 0. 05); the number of DCX-EGFP positive neonatal neurons in the DG area of the hippocampus of mice in the TBI + ChR2 group was significantly higher than that in the sham operation group at 5,7,9,12 days after modeling( P < 0. 05,P < 0. 01). The number of DCX-EGFP positive neonatal neurons in the DG area of the hippocampus of mice in TBI + ChR2 group at 9 days after modeling was significantly higher than other time points( P < 0. 05,P < 0. 01). There was no significant difference in the latency for searching hidden platform of mice among the four groups at 21 days after modeling( P > 0. 05). At 22-26 days after modeling,the latency for searching hidden platform of mice in TBI groupand TBI + EGFP group was significantly longer than that in the sham operation group( P < 0. 01);there was no significant difference in the latency for searching hidden platform of mice between TBI + ChR2 group and sham operation group( P > 0. 05); there was no significant difference in the latency for searching hidden platform of mice between TBI + EGFP group and TBI group( P > 0. 05); the latency for searching hidden platform of mice in TBI + ChR2 group was significantly shorter than that in the TBI group and TBI + EGFP group( P < 0. 01). The target quadrant staying time and the number of crossing platforms of mice in TBI group and TBI + EGFP group were significantly lower than those in the sham operation group( P < 0. 01); there was no significant difference in the target quadrant staying time and the number of crossing platforms of mice between TBI + ChR2 group and sham operation group( P > 0. 05); the target quadrant staying time and the number of crossing platforms of mice in TBI + ChR2 group were significantly higher than those in the TBI group and TBI + EGFP group( P < 0. 05); there was no significant difference in the target quadrant staying time and the number of crossing platforms of mice between TBI + EGFP group and TBI group( P > 0. 05). At 28 days after modeling,the number of neonatal mature neurons in the DG area of the hippocampus of mice in TBI + ChR2 group was significantly higher than that in the TBI group and TBI +EGFP group( P < 0. 01); there was no significant difference in the number of neonatal mature neurons in the DG area of the hippocampus of mice between TBI group and TBI + EGFP group( P > 0. 05). Conclusion Optical regulation depolarization of cells expressing DCX-EGFP can enhance the survival and maturation of newborn neurons in hippocampus dentate gyrus and significantly improve the cognitive deficits of mice after TBI.
引文
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[18]KARL C,COUILLARD-DESPRES S,PRANG P,et al.Neuronal precursor-specific activity of a human doublecortin regulatory sequence[J].J Neurochem,2005,92(2):264-282.
[19]姜春莲,汪艳璐,罗玉萍.成年哺乳动物神经发生的研究进展[J].中国生物工程杂志,2017,37(5):107-112.
[20]ZHANG J,GIESERT F,KLOOS K,et al.A powerful transgenic tool for fate mapping and functional analysis of newly generated neurons[J].BMC Neurosci,2010,11:158.
[2]王海涛,王锦波.重型脑损伤患者早期肝肾功能变化临床研究[J].新乡医学院学报,2016,33(5):421-424.
[3]KERNIE S G,PARENT J M.Forebrain neurogenesis after focal Ischemic and traumatic brain injury[J].Neurobiol Dis,2010,37(2):267-274.
[4]杨伟锋,金保哲,张新中,等.丙戊酸对颅脑损伤大鼠海马成体神经干细胞原位激活的作用[J].新乡医学院学报,2016,33(6):469-472.
[5]ZHAO W Y,CHEN S B,WANG J J,et al.Establishment of an ideal time window model in hypothermic-targeted temperature management after traumatic brain injury in rats[J].Brain Res,2017,1669:141-149.
[6]HSIEH J,SCHNEIDER J W.Neuroscience.Neural stem cells,excited[J].Science,2013,339(6127):1534-1535.
[7]MING G L,SONG H.Adult neurogenesis in the mammalian brain:significant answers and significant questions[J].Neuron,2011,70(4):687-702.
[8]CHEN C,MA T Z,WANG L N,et al.Mild hypothermia facilitates the long-term survival of newborn cells in the dentate gyrus after traumatic brain injury by diminishing a pro-apoptotic microenvironment[J].Neuroscience,2016,335:114-121.
[9]SONG J,ZHONG C,BONAGUIDI M A,et al.Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision[J].Nature,2012,489(7414):150-154.
[10]SPITZER N C.Electrical activity in early neuronal development[J].Nature,2006,444(7120):707-712.
[11]路承彪.基于光敏蛋白的光控技术对神经细胞活动的时空定向控制[J].新乡医学院学报,2012,29(3):161-163.
[12]NAKANISHI S,OKAZAWA M.Membrane potential-regulated Ca2+signalling in development and maturation of mammalian cerebellar granule cells[J].J Physiol,2006,575(Pt 2):389-395.
[13]EL-SHAMAYLEH Y,KOJIMA Y,SOETEDJO R,et al.Selective optogenetic control of purkinje cells in monkey cerebellum[J].Neuron,2017,95(1):51-62.
[14]BORDIA T,ZHANG D,PEREZ X A,et al.Striatal cholinergic interneurons and D2 receptor-expressing GABAergic medium spiny neurons regulate tardive dyskinesia[J].Exp Neurol,2016,286:32-39.
[15]MELCHIOR J R,FERRIS M J,STUBER G D,et al.Optogenetic versus electrical stimulation of dopamine terminals in the nucleus accumbens reveals local modulation of presynaptic release[J].J Neurochem,2015,134(5):833-844.
[16]YIZHAR O,FENNO L E,DAVIDSON T J,et al.Optogenetics in neural systems[J].Neuron,2011,71(1):9-34.
[17]GU Y,ARRUDA-CARVALHO M,WANG J,et al.Optical controlling reveals time-dependent roles for adult-born dentate granule cells[J].Nat Neurosci,2012,15(12):1700-1706.
[18]KARL C,COUILLARD-DESPRES S,PRANG P,et al.Neuronal precursor-specific activity of a human doublecortin regulatory sequence[J].J Neurochem,2005,92(2):264-282.
[19]姜春莲,汪艳璐,罗玉萍.成年哺乳动物神经发生的研究进展[J].中国生物工程杂志,2017,37(5):107-112.
[20]ZHANG J,GIESERT F,KLOOS K,et al.A powerful transgenic tool for fate mapping and functional analysis of newly generated neurons[J].BMC Neurosci,2010,11:158.
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