猪中枢神经系统的组织学分析的曝光：一个手册斩首，颅骨打开，并去除脑
Summary
本文和视频教学的目的是描述如何揭露和删除死后猪脑和垂体处于完整状态，适合后续宏观和组织学分析。
Abstract
猪有大型动物平移神经科学的研究越来越受欢迎作为一个经济上和道德上可行的替代品，以非人类的灵长类动物。猪的大脑神经尺寸允许使用常规临床脑成像器和直接使用和神经外科手术和设备测试从人的诊所。进一步宏观和组织学分析，但是，需要的猪中枢神经系统（CNS）和脑随后去除死后曝光。这不是一件容易的事，因为猪的中枢神经系统是由厚，骨头骨和脊柱封装。本文和视频教学的目的是描述如何揭露和删除死后猪脑和完整状态的脑垂体，适合后续的宏观和组织学分析。
Introduction
猪转化神经科学的研究在过去二十年中已经变得越来越流行。猪大脑的大尺寸使得能够使用常规临床脑成像器和直接使用和来自人诊所1，2，3，4，5，6，7，8神经外科手术和设备测试。在过去的20年里，猪，迷你猪特别是（ 例如，哥廷根小型猪），已被用于检查神经调节的治疗方式，如造血干细胞移植;病毒载体转染;和深部脑刺激针对帕金森病，肥胖症，抑郁症，和阿尔茨海默病2，6，= “外部参照”> 9，10，11，12，13，14，15，16，17。这一直跟着的立体性和手术方法的发展，操纵小型猪CNS 3，18，19，20，21。的提起CNS变化在使用脑成像（PET 10，13，22，24和MR 23），膀胱测压11，12，25活体动物被评估，步态分析17，神经评价9，17，和基于组织学和体视分析14，15，17，26，27，31尸检。然而，事后分析需要曝光和去除猪脑，这不是一件容易的事，作为一个厚，骨颅骨和硬脑膜纤维覆盖包围猪脑子。
本文和视频教学的目的是描述如何在死后猪脑和垂体可在15-20分钟使用非机动手术工具进行曝光和去除的完整状态。教学视频和摄影插图显示雄性迷你猪（年龄：20-25公斤：6个月，体重），用于在小型猪脑垂体解剖研究。
Protocol
动物麻醉和euthanesia是按照（NIH出版号86-23，1985年修订版）和丹麦议会动物研究伦理认可的“实验动物管理的原则”进行。 1.仪器收集的视频介绍和材料表中所列的仪器。 2.斩首注：麻醉诱导通过5mL的咪达唑仑（5毫克/毫升）和5毫升氯胺酮（25毫克/毫升）的肌内注射。 5-10分钟后，当动物被深深地镇静剂，耳静?…
Representative Results
为了防止干燥的组织材料，建议去除的脑和垂体存储在填充有固定剂或已经执行宏观分析之后立即等渗盐水罐。组织材料可以被存储在固定剂多年，而存储在等渗盐水，即使在冰箱中，会导致组织随时间衰减。 除去的垂体也可以通过浸入干冰冷却液体的2-甲基丁烷中直接冷冻，而完整猪脑为直接冷冻28过大。?…
Discussion
大多数实验研究神经科学在小动物物种，如小鼠和大鼠，在那里访问CNS由薄skull-和硬脑膜厚度容易进行。然而，在较大的实验动物如猪1，4，8，羊32，以及非人类灵长类动物中，相当大的厚度这些结构需要使用坚固的仪器（材料表）和适当的入口点头骨骨去除（ 图的2）。需要限制硬?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感激地承认特林·W·米尔森太太的熟练援助，莉莎M.夫人配件，并在Påskehøjgaard员工。丹麦医学研究委员会，Lundbeck公司基金会和诺和诺德基金会提供资助的研究。
Materials
| Heavy Scalpel Handle #4 | FST (Fine Science Tools) | 10008-13 | Good for skin incision and soft tissue removal |
| Non-Sterile Scalpel Blades #23 | FST | 10023-00 | |
| Scalpel Handle #7 | FST | 10007-12 | Optimal for dural incision and precision work |
| Non-Sterile Scalpel Blades #11 | FST | 10011-00 | |
| Surgical Forceps | FST | 11024-18 | The tip of the surgical forceps ensure a firm grip |
| Kerrison Bone Punch | Aesculap Neurosurgery | FF713R | Must be robust, bite size 3-5 mm |
| Bone Rongeur | Aesculap Neurosurgery | MD615 | Must be robust, bite size 15 x 5 mm |
| Bone Rongeur | Aesculap Neurosurgery | FO551R | Must be robust, bite size 25 x 15 mm |
| Bone Chisel | Lawton | 67-0335 | The size of the chisel head should not exceed 20 mm |
| Mallet (Hammer) | Millarco | 5624108 | Weigth 300 g, length 30 cm, head hit area size 2 x 2 cm |
| Micro-Scissor | FST | 14002-14 | |
| Dissector | Aesculap Neurosurgery | OL165R | |
| Göttingen minipigs | Ellegaard Göttingen Minipigs A/S, Denmark | ||
| Euthanimal | pentobarbital | ||
| Ketamine | Pfizer | ||
| Midazolam | Hameln Pharmaceuticals |
References
- Lind, M. N., Moustgaard, A., Jelsing, J., Vajta, G., Cumming, P., Hansen, A. K. The use of pigs in neuroscience: Modeling brain disorders. Neurosci Biobehav Rev. 31, 728-751 (2007).
- Bjarkam, C. R., et al. Neuromodulation in a minipig model of Parkinson disease. British J Neurosurg. 22 (Suppl. 1), S9-S12 (2008).
- Bjarkam, C. R., Cancian, G., Glud, A. N., Ettrup, K. S., Østergaard, L., Sørensen, J. C. MRI-guided stereotaxic targeting in pigs based on a stereotaxic localizer box fitted with an isocentric frame and use of SurgiPlan computer-planning software. J Neurosci Methods. 183 (2), 119-126 (2009).
- Sauleau, P., Lapouble, E., Val-Laillet, D., Malbert, C. H. The pig model in brain imaging and neurosurgery. Animal. 3 (8), 1138-1151 (2009).
- Bjarkam, C. R., et al. Safety and function of a new clinical intracerebral microinjection instrument for stem cells and therapeutics examined in the Göttingen minipig. Stereotact Funct Neurosurg. 88 (1), 56-63 (2010).
- Fjord-Larsen, L., et al. Long-term delivery of nerve growth factor by encapsulated cell biodelivery in the minipig basal forebrain. Mol Therapy. 18 (12), 2164-2172 (2010).
- Sørensen, J. C., et al. Development of neuromodulation treatments in a large animal model – Do neurosurgeons dream of electric pigs?. Prog Brain Res. 194, 97-103 (2011).
- Dolezalova, D., et al. Pig models of neurodegenerative disorders: utilization in cell replacement-based preclinical safety and efficacy studies. J Comp Neurol. 522 (12), 2784-2801 (2014).
- Mikkelsen, M., Moller, A., Jensen, L. H., Pedersen, A., Harajehi, J. B., Pakkenberg, H. MPTP-induced Parkinsonism in minipigs: A behavioral, biochemical, and histological study. Neurotoxicol Teratol. 21, 169-175 (1999).
- Danielsen, E. H., et al. The DaNEX study of embryonic mesencephalic, dopaminergic tissue grafted to a minipig model of Parkinson’s disease: Preliminary findings of effect of MPTP poisoning on striatal dopaminergic markers. Cell Transplant. 9 (2), 247-259 (2000).
- Dalmose, A., Bjarkam, C. R., Sørensen, J. C., Jørgensen, T. M., Djurhuus, J. C. Effects of high frequency deep brain stimulation on urine storage and voiding function in conscious minipigs. Neurourol Urodyn. 23 (3), 265-272 (2004).
- Dalmose, A., Bjarkam, C. R., Djurhuus, J. C. Stereotactic electrical stimulation of the pontine micturition center in the pig. Br J Urol. 95, 886-889 (2005).
- Andersen, F., Watanabe, H., Bjarkam, C. R., Danielsen, E. H., Cumming, P. Pig brain stereotaxic standard space: Mapping of cerebral blood flow normative values and effect of MPTP-lesioning. Brain Res Bull. 66 (1), 17-29 (2005).
- Glud, A. N., et al. Direct gene transfer in the minipig CNS using stereotaxic lentiviral microinjections. Acta Neurobiol Exp. 70 (3), 1-8 (2010).
- Glud, A. N., et al. Direct MRI-guided stereotaxic viral mediated gene transfer of alpha-synuclein in the minipig CNS. Acta Neurobiol Exp. 71 (4), 508-518 (2011).
- Ettrup, K. S., Sørensen, J. C., Rodell, A., Alstrup, A. K. O., Bjarkam, C. R. Hypothalamic deep brain stimulation influences autonomic and limbic circuitry involved in the regulation of aggression and cardiocerebrovascular control in the minipig. Stereotact Funct Neurosurg. 90 (5), 281-291 (2012).
- Nielsen, M. S., et al. Continuous MPTP intoxication in the minipig results in chronic parkinsonian deficits. Acta Neurobiol Exp. 76, 198-210 (2016).
- Bjarkam, C. R., et al. A MRI-compatible stereotaxic localizer box enables high-precision stereotaxic procedures in pigs. J Neurosci Methods. 139 (2), 293-298 (2004).
- Bjarkam, C. R., Jorgensen, R. L., Jensen, K. N., Sunde, N. A. A., Sørensen, J. C. H. Deep brain stimulation electrode anchoring using BioGlue®, a protective electrode covering, and a titanium microplate. J Neurosci Methods. 168, 151-155 (2008).
- Ettrup, K. S., et al. Basic Surgical Techniques in the Minipig: Intubation, Transurethral Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion. J Vis Exp. (52), e2652 (2011).
- Ettrup, K. S., Tornøe, J., Sørensen, J. C., Bjarkam, C. R. A surgical device for minimally invasive implantation of experimental deep brain stimulation leads in large research animals. J Neurosci Methods. 200 (1), 41-46 (2011).
- Danielsen, E. H., et al. Positron emission tomography of living brain in minipigs and domestic pigs. Scand J Lab Anim Sci Suppl. 25 (1), 127-135 (1998).
- Røhl, L., et al. Time evolution of cerebral perfusion and ADC measured by MRI in a porcine stroke model. J Magn Reson Imaging. 15 (2), 123-129 (2002).
- Cumming, P., Gillings, N. M., Jensen, S. B., Bjarkam, C. R., Gjedde, A. Kinetics of the uptake and distribution of the dopamine D2/3 agonist (R)-N-[1-11C]n-propylnorapomorphine in brain of healthy and MPTP-poisoned Gottingen miniature pigs. Nucl Med Biol. 30 (5), 547-553 (2003).
- Jensen, K. N., Deding, D., Sørensen, J. C., Bjarkam, C. R. Long-term implantation of deep brain stimulation electrodes in the pontine micturition centre of the minipig. Acta Neurochir. 151 (7), 785-794 (2009).
- Rosendal, F., et al. Does chronic low dose treatment with ciclosporin influence the brain? A histopathological study in pigs. Transplantation Proc. 37 (8), 3305-3308 (2005).
- Nielsen, M. S., Sørensen, J. C., Bjarkam, C. R. The substantia nigra pars compacta of the minipig: An anatomical and stereological study. Brain Struct Funct. (4-5), 481-488 (2009).
- Sørensen, J. C., Bjarkam, C. R., Simonsen, C. Z., Danielsen, E., Geneser, F. A. Oriented sectioning of irregular tissue blocks in relation to computerized scanning modalities. Results from the domestic pig brain. J Neurosci Methods. 104, 93-98 (2000).
- Bjarkam, C. R., Pedersen, M., Sørensen, J. C. New strategies for embedding, orientation and sectioning of small brain specimens enable direct correlation to MR-images, brain atlases, or use of unbiased stereology. J Neurosci Methods. 108, 153-159 (2001).
- Bjarkam, C. R., Sørensen, J. C., Geneser, F. A. Distribution and morphology of serotonin-immunoreactive axons in the hippocampal region of the New Zealand white rabbit. I. Area dentata and hippocampus proper. Hippocampus. 13 (1), 21-37 (2003).
- Bjarkam, C. R., Glud, A. N., Orlowski, D., Sørensen, J. C., Palomero-Gallagher, N. The telencephalon of the minipig, cytoarchitecture and cortical surface anatomy. Brain Struct Funct. , (2016).
- Boltze, J., Nitzsche, B., Geiger, K. D., Schoon, H. A. Histopathological investigation of different MCAO modalities and impact of autologous bone marrow mononuclear cell administration in an ovine stroke model. Transl Stroke Res. 2, 279-293 (2011).
- Jortner, B. S. The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation. Neurotoxicology. 27, 628-634 (2006).
Cite This Article
Bjarkam, C. R., Orlowski, D., Tvilling, L., Bech, J., Glud, A. N., Sørensen, J. H. Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal. J. Vis. Exp. (122), e55511, doi:10.3791/55511 (2017).