在普通马莫塞特中植入微电极阵列的立体外科（Callithrix jacchus）
Summary
这项工作提出了一个协议，以执行立体，神经外科植入微电极阵列在常见的马莫塞特。这种方法特别允许在自由行为的动物进行电生理记录，但可以很容易地适应该物种的任何其他类似的神经外科干预（例如，用于药物给药的管状或用于大脑刺激的电极）。
Abstract
马莫采斯（Callithrix jacchus）是小型非人类灵长类动物，在生物医学和临床前研究，包括神经科学中越来越受欢迎。从遗传学上讲，这些动物比啮齿动物更接近人类。它们还显示复杂的行为，包括广泛的发声和社交互动。这里，描述了一种有效的立体神经外科手术程序，用于在普通马莫塞特中植入记录电极阵列。该协议还详细说明了成功实施此类手术所需的动物护理术前和术后步骤。最后，该协议显示了一个在手术后1周内自由行为的人造肿瘤中局部场势和峰值活动记录的例子。总体而言，这种方法提供了一个机会，研究大脑功能在清醒和自由行为的黑猩猩。研究其他小型灵长类动物的研究人员可以很容易地使用同样的协议。此外，它可以很容易地修改，以允许其他需要植入物的研究，如刺激电极，显微注射，植入的异体或导管，或离散组织区域消融。
Introduction
常见的黑猩猩（Callithrix jacchus）正在作为包括神经科学在内的许多研究领域的重要模型有机体获得认可。这些新世界灵长类动物是啮齿动物和其他非人类灵长类动物（如风河猴）的重要补充动物模型。与啮齿动物一样，这些动物体积小，易于操作，与较大的NHP相比，照顾和繁殖1、2、3、4相对经济。此外，这些动物与其他NHPs1、2、3相比，具有结对和高肥度的倾向。与许多其他灵长类动物的一个优势是，现代分子生物学工具3，4，5，6，7和测序基因组2 ，3，4，5，8已经被用来基因改造它们。使用慢病毒5的敲门动物和使用锌指核酸酶（ZFNs）和转录活化剂样核酸酶（TALENS）7的淘汰动物都产生了可行的成因动物。
与啮齿动物相比，一个优点是，作为灵长类动物，黑猩猩在基因上更接近人类3，5，6，9，10，11。和人类一样，动物是白天的动物，它们依靠高度发达的视觉系统来指导它们的大部分行为。此外，黑猩猩表现出行为的复杂性，包括广泛的社会行为，如使用不同的发声3，使研究人员能够解决其他物种无法解决的问题。从神经科学的角度来看，黑猩猩有脑血管，不像更常用的河河猴9。此外，黑猩猩的中枢神经系统与人类相似，包括更发达的前额叶皮质9。总之，所有这些特征将黑猩猩定位为研究大脑在健康和疾病中功能的宝贵模型。
研究大脑功能的常用方法是通过立体神经外科在解剖特定位置植入电极。这允许在清醒和自由行动的动物12，13的不同目标区域的神经活动进行慢性记录。立体神经外科是许多研究领域中不可或缺的技术，因为它允许精确定位神经解剖区域。与猴和啮齿动物文献相比，描述动物的立体神经外科的已发表的研究较少，它们往往提供手术中涉及的步骤的稀疏细节。此外，那些更详细的重点是电生理记录的程序在头部约束动物14，15，16，17。
为了促进在神经科学研究中更广泛地采用黑猩猩作为模型有机体，目前的方法定义了该物种中成功的立体神经外科所需的具体步骤。除了植入记录阵列（如本方法所述），同样的技术可以适用于许多其他实验目的，包括植入刺激电极用于治疗疾病 18或因果驱动电路行为19;植入导管用于提取和定量神经递质20，注射试剂，包括诱导疾病模型12或电路追踪研究15;离散组织区域消融21;为光遗传学研究植入选择22;植入光学窗口进行皮质显微分析23;和植入电皮质学（ECoG）阵列24。因此，这个程序的总体目标是概述在自由行为的人造肿瘤中植入用于慢性电生理记录的微电极阵列的手术步骤。
Protocol
动物实验是根据国家卫生研究院《实验室动物护理和使用指南》进行的，并经桑托斯杜蒙特研究所伦理委员会批准（第02/2015AAS议定书）。 1. 手术准备 将每个电极阵列连接到与要使用的立体框架兼容的电极支架上。 将一个电极支架连接到立体微操作器，并将一根微线设置为耳间坐标。如有必要，对附加电极阵列和支架重复此操作。注：任何微线的间坐标可?…
Representative Results
本研究的目的是描述一个立体神经外科手术程序，用于在普通马莫塞特植入微电极阵列进行电生理记录。典型的手术（从麻醉诱导到麻醉恢复）将持续约5~7小时，具体取决于植入的阵列数量。在这里，两个阵列是对称地植入的，一个在每个大脑半球。每个阵列包含 32 条不锈钢微线，这些微线排列在 7 个束中，针对基底神经质-皮质瘤电路的多个结构（图 1），但电极设计和目…
Discussion
这项工作提供了在马莫塞特大脑中植入微电极记录阵列所涉及的过程的详细描述。当在其他小型灵长类动物中植入电极时，无论是自制的还是商用的电极，都可以很容易地使用同样的方案。此外，它可以很容易地适应其他实验目的，需要精确定位大脑结构。因此，该协议在立体坐标和颅骨钻孔技术方面故意含糊不清，因为这些方面可能变化最大。例如，为了植入手术中使用的阵列，颅骨切除术在?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢贝尔纳多·路易斯在拍摄和编辑方面提供的技术协助。这项工作得到了桑托斯杜蒙特研究所、巴西教育部和高等高级学院的支持。
Materials
| Equipments | |||
| 683 Small Animal Ventilator | Harvard Apparatus, Inc. | 55-0000 | |
| Anesthesia Assembly | BRASMED | COLIBRI | |
| Barber Clippers | Mundial | HC-SERIES | |
| Dental Drill | Norgen | B07-201-M1KG | |
| Homeothermic Heating Pad and Monitor | Harvard Apparatus, Inc. | 50-7212 | |
| Marmoset Stereotaxic Frame | Narishige Scientific Instrument Lab | SR-6C-HT | |
| Patient Monitor and Pulse Oximeter | Bionet Co., Ltd | BM3 | |
| Stereotaxic Micromanipulator | Narishige Scientific Instrument Lab | SM-15R | |
| Surgical Microscope | Opto | SM PLUS IBZ | |
| Instruments | |||
| Allis tissue forceps | Sklar | 36-2275 | |
| Alm Retractor, rounded point, 4×4 teeth | Rhosse | RH11078 | |
| Angled McPherson Forceps | Oftalmologiabr | 11301A | |
| Curved Surgial Scissors | Harvard Apparatus, Inc. | 72-8422 | |
| Curved Tissue Forceps | Sklar | 47-1186 | |
| Delicate Dissection forceps | WPI | WP5015 | |
| Dental Drill Bit | Microdont | ISO.806.314.001.524.010 | |
| Essring Tissue Forceps | Sklar | 19-2460 | |
| FG 1/4 Dental Drill Bit | Microdont | ISO.700.314.001.006.005 | |
| Halsey Needle Holder | WPI | 15926-G | |
| Halstead Mosquito forceps | WPI | 503724-12 | |
| Hemostatic Forceps, Straight | Sklar | 17-1260 | |
| Jewler Forceps | Sklar | 66-7436 | |
| McPherson-Vannas Optathalmic microscissor, 3 mm point | Argos Instrumental | ARGOS-4004 | |
| Pereosteal Raspatory | Golgran | 38-1 | |
| Scalpal Handle | Harvard Apparatus, Inc. | 72-8354 | |
| Screwdrivers | Eurotool | SCR-830.00 | |
| Sodering Iron | Hikari | 21K006 | |
| Surgical Scissor | Harvard Apparatus, Inc. | 72-8400 | |
| Toothed forceps | WPI | 501266-G | |
| Disposables/Single Use | |||
| 1 ml sterile syringe with 26 G needle | Descarpack | 7898283812785 | |
| 130 cm x 140 cm surgical field, presterilized | ProtDesc | 7898467276344 | |
| 24G Needle, presterilized | Descarpack | 7898283812846 | |
| 50 cm x 50 cm surgical field, presterilized | Esterili-med | 110100236 | |
| Cotton Tipped Probes, Presterilized | Jiangsu Suyun Medical Materials Co. LTD | 23007 | |
| Cotton tipped Qutips | Higie Topp | 7898095296063 | |
| Electrode Array | Home made | ||
| Endotracheal tube without cuff, internal diameter 2.0 mm, outer diameter 2.9 mm | Solidor | 7898913077201 | |
| Tinned copper wire, 0.15 mm diameter | |||
| M1.4×3 Stainless steel screws | USMICROSCREW | M14-30M-SS-P | |
| Medical Tape | Missner | 7896544910102 | |
| Nylon surgical sutures | Shalon | N540CTI25 | |
| Scalpal Blade, presterilized | AdvantiVe | 1037 | |
| solder | Kester | SN63PB37 | |
| Sterile Saline 0.9% | Isofarma | 7898361700041 | |
| Sterile Surgical Gloves | Maxitex | 7898949349051 | |
| Sterile Surgical Gown | ProtDesc | 7898467281208 | |
| Surgical Gauze, 15 cm x 26 cm presterilized | Héika | 7898488470315 | |
| Gelfoam | Pfizer | ||
| Drugs/Chemicals | |||
| 0.25mg/ml Atropine | Isofarma | ||
| 10% Lidocaine Spray | Produtos Químicos Farmacêuticos Ltda. | 7896676405644 | |
| 2.5% Enrofloxacino veterinary antibiotic | Chemitec | 0137-02 | |
| Dexametasona Veterinary Anti inflammatory | MSD | R06177091A-00-15 | |
| Hydrogen Peroxide | Farmax | 7896902211537 | |
| Isoflourane | BioChimico | 7897406113068 | |
| Jet Acrylic polymerization solution | Artigos Odontológicos Clássico | ||
| Jet Auto Polymerizing Acrylic | Artigos Odontológicos Clássico | ||
| Ketamine 10% | Syntec | ||
| Lidocaine and Phenylephrine 1.8 ml local anesthetic | SS White | 7892525041049 | |
| Povidone-Iodine solutiom | Farmax | 7896902234093 | |
| Riohex 2% surgical Soap | Rioquímica | 7897780209418 | |
| Silver Paint | SPI Supplies | 05002-AB | |
| Tramadol chloride 50 mg/ml | União Química | 7896006245452 | |
| Refresh gel (polyacrylic acid) | Allergan |
Referências
- Okano, H., Hikishima, K., Iriki, A., Sasaki, E. The common marmoset as a novel animal model system for biomedical and neuroscience research applications. Seminars in Fetal and Neonatal Medicine. 17 (6), 336-340 (2012).
- Harris, R. A., et al. Evolutionary genetics and implications of small size and twinning in callitrichine primates. Proceedings of the National Academy of Sciences. 111 (4), 1467-1472 (2014).
- Kishi, N., Sato, K., Sasaki, E., Okano, H. Common marmoset as a new model animal for neuroscience research and genome editing technology. Development, Growth & Differentiation. 56 (1), 53-62 (2014).
- Sasaki, E. Prospects for genetically modified non-human primate models, including the common marmoset. Neuroscience Research. 93, 110-115 (2015).
- Sasaki, E., et al. Generation of transgenic non-human primates with germline transmission. Nature. 459 (7246), 523-527 (2009).
- Sasaki, E. Creating Genetically Modified Marmosets. The Common Marmoset in Captivity and Biomedical Research. , 335-353 (2019).
- Sato, K., et al. Generation of a Nonhuman Primate Model of Severe Combined Immunodeficiency Using Highly Efficient Genome Editing. Cell Stem Cell. 19 (1), 127-138 (2016).
- Sato, K., et al. Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis. Scientific Reports. 5, 16894 (2015).
- Chaplin, T. A., Yu, H. H., Soares, J. G. M., Gattass, R., Rosa, M. G. P. A Conserved Pattern of Differential Expansion of Cortical Areas in Simian Primates. Journal of Neuroscience. 33 (38), 15120-15125 (2013).
- Mitchell, J. F., Leopold, D. A. The marmoset monkey as a model for visual neuroscience. Neuroscience Research. 93, 20-46 (2015).
- Brok, H. P. M., et al. Non-human primate models of multiple sclerosis: Non-human primate models of MS. Immunological Reviews. 183 (1), 173-185 (2001).
- Santana, M. B., et al. Spinal Cord Stimulation Alleviates Motor Deficits in a Primate Model of Parkinson’s disease. Neuron. 84 (4), 716-722 (2014).
- MacDougall, M., et al. Optogenetic manipulation of neural circuits in awake marmosets. Journal of Neurophysiology. 116 (3), 1286-1294 (2016).
- Wakabayashi, M., et al. Development of stereotaxic recording system for awake marmosets (Callithrix jacchus). Neuroscience Research. 135, 37-45 (2018).
- Johnston, K. D., Barker, K., Schaeffer, L., Schaeffer, D., Everling, S. Methods for chair restraint and training of the common marmoset on oculomotor tasks. Journal of Neurophysiology. 119 (5), 1636-1646 (2018).
- Sedaghat-Nejad, E., et al. Behavioral training of marmosets and electrophysiological recording from the cerebellum. Journal of Neurophysiology. , (2019).
- Kringelbach, M. L., Owen, S. L., Aziz, T. Z. Deep-brain stimulation. Future Neurology. 2 (6), 633-646 (2007).
- Talakoub, O., Gomez Palacio Schjetnan, A., Valiante, T. A., Popovic, M. R., Hoffman, K. L. Closed-Loop Interruption of Hippocampal Ripples through Fornix Stimulation in the Non-Human Primate. Brain Stimulation. 9 (6), 911-918 (2016).
- Oddo, M., Hutchinson, P. J. Understanding and monitoring brain injury: the role of cerebral microdialysis. Intensive Care Medicine. 44 (11), 1945-1948 (2018).
- Metz, G. A., Whishaw, I. Q. Cortical and subcortical lesions impair skilled walking in the ladder rung walking test: a new task to evaluate fore- and hindlimb stepping, placing, and co-ordination. Journal of Neuroscience Methods. 115 (2), 169-179 (2002).
- Gradinaru, V., Mogri, M., Thompson, K. R., Henderson, J. M., Deisseroth, K. Optical Deconstruction of Parkinsonian Neural Circuitry. Science. 324, 354-359 (2009).
- Hammer, D. X., et al. Longitudinal vascular dynamics following cranial window and electrode implantation measured with speckle variance optical coherence angiography. Biomedical Optics Express. 5 (8), 2823-2836 (2014).
- Komatsu, M., Kaneko, T., Okano, H., Ichinohe, N. Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset. Journal of Visualized Experiments. (144), (2019).
- Oliveira, L. M. O., Dimitrov, D. . Surgical Techniques for Chronic Implantation of Microwire Arrays in Rodents and Primates. , (2008).
- Santana, M. B., et al. Spinal Cord Stimulation Alleviates Motor Deficits in a Primate Model of Parkinson’s disease. Neuron. 84 (4), 716-722 (2014).
- Santana, M., Palmér, T., Simplício, H., Fuentes, R., Petersson, P. Characterization of long-term motor deficits in the 6-OHDA model of Parkinson’s disease in the common marmoset. Behavioural Brain Research. 290, 90-101 (2015).
- Misra, S., Koshy, T. A review of the practice of sedation with inhalational anaesthetics in the intensive care unit with the AnaConDa device. Indian Journal of Anaesthesia. 56 (6), 518-523 (2012).
- Freire, M. A. M., et al. Distribution and Morphology of Calcium-Binding Proteins Immunoreactive Neurons following Chronic Tungsten Multielectrode Implants. PLOS ONE. 10 (6), 0130354 (2015).
- Budoff, S., et al. Astrocytic Response to Acutely- and Chronically Implanted Microelectrode Arrays in the Marmoset (Callithrix jacchus) Brain. Brain Sciences. 9 (2), 19 (2019).
- Dzirasa, K., Fuentes, R., Kumar, S., Potes, J. M., Nicolelis, M. A. L. Chronic in vivo multi-circuit neurophysiological recordings in mice. Journal of Neuroscience Methods. 195 (1), 36-46 (2011).
- Nicolelis, M. A. L., et al. Chronic, multisite, multielectrode recordings in macaque monkeys. Proceedings of the National Academy of Sciences. 100 (19), 11041-11046 (2003).
- Lehew, G., Nicolelis, M. A. L. . State-of-the-Art Microwire Array Design for Chronic Neural Recordings in Behaving Animals. , (2008).
- Paxinos, G., Watson, C., Petrides, M., Rosa, M., Tokuno, H. . The Marmoset Brain in Stereotaxic Coordinates. , (2012).
- Brown, M. J., Pearson, P. T., Tomson, F. N. Guidelines for animal surgery in research and teaching. American Journal of Veterinary Research. 54 (9), 1544-1559 (1993).
- Flecknell, P. A. Anaesthesia of Animals for Biomedical Research. British Journal of Anaesthesia. 71 (6), 885-894 (1993).
- Kurihara, S., et al. A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus). Journal of Visualized Experiments. (132), (2018).
- Boer, R. A., de Vries, A. M. O., Louwerse, A. L., Sterck, E. H. M. The behavioral context of visual displays in common marmosets (Callithrix jacchus). American Journal of Primatology. 75 (11), 1084-1095 (2013).
- Kudo, C., Nozari, A., Moskowitz, M. A., Ayata, C. The impact of anesthetics and hyperoxia on cortical spreading depression. Experimental Neurology. 212 (1), 201-206 (2008).
- Ghomashchi, A., et al. A low-cost, open-source, wireless electrophysiology system. 2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. , 3138-3141 (2014).
- Fu, T. M., Hong, G., Viveros, R. D., Zhou, T., Lieber, C. M. Highly scalable multichannel mesh electronics for stable chronic brain electrophysiology. Proceedings of the National Academy of Sciences. 114 (47), 10046-10055 (2017).
Citar este artigo
Budoff, S. A., Rodrigues Neto, J. F., Arboés, V., Nascimento, M. S. L., Kunicki, C. B., Araújo, M. F. P. d. Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus). J. Vis. Exp. (151), e60240, doi:10.3791/60240 (2019).