Effectiveness of Low Intensity Focused Ultrasound Stimulation of Nucleus Accumbens in Mice
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摘要:
目的 使用低强度聚焦超声对正常小鼠的伏隔核进行超声刺激，从行为学、病理改变以及神经生物化学方面，探讨超声刺激进行神经调控的有效性。 方法 100只C57BL/6小鼠随机分为实验组和对照组各50只，实验组给予经颅低强度聚焦超声对伏隔核进行超声刺激，每天2次，每次10 min，连续刺激7 d；对照组给予假刺激。干预结束后，2个大组内又随机分为行为学评估30只（分别进行强迫游泳、旷场和水迷宫实验）、电镜检测10只和神经递质检测10只，立刻进行相关检测。强迫游泳实验评估小鼠的抑郁程度，旷场实验评估小鼠的焦虑程度，水迷宫实验评估小鼠的学习记忆能力。透射电镜观察小鼠伏隔核神经元超微结构的改变，ELISA法测定小鼠NAc中单胺类神经递质（去甲肾上腺素、多巴胺、5-羟色胺）的含量。 结果 （1）行为学评价结果：与空白对照组比较，超声刺激组在强迫游泳实验中不动时间明显延长（P < 0.001），在旷场中行进总距离、行进平均速度、中央区活动距离、中央区停留时间和中央区穿越次数均显著减少（ P < 0.05， P < 0.01），在水迷宫实验中逃避潜伏时间明显延长，穿越平台次数明显减少，在目标象限停留时间明显缩短（ P < 0.05， P < 0.001）；（2）电镜结果：对照组NAc神经元超微结构基本正常，超声刺激组神经元肿胀，神经纤维溶解坏死，胞浆内细胞器减少或消失，线粒体空泡化，内质网扩张；（3）神经递质测定结果：与对照组比较，超声刺激组小鼠NAc单胺类神经递质去甲肾上腺素、多巴胺、5-羟色胺的含量均显著降低（ P < 0.0001）。 结论 低强度聚焦超声对小鼠可产生抑制性的神经调节作用，并造成NAc神经元形态学以及神经生物化学改变，这可能是超声神经调控的机制之一。 Abstract:Objective To investigate the effectiveness of ultrasound stimulation for neural regulation from the aspects of behavioral pathology and neurobiochemistry by using low intensity focused ultrasound to stimulate the nucleus accumbens of normal mice. Methods 100 C57BL/6 mice were randomly divided into experimental group and control group with 50 mice in each group. The control group was given sham stimulation, and the experimental group was given transcranial low-intensity focused ultrasound to stimulate the NAc, twice a day, 10 min each time, for 7 consecutive days. After the intervention, 30 mice were randomly assigned to behavioral assessment （forced swimming, open field, and water maze）, 10 mice were assigned to electron microscopy （EM）, and 10 mice were assigned to neurotransmitter test.The depression, anxiety and learning and memory ability of the mice were evaluated by forced swimming, open field and water maze tests.The ultrastructural changes of neurons in the NAcof mice were observed by transmission electron microscopy. The contents of monoamine neurotransmitters （norepinephrine, dopamine, 5-hydroxytryptamine） in NAc of mice were determined by ELISA. Results 1. Behavioral evaluation results: Compared with the control group, the immobile time in the ultrasonic stimulation group was significantly longer （P < 0.01）, the total distance travelled, the average speed travelled, the distancetravelled of the central area, the time of the central area and the number of exits from the central area in the open field all decreased significantly （ P < 0.05, P < 0.01）, In the water maze experiment, the avoidance latency time was significantly longer, the number of crossing the platform was significantly reduced, and the residence time in the target quadrant was significantly shorter （ P < 0.05, P < 0.01）. 2. Electron microscopy results: The ultrastructure ofNAc neurons in the control group was basically normal. In the ultrasound stimulation group, the neurons were swelling, nerve fiber dissolution and necrosis, mitochondrial vacuolation, endoplasmic reticulum dilation, and intracytoplasmic organelles were reduced or disappeared.3. Measurement results of neurotransmitters: Compared with the control group, the contents of NAc monoamine neurotransmitters NE, DA and 5-HT were significantly decreased in the ultrasonic stimulation group （ P < 0.05）. Conclusion Low intensity focused ultrasound can induce inhibitory neuroregulation in mice, and cause morphological and neurobiochemical changes in NAC neurons, which may be one of the mechanisms of ultrasound neuromodulation. -
Key words:
- Low intensity focused ultrasound /
- Neuromodulation /
- Nucleus accumbens /
- Effectiveness
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图 1 LIFU刺激小鼠NAc图
A：超声神经刺激仪实物图；B：LIFU刺激小鼠NAc工作图。
Figure 1. Image of NAC stimulated by LIFU
图 2 2组小鼠的游泳路径图
A：超声刺激组小鼠游泳路径图；B：对照组小鼠游泳路径图。
Figure 2. Swimming paths of the two groups of mice
图 3 小鼠伏隔核神经元的超微结构
A、B：对照组；C、D：超声刺激组。
Figure 3. Ultrastructure of mouse nucleus accumbens neurons
表 1 LIFU对小鼠强迫游泳实验漂浮不动时间的影响（
$\bar x \pm s$ ）Table 1. Effect of LIFU on floating immobility time of mice in forced swimming experiment （
$\bar x \pm s$ ）组别 漂浮不动时间（s） t P 超声刺激组 46.29 ± 18.40 4.71 < 0.001 *** 对照组 16.67 ± 7.56 与对照组比较，***P < 0.001。 
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表 2 LIFU对小鼠旷场实验各项指标的影响（
$\bar x \pm s$ ）Table 2. Effects of LIFU on various indicators of open field experiment in mice （
$\bar x \pm s$ ）组别 行进总距离
（cm）行进平均速度（cm/s） 中央区活动距离（cm） 中央区停留时间（s） 中央区穿越次数（次） 超声刺激组 44.00 ± 17.81 0.15 ± 0.06 4.81 ± 3.06 16.35 ± 9.20 7.30 ± 4.08 对照组 67.79 ± 25.80 0.23 ± 0.09 11.11 ± 4.60 32.36 ± 11.15 17.10 ± 7.55 t −2.40 −2.40 −3.60 −3.50 −3.61 P < 0.05 * < 0.05 * < 0.01 ** < 0.01 ** < 0.01 ** 与对照组比较，*P < 0.05， **P < 0.01。
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表 3 LIFU对小鼠水迷宫实验各项指标的影响（
$\bar x \pm s$ ）Table 3. Effects of LIFU on various indicators of water maze experiment in mice （
$\bar x \pm s$ ）组别 逃避潜伏时间（s） 穿越平台次数（次） 目标象限停留时间（s） 超声刺激组 32.50 ± 20.14 1.08 ± 0.72 22.40 ± 7.50 对照组 16.04 ± 4.55 2.38 ± 0.74 31.37 ± 6.63 t 2.52 −4.00 −2.83 P < 0.05 * < 0.001 *** < 0.05 * 与对照组比较，*P < 0.05， ***P < 0.001。
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表 4 LIFU对小鼠单胺类神经递质含量的影响（
$\bar x \pm s$ ）Table 4. Effects of LIFU on monoamine neurotransmitters in mice （
$\bar x \pm s$ ）组别 DA（pg/mL） NE（ng/mL） 5-HT（ng/mL） 超声刺激组 25.69 ± 3.22 1.95 ± 0.19 46.83 ± 5.38** 对照组 33.52 ± 1.82 2.73 ± 0.25 78.19 ± 5.47 t −6.69 −7.41 −15.37 P < 0.0001 ** < 0.0001 ** < 0.0001 ** 与对照组比较，**P < 0.0001。
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