支持DK065872（ENP），一个卓越的史密斯一家基金会在生物医学研究奖（ENP），F31 DA023760。

摘要两个月的平均年龄C57BL/6J小鼠或同等或3个月岁，平均年龄SD大鼠或同等学历麻醉与氯胺酮（60毫克/公斤大鼠的IP; 100毫克/公斤对小鼠的IP）和甲苯噻嗪（10毫克/公斤，对于任何一个物种的IP）。镇静是监测使用一个温柔的脚趾捏撤回反射<a href="/index/details.stp?ID=236">证明Walantus等</a> 。（朱庇特，6，2007年） <a href="/index/details.stp?ID=404">和Szot</a>等（朱庇特，9日，2007） 。体温可提供通过thermostatregulated加热垫（ALA仪器公司）和通过直肠温度计监视。是剃光头的皮毛和碘与切口前清洗。切皮后（大鼠，小鼠长1厘米2厘米长的）和头骨表面清除所有的软组织，指导导管的位置是确定有关前囟。 6毫米的孔是通过颅骨钻灭鼠手术设计了一个电池供电钻（精细的科学工具，公司）。护理是使钻头不穿透脑膜膜或血管​​。颅骨植入双边5毫米21压力表不锈钢指南领先后的伏隔核，背侧纹状体或前额叶皮质轴。脑立体定位坐标每富兰克林和Paxinos，1997年成立（在鼠脑立体定位坐标，学术出版社）或Paxinos和Watson，2006年（在大鼠脑立体定位坐标，科学出版社）。植入物是安全的牙科水泥。乳酸林格的0.9％生理盐水静脉推注是在手术结束时（5mls资深大律师在大鼠和小鼠的1毫升SC后的液体加热到体温正常），以防止脱水。丁丙诺啡（0.1-0.5mg/kg SC）管理，每日两次，然后，根据需要，如果动物出现在痛苦中。局部抗生素治疗（杆菌肽软膏）和全身性抗生素治疗（青霉素10万IU /公斤，每12小时即时的第一个48小时后运）的管理如果发生术后感染。 手术后，动物被单独安置与食物和水可用的广告随意。允许至少一个星期之前，微透析和安乐死的复苏。从手术中恢复之后，动物们转移到一个微透析笼和微透析探针插入，并在手术过程中已安装在引导轴凝成。探针插入不会引起疼痛或不适，因为探头是通过引导轴绕过皮肤，肌肉和脑膜组织。因此，探针插入无需麻醉和神经化学或行为上的任何麻醉诱导效应是可以避免的。我们让探头稳定12小时，然后我们开始根据实验另一个8-12小时，每30分钟取样。我们监控的运动动物的行为，通过光电池或手动录像实验者运动。 Microdialysate样品被注入到一个高性能液相色谱电化学检测法（HPLC - EC）神经化学物质的检测和分析仪器。我们期待为基底神经化学和运动行为的影响。在实验结束后动物安乐死过量全身氯胺酮（200毫克/公斤的IP）和甲苯噻嗪（20毫克/公斤，IP）。心脏灌注4％多聚甲醛的0.9％生理盐水。大脑被删除，冻结和削减沿微透析探针道，以验证准确的探头安置。 程序<ol><li>成立脑立体定位仪和所需的所有材料。确保面积和工具清洗和消毒。 </li><li>电动剃须刀剃去毛皮。从耳朵转到刚刚在两眼之间，在不同的方向移动的剃须刀，有效地清理面积的皮草。 povidine /碘剃光区，但保护它的眼睛。 </li><li>装载动物的立体定位仪上，放入耳道，耳棒和收紧到位。首先装载在耳道的耳栏，然后举行到位，并在另一只耳朵栏幻灯片。你知道你在正确的位置时，头部可以不再移动一边到另一边。立体前安装安全口，确保头部是用尺子水平。在脑立体定位仪平台的垂直位置的标尺，并为90 °角之间的统治者和动物的头皮，中间检查）。通过脑立体定位仪确认，如果提供这种能力。 </li><li>前/后切口在头皮上的lambda只是在动物之间的眼睛，用无菌手术刀。使用消毒止血，捏皮肤和保持切口开放。使用几个无菌棉签，干燥暴露颅骨表面。 <li>摆上其装载的引导导管，发现在头骨的前囟门和位置引导导管在这个位置的权利。写下的前/后和横向坐标。从囟门，探头安置立体图集援助需要找到正确的坐标。引导导管正确的坐标位置通过添加或减去从囟。把你的引导导管，直到它触及颅骨，然后记录这个腹侧协调。在头骨上的这个位置的无菌铅笔铅笔标记，这是在那里你会被钻。 </li><li>取出引导的套管和消毒钻头。小心地钻一个孔铅笔标记，直到你通过颅骨的宽度。指导导管检查，看看它是否会明确双方接触孔。保持钻井和检查，直到套管可以在一条直线路径明确。一旦孔，使用消毒针头，轻轻一拳脑膜，以便通畅的导管插入。 </li><li>接下来，使用手电钻，使颅骨螺钉：两个导管孔，两个外侧的导管孔，和两个后向两侧的前六洞。消毒六颗螺丝，并放置到他们的头骨，直到它们被紧紧地固定在。 </li><li>用乙醇和生理盐水，装入清洁的引导导管，慢慢降低到正确的腹协调。确保双方没有接触，这是完全垂直。 </li><li>将固定螺丝内侧和后面的后颅骨螺钉和它举行的地方用镊子。混合成薄薄的液体牙科水泥批次和覆盖引导套管，螺丝，和头骨的其余部分用无菌压舌板。另一批，较厚的这段时间，完全覆盖面积和足够的套管和固定螺丝，以确保它。 </li><li>由于水泥较厚，凝固前，单独从水泥杯皮肤和模具用锅铲水泥杯，以确保水泥盖周围是光滑的，不刺激皮肤后。 </li><li>允许牙科水泥完全干透后再从设备中取出的动物。取出的止血。申请杆菌肽周围的水泥盖的方式。 </li><li>一旦关闭动物脑立体定位仪，注射0.25毫升1毫升生理盐水SC（皮下注射）青霉素IM（肌肉内）。 </li><li>放置在自己的笼子里的动物和监视它，直到它变成自觉，然后返回到它的房间恢复。 </li><li>监控动物，直到他们从麻醉中恢复的手术和日常运算后的一天，直到实验结束后，感染和疼痛/窘迫的评价迹象。这包括周末和节假日。低自发运动，遇险后处理发声，弯腰驼背的姿势，腹泻，肿胀和排放的headmount区，缺乏喂养/饮用水所有的痛苦和苦恼的迹象。丁丙诺啡（0.1-0.5mg/kg SC）的管理，每天两次，然后，根据需要，如果动物出现在痛苦中。局部抗生素治疗（杆菌肽软膏）和全身性抗生素治疗（青霉素10万IU /公斤，每12小时即时的第一个48小时后运）的管理如果发生术后感染。如果任何这些症状坚持以下丁丙诺啡管理，补充液，并在12个小时的手术内的抗生素治疗，动物安乐死。 </li></ol>

<ol>
	<li>Bungay, P. M., Newton-Vinson, P., Isele, W., Garris, P. A., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+of+dopamine+interpreted+with+quantitative+model+incorporating+probe+implantation+trauma.">Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma.</a> J. Neurochem. 86, 932-946 (2003).</li><li>Chen, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+tissue+trauma+on+the+characteristics+of+microdialysis+zero-net-flux+method+sampling+neurotransmitters.">Effects of tissue trauma on the characteristics of microdialysis zero-net-flux method sampling neurotransmitters.</a> Journal of Theor. Biology. 238, 863-881 (2006).</li><li>Geiger, B. M., Behr, G. G., Frank, L., Caldera-Siu, A. D., Beinfeld, M. C., Kokkotou, E. G., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+defective+mesolimbic+dopamine+exocytosis+in+obesity-prone+rats.">Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats.</a> FASEB Journal. 8, 2740-2746 (2008).</li><li>Pothos, E. N., Creese, I., Hoebel, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+eating+with+weight+loss+selectively+decreases+extracellular+dopamine+in+the+nucleus+accumbens+and+alters+dopamine+response+to+amphetamine,+morphine+and+food+intake.">Restricted eating with weight loss selectively decreases extracellular dopamine in the nucleus accumbens and alters dopamine response to amphetamine, morphine and food intake.</a> The Journal of Neuroscience. 15, 6640-6650 (1995).</li></ol>

The authors have nothing to disclose.

在体内微透析技术的首选工具，衡量一个活生生的动物不同脑网站的多个神经递质及代谢产物。但是，它只监视外的神经化学物质的水平，并没有提供时间分辨率实时监控胞外分泌的神经递质。通过所谓的“净流量”的技术版本，可以在一个给定网站的实际的神经递质浓度计算，这反过来又可以通过质膜转运给神经递质的再摄取率的准确测量。 微透析是在说明在动物之间的不同群体（即不同基因型）和破?...

survivable stereotaxic surgery in rodents 

能够测量大脑清醒动物神经递质外基础水平可确定不同的体制性挑战（药理或生理）中枢神经系统的影响。例如，一个可以直接测量动物的中脑多巴胺预测如何应对像D -苯丙胺或自然的刺激，如食品药物多巴胺的释放。在这段视频中，我们告诉你如何引导套管针对大鼠大脑中的特定的网站植入，如何插入和植入微透析探针，以及如何使用高效液相色谱电化学检测法（HPLC - EC）来衡量外水平可氧化神经递质及代谢产物。通过微透析探针局部药物精确的介绍，让现场特异性的提炼工作，在一个化合物的S作用机制。这种技术具有优良的解剖和化学决议，但只有温和微透析样品的时间分辨率通常处理，每次20-30分钟，以确保检测的神经递质水平。可以帮助补充体内的工具（例如，切片和细胞培养电）监测实时的神经递质。

Watch this Scientific Journal Video about Survivable Stereotaxic Surgery in Rodents at JoVE.com

Survivable Stereotaxic Surgery in Rodents

自由移动的动物不同的大脑区域外的神经递质水平的监测神经递质的释放和行为之间的联系提供了见解。在体内微透析技术结合电化学检测提供了极好的解剖和化学的决议;和基底神经传递是如何由药理或生理操作改变的信息。

在啮齿类动物的存活立体定向手术

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

survivable-stereotaxic-surgery-in-rodents

Research

JoVE Journal

Biology

Survivable Stereotaxic Surgery in Rodents 

68.1K 次观看.  Tufts University. 自由移动的动物不同的大脑区域外的神经递质水平的监测神经递质的释放和行为之间的联系提供了见解。在体内微透析技术结合电化学检测提供了极好的解剖和化学的决议;和基底神经传递是如何由药理或生理操作改变的信息。

视频: Survivable Stereotaxic Surgery in Rodents

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

一种慢性脑功能成像开颅手术过程

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

科研

生物学

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

剖析在非人类灵长类动物脑立体定向空间

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

建立从小学成人成纤维细胞培养鼠害

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

鼠类外科的原则，为新的外科医生

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Intracavernosal 压力记录对啮齿动物勃起功能的评价

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and disease. EIM is obtained by applying a low-intensity, directionally focused, electrical current to a muscle of interest across a range of frequencies (i.e., from 1 kHz to 10 MHz) and recording the resulting voltages. From these, several standard impedance components, including the reactance, resistance, and phase, are obtained. When performing ex vivo measurements on excised muscle, the inherent passive electrical properties of the tissue, namely the conductivity and relative permittivity, can also be calculated. EIM has been used extensively in animals and humans to diagnose and track muscle alterations in a variety of diseases, in relation to simple disuse atrophy, or as a measure of therapeutic intervention. Clinically, EIM offers the potential to track disease progression over time and to assess the impact of therapeutic interventions, thus offering the opportunity to shorten the clinical trial duration and reduce sample size requirements. Because it can be performed noninvasively or minimally invasively in living animal models as well as humans, EIM offers the potential to serve as a novel translational tool enabling both preclinical and clinical development. This article provides step-by-step instructions on how to perform in vivo and ex vivo EIM measurements in mice and rats, including approaches to adapt the techniques to specific conditions, such as for use in pups or obese animals.

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

This article details how to perform in vivo (using surface and needle electrode arrays) and ex vivo (using a dielectric cell) electrical impedance myography on the rodent gastrocnemius muscle. It will demonstrate the technique in both mice and rats and detail the modifications available, (i.e., obese animals, pups).

在啮齿动物中进行体内和离体电阻抗肌图

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and ...

通过脱细胞定量骨骼肌脂肪浸润

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

作者聚焦：基于脱细胞化的骨骼肌脂肪浸润定量  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

基于脱细胞的骨骼肌脂肪浸润定量

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

饲养无菌 Nasonia 黄蜂

Optimizing the Rearing Procedure of Germ-Free Wasps

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of external microorganisms on insect microbiota and thus promote the rapid development of insect microbiota research. Nasonia (wasp genus) is a parasitic wasp insect that has many advantages, such as a short lifespan, high genetic variation, easy operation, etc., and is widely used as an insect model system. Unlike antibiotic treatment, which can only reduce the number of microorganisms in animals, aseptic rearing techniques can control both the composition and quantity of microorganisms in animals, further facilitating the study of host-microbe interactions. However, previous versions of Nasonia rearing medium (NRM) have some defects and problems, such as a complex and time-consuming preparation process, easy contamination by bacteria or fungi, and short storage time. Therefore, this study solves these problems by optimizing the tools used in the NRM preparation process, storage conditions, and component ratios. The optimized medium could allow storage at -20 &#176;C for at least 3 months and eliminate the possibility of NRM contamination during feeding sterile wasps. This further improves the survival rate and health level of aseptic Nasonia, which is important for using Nasonia as a model for microbial research.

作者聚焦：优化无菌黄蜂的饲养程序  

Nasonia wasp embryos were dissected from Lucillia sericata pupae after parasitization for 12-24 h and washed with alcohol and 10% sodium hypochlorite solution to obtain germ-free embryos. After rearing the germ-free embryos&#160;and supplying them with Nasonia rearing medium to grow and develop in vitro, germ-free Nasonia adults were obtained.

优化无菌黄蜂的饲养程序

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of ...