癫痫Electrocorticograms（ECoGs）的操作，并睡在针刺大鼠和小鼠
Summary
This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.
Abstract
Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.
Introduction
癫痫是在整个病人的寿命反复发作会出现一种常见的神经系统疾病。大多数癫痫复发可以通过抗癫痫药物（AEDs）良好的控制。然而，癫痫患者约30％的开发难治性癫痫1。癫痫原因睡眠干扰，这可能进一步加剧复发。证据表明，癫痫患者既可以在夜间扰乱睡眠或导致白天过度嗜睡2,3。我们先前的研究进一步表明，癫痫在时间授时（ZT）0，即发生在光的光周期的开始：黑暗循环，减少睡眠;这是由促肾上腺皮质激素释放激素（CRH），一个稳态因子介导的。癫痫在ZT13（暗期的开始）增强的另一稳态因子，白介素-1（IL-1），这增加睡眠的表达。当癫痫发生在ZT6，的中间睡眠昼夜节律发生改变光周期4,5。另一方面，睡眠问题进一步加剧癫痫6的进展和复发。根据上述证据，我们力图揭示一个最佳的治疗方法同时控制癫痫和防止干扰睡眠癫痫患者。我们以前发现，电针与10赫兹的刺激频率，其中的电流在一定量通过一个不锈钢针递送到穴位，成功地抑制了电图（脑电图）癫痫活动和癫痫引起的睡眠障碍7 。 EA用100赫兹的刺激频率进一步降低癫痫活动和大鼠8,9-睡眠中断。这个实验成功取决于三个因素：首先，一个可行的癫痫动物模型;其次，对于在啮齿类动物睡眠记录和分析的方法;第三，针灸的准确性能和穴位位置的服务的准确性附件。
癫痫病一直分为两大类型：局灶性癫痫和全身性癫痫。我们感兴趣的是焦距颞叶癫痫（TLE），全身性癫痫，癫痫持续状态（SE），和自发全身性癫痫的复发。因此，不同的操作应用到我们的实验，创造出适合癫痫模型。建立联络的TLE，毛果芸香碱的低剂量给予到杏仁核（CEA）的左中央核。为了验证此模型，六脑电图电极植入在额叶（F1＆F2），顶叶（P1＆P2）和枕骨（O1＆O2）裂片在左和右半球两者，和另外两个参考电极（R 1 R2）被放置在两个半球，小脑。一个附加的显微注射导向插管手术植入到左杏仁（AP，来自前囟2.8毫米; ML，4.2毫米; DV，相对于前囟7.8毫米）。坐标适于从Paxinos和Watson鼠图谱10。如果焦点TLE被成功地诱导，只从（P1）上左顶叶的电极，其是邻近左侧杏仁记录，应获得主导癫痫ECoGs，与来自其他脑电图电极记录无显著痫ECoGs。毛果芸香碱的腹膜内（IP）注射入大鼠诱发全身性癫痫和SE，但是这可能是致命的。五戊四氮（PTZ）每次注射之间的为期一天的间隔注射IP诱导成功自发全身性癫痫小鼠和也保证了小鼠的生存。两线脑电图电极植入在小鼠额叶和顶叶皮质接受脑电图信号，并核实自发反复癫痫。
多导睡眠图（PSG）是记录睡眠过程中发生的生理变化的综合方法，它可以客观地划分成睡眠非快速眼动（NREM）和r的不同阶段APID眼动（REM）睡眠。 PSG记录的身体功能参数，包括脑电波（脑电图，EEG），眼球运动（眼电，EOG），骨骼肌音（肌电图，EMG），心脏节律（心电图，心电图）和血氧水平和呼吸参数。在大鼠中，我们记录ECoGs，肌电图，皮质下，和运动活动的警惕状态分为清醒，NREM睡眠和快速眼动睡眠。在小鼠睡眠分析使用ECoGs，肌电图，和自主活动的结果进行。大鼠手术有三个脑电图立体定向手术拧在额叶，顶叶电极，对侧小脑皮层植入。警戒状态（觉醒，NREM睡眠和REM睡眠）的采集后判断根据来自ECoGs，肌电图，脑温，和运动活动所获得的参数进行。进行分类在大鼠和小鼠的动物的行为的详细条件在叔描述他协议。
大鼠和小鼠需要用低剂量zoletil的（25毫克/千克），它是立体手术过程中通常施用麻醉剂的一半剂量被麻醉，执行手动针刺或EA之前。这一剂量可以向动物注射后醒来30至35分钟。无论手动针刺或EA在黑暗时期的开始进行的，以30分钟的恒定的时间段，并且每个动物被连续为两到三天处理。刺激EA电流通过被插入到穴的不锈钢针递送到特定穴位。刺激电流是双相方脉冲序列，其中脉冲持续时间为150毫秒和刺激强度为1毫安。如果干针用于手动针刺，插入穴位针头抽动每隔5分钟10次。手动针刺或EA的困难的部分是定位取穴在啮齿类动物。组委会在大鼠或小鼠穴位通货膨胀类似于在人体内的解剖学位置。例如，双侧凤池穴位于从颈部后部中线3毫米远，两个耳朵，这类似于在人体11的解剖位置之间。此外，在皮肤上的低阻抗的穴位，可以进一步证实。假针灸或假EA操作是必要的针灸或EA实验。假针刺或假的EA应在靠近穴，如腋下12附近的非穴位进行。
要成功地观察针刺或电针的癫痫和癫痫引起的睡眠中断的影响，以下因素必须到位：一个可行的癫痫动物模型，癫痫ECoGs的精准分析和癫痫的复发，要警惕状态进行分类的方法和在啮齿类动物针刺或EA的精确性能。
Protocol
Representative Results
Discussion
选择一个可行癫痫的动物模型为每个实验目的是至关重要的。我们的目标之一是阐明电针对癫痫抑制的作用。 EA是一种替代药物，可能在癫痫表现出的治疗效果，并在中国古代文献中已有记载。然而，有一个缺乏科学证据来证明这一点。为了确定EA对癫痫的影响，我们主要侧重于EA的影响轻度局灶性癫痫，而不是严重的癫痫大发作或SE。我们以前的研究中使用大鼠的焦点TLE模型来证明对毛果芸香碱?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by Ministry of Science and Technology (MOST) grants MOST104-2410-H-002-053 & NSC99-2320-B-002-026-MY3. This manuscript was edited and proofread by Mr. Brian Chang, who has experience revising professional documents.
Materials
| Drugs | |||
| Zoletil | Virbac | 50 mg/kg i.p. | |
| pilocarpine | Sigma-Aldrich | P6503 | 300 mg/kg i.p.; 1.2 mg microinjection |
| PTZ | Sigma-Aldrich | P6500 | 0.035 mg/mouse |
| polysporin | Pfizer | ||
| Surgery | |||
| ECoG electrode | Plastics One | E363/20 | screw electrode for rats |
| Pedestal | Plastics One | MS363 | |
| Cannula | Plastics One | C315G/spc | |
| Thermistor | Omega Engineering | 44008 | |
| Dental acrylic | Tempron | ||
| Stereotaxic Instrument | Stoelting | Dural arms | |
| Recording equipments | |||
| ECoG amplifier | Colbourn Instruments | V75-01 | |
| A/D Board | National Instruments | NI PCI-6033E | |
| Infrared-based motion detectors | Biobserve GmbH | custom-made | |
| ICELUS | G-System | ||
| AxoScope 10 Software | Molecular Devices | ||
| Acupuncture needs | |||
| Stainless needles | Shanghai Yanglong Medical Articles Co. | 32 gauge x 1” | |
| Functions Electrical Stimulator | I.T.O., Japan | Trio 300 | |
| AcuPen | Lhasa OMS | Pointer Excel II |
Referências
- Regesta, G., Tanganelli, P. Clinical aspects and biological bases of drug-resistant epilepsies. Epilepsy Res. 34 (2-3), 109-122 (1999).
- Malow, B. A., Bowes, R. J., Lin, X. Predictors of sleepiness in epilepsy patients. Sleep. 20 (12), 1105-1110 (1997).
- Stores, G., Wiggs, L., Campling, G. Sleep disorders and their relationship to psychological disturbances in children with epilepsy. Child Care Health Dev. 24 (1), 5-19 (1998).
- Yi, P. L., Tsai, C. H., Lin, J. G., Lee, C. C., Chang, F. C. Kindling stimuli delivered at different times in the sleep-wake cycle. Sleep. 27 (2), 203-212 (2004).
- Yi, P. L., Chen, Y. J., Lin, C. T., Chang, F. C. Occurrence of epilepsy at different zeitgeber times alters sleep homeostasis differently in rats. Sleep. 35 (12), 1651-1665 (2012).
- Bazil, C. W. Sleep and epilepsy. Semin Neurol. 22, 321-327 (2002).
- Yi, P. L., Lu, C. Y., Jou, S. B., Chang, F. C. Low frequency electroacupuncture suppress focal epilepsy and improves epilepsy-induced sleep disruptions. J Biomed Sci. 22, 49 (2015).
- Yi, P. L., Lu, C. Y., Cheng, C. H., Tsai, Y. F., Lin, C. T., Chang, F. C. Activation of amygdala opioid receptors by electroacupuncture of Feng-Chi (GB20) acupoints exacerbates focal epilepsy. BMC Complement Altern Med. 13, 290 (2013).
- Yi, P. L., Lu, C. Y., Cheng, C. H., Tsai, Y. F., Lin, C. T., Chang, F. C. Amygdala opioid receptors mediate the electroacupuncture-induced deteriortation of sleep disruptions in epilepsy rats. J Biomed Sci. 20, 85 (2013).
- Paxinos, G., Watson, W. . The Rat Brain in Stereotaxic Coordinates (4th edition). , (1998).
- Siu, F. K. W., Lo, S. C. L., Leung, M. C. P. Electro-acupuncture potentiates the disulphide-reducing activities of thioredoxin system by increasing thioredoxin expression in ischemia-reperfused rat brains. Life Sci. 77 (4), 386-399 (2005).
- Yi, P. L., Tsai, C. H., Lin, J. G., Liu, H. J., Chang, F. C. Effects of electroacupuncture at ‘Anmian’ (extra) acupoints on sleep activities in rats: the implication of the caudal nucleus tractus solitaries. J Biomed Sci. 11 (5), 579-590 (2004).
- Jou, S. B., Kao, I. F., Yi, P. L., Chang, F. C. Electrical stimulation of left anterior thalamic nucleus with high-frequency and low-intensity currents reduces the rate of pilocarpine-induced epilepsy in rats. Seizure. 22 (3), 221-229 (2012).
- Chang, F. C., Opp, M. R. Blockade of corticotropin-releasing hormone receptors reduces spontaneous waking in the rat. Am J Physiol Regul Integr Comp Physiol. 275 (3), 793-802 (1998).
- Cheng, C. H., Yi, P. L., Lin, J. G., Chang, F. C. Endogenous opiates in the nucleus tractus solitaries mediate electroacupuncture-induced sleep activities in rats. Evid Based Complement Alternat Med. 2011, 159209 (2011).
- Tsui, P., Leung, M. C. Comparison of the effectiveness between manual acupuncture and electro-acupuncture on patients with tennis elbow. Acupunct Electrother Res. 27 (2), 107-117 (2002).
- Napadow, V., Makris, N., Liu, J., Kettner, N. W., Kwong, K. K., Hui, K. K. Effects of electroacupuncture versus manual acupuncture on the human brain as measured by fMRI. Hum Brain Mapp. 24 (3), 193-205 (2005).
