这项研究是由Vanderbilt - Ingram癌症中心支援津贴（P30 CA068485），儿童脑瘤基金会和美国国立卫生研究院（NS042205）的赠款支持。

1。荷瘤小脑和肿瘤组织中的分离的显微解剖<ol><li> 检索肿瘤组织 <ol><li>轴承髓母细胞瘤的患病老鼠往往runted，并显示脑积水和典型的神经症状，包括瘫痪后未能收复当朝天的姿势。要检索肿瘤组织，安乐死小鼠吸入二氧化碳。这是项重要的，不执行颈椎脱位，程序产生的压力后的头骨，并能破坏肿瘤组织的完整性。 </li><li>斩首是死亡后，立即用一把剪刀，头发和肌肉组织，尽可能消除良好的可视化的头骨。清洁的头骨表面用95％乙醇浸泡Kimwipe。 </li><li>用细剪刀削减开放沿着头骨中线，然后取出用细镊子，在这一点荷瘤全脑包括小脑暴露颅骨组织。 </li><li>虽然健康成人小脑显示定义的半球和蚓部，荷瘤小鼠的小脑往往是一个光滑的表面和突出的血管扩大，无定形。使用无菌技术，检索使用镊子在冰冷的磷酸盐和地方的小脑肿瘤缓冲无镁液（PBS）+和Ca 2 +。 </li></ol> 注：所有的仪器都在95％的乙醇，并在使用前高压灭菌蒸汽进行消毒。 </li><li> 肿瘤组织离解 <ol><li>转移的肿瘤组织从PBS，以50％的Accutase（PBS稀释）的4倍左右的肿瘤组织的体积，组织讳言罚款剪刀为3分钟，在室温下孵育其次，在37 ° C，4分钟之后，该组织经过额外3分钟1毫升Pipetman重复pipeting。这种方法应该产生一个单细胞和小细胞聚集的混合物。 </li><li>稀释的细胞悬浮在1000xg 5分钟沉淀细胞，用PBS离心3倍。 （上述程序已被形容“髓母细胞瘤干细胞的分离，浓缩和维护”，朱庇特在新闻） </li> 重悬细胞沉淀在新鲜配制的神经干细胞Neurobasal培养基的组成与谷氨酰胺，青霉素，链霉素，氮，B27，人类表皮生长因子（25毫微克/毫升）和基本的FGF（25毫微克/毫升）媒介。计算细胞密度的解决方案，并作出适当的进一步稀释，与其他神经干细胞的培养基可以载入适当数量的游离肿瘤细胞，如5X10 5 4μL的解决方案，成为一个无菌的锥尖10μL注射器。接种细胞的确切数目，应确定由研究人员根据具体的实验要求。保持在一个小型的200μL的离心管的细胞溶液（PCR管）在冰上。 </ol></li></ol> 2。麻醉剂受体小鼠的程序对于外科手术，消毒面积灭鼠手术是需要过程的持续时间。理想的情况下，为动物准备，经营领域和动物恢复独立，但毗邻地区成立一个长板凳。外科手套，手套不考试，需要手术。保持无菌技术操作，戴手套的手，腰部以上的，仅用于干燥无菌表面处理无菌对象。 <ol><li>用于麻醉氯胺酮（鼠标重量每公斤100毫克氯胺酮）和甲苯噻嗪（鼠标的重量每公斤10毫克甲苯噻嗪）的混合物。管理麻醉药腹腔。 </li><li>它一般需要3-5分钟麻醉药充分的效果。判断鼠标是否完全麻醉脚趾/尾掐。将少量眼药膏对鼠标的眼睛。带来的完全麻醉的鼠标操作区，不断监测，以确保鼠标正常呼吸。 </li></ol> 3。颅内移植的肿瘤细胞<ol><li>剃须鼠标的背后的头地区，使用推剪，来揭示脑和小脑以上后的头骨。 </li><li>申请使用棉花尖端涂药，用酒精垫擦拭就暴露出来了头皮优碘的解决方案。重复这两个步骤消毒两次。 </li><li>请在四分之一英寸的切口后用消毒手术刀头皮。钻一个0.5毫米的毛刺孔的权利2毫米和2毫米，后用消毒的牙钻的lambda。 </li><li>鼠标放置到一个立体的框架，通过连接到帧保持其门牙。仔细下降和定位锥尖10μL注射器装载4μL到毛刺洞肿瘤细胞的解决方案。一旦注射器针头斜角低于颅骨表面，Descend另外3毫米，然后登上0.5毫米。慢慢地注入肿瘤细胞所需的金额，在30秒的时间框架的稳定力量，进入收件人的小脑。完成增加一个两分钟的注射针后，留在其地方。然后取出针头和注射器。使用autoclip剪辑的伤口。 </li><li>在手术后的升温毯保持鼠标，以帮助保持其体温。移动和呼吸模式将持续观察。一旦小鼠从麻醉中复苏，放置在无菌住房笼。监察日常食物和水的摄入量，行为，仪容，眼睛红染色。检查切口部位感染的迹象。取出手术后7天autoclips。 为2-6个月的平均保持在小鼠和牺牲者作进一步的研究显示典型的髓母细胞瘤的症状。 </li></ol> 4。代表性的成果在受体小鼠中开发的二次髓母细胞瘤非常相似的原发肿瘤的。轴承继发性肿瘤的小脑时往往扩大异位血管明显的无定形全坐骑观看。继发性肿瘤组织的免疫组织化学分析显示，肿瘤细胞高度增生，对此表示强烈SmoM2 - YFP和小脑颗粒神经元前体，如Math1和PAX6标志物。当分离和培养使用方法（“分离，浓缩和维护髓母细胞瘤干细胞”，按朱庇特 ），二级髓母细胞瘤肿瘤细胞可迅速扩大，表达多种干细胞标记物，克隆形成，并可以进一步启动肿瘤形成时移植到其他免疫功能低下受助。 <img src="/files/ftp_upload/2153/2153fig1.jpg" alt="图1" /> 图1。典型的整体式和分段一所中学的髓母细胞瘤组织的意见 一个典型的例子中学髓母细胞瘤组织开发的5X10 5原发肿瘤细胞注射后26天。苏木素染色，揭示了典型的髓母细胞瘤细胞形态，包括高核/质比和核多态性。这些继发性肿瘤细胞高度增生，Ki67表达表示，他们也有力表达膜SmoM2 - YFP。 <img src="/files/ftp_upload/2153/2153fig2.jpg" alt="图2" /> 图2。中学髓母细胞瘤细 ​​胞可以在体外培养和扩大 使用鼠标主髓母细胞瘤细胞（“分离，浓缩和维护髓母细胞瘤干细胞”，按朱庇特）所描述的协议，这些中学的髓母细胞瘤细胞可以培养和扩大，为多个段落。图为代表在4天，14天培养的肿瘤细胞集落。培养的肿瘤细胞，双极性，用很少的细胞质和辐射往往从一个细胞总量的致密核心拉长。他们没有表现出生长接触抑制。小圆细胞是红细胞。

<ol>
	<li>Schuller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+granule+neuron+precursor+identity+is+a+critical+determinant+of+progenitor+cell+competence+to+form+Shh-induced+medulloblastoma.">Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma.</a> Cancer Cell. 14, 123-134 (2008).</li><li>Yang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+alters+the+fate+of+preneoplastic+cells+in+a+mouse+model+of+medulloblastoma.">N-myc alters the fate of preneoplastic cells in a mouse model of medulloblastoma.</a> Genes Dev. 23, 157-170 (2009).</li><li>Flora, A., Klisch, T. J., Schuster, G., Zoghbi, H. Y., Y, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Atoh1+disrupts+Sonic+Hedgehog+signaling+in+the+developing+cerebellum+and+prevents+medulloblastoma.">Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma.</a> Science. 326, 1424-1427 (2009).</li><li>Salsano, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+neurogenic+basic+helix-loop-helix+transcription+factor+NEUROG1+identifies+a+subgroup+of+medulloblastomas+not+expressing+ATOH1.">Expression of the neurogenic basic helix-loop-helix transcription factor NEUROG1 identifies a subgroup of medulloblastomas not expressing ATOH1.</a> Neuro Oncol. 9, 298-307 (2007).</li></ol>

几个关键因素，以确保成功的髓母细胞瘤细胞allografting，产生继发性肿瘤的形成如下：首先，用于组织解离，只有50％Accutase和不过消化的组织，作为长期的酶处理细胞活力显着降低。另外，只使用1毫升的大小Pipetman较小的提示，还可以生成游离细胞的物理损坏。它是好的，有单个细胞和小allografting聚集的混合物。二，混合和平衡的肿瘤细胞的解决方案，每次装货前汉密尔顿注射器。不要使用超过...

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<td>Neurobasal medium</td>
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<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0311</td>
<td>25ng/ml</td>
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<td>bFGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0023</td>
<td>25ng/ml</td>
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<td>N2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502048</td>
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<td>B27(-RA)</td>
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intracranial orthotopic allografting of medulloblastoma cells in immunocompromised mice

髓母细胞瘤是最常见的小儿神经系统的肿瘤。小脑颗粒神经元前体（CGNPs）1-4为髓母细胞瘤细 ​​胞的原产地集中于动物研究的一个庞大的身躯。然而，在人类患者（结节，促纤维增生，古典和大细胞/间变性），而事实上，在没有CGNP标记5的异位表达的人类患者的一个子集发现髓母细胞瘤是，髓母细胞瘤亚型的不同临床表现表明，髓母细胞瘤细胞和分子的起源更复杂，远未完全破译。因此，它是必不可少的，以确定是否有替代髓母细胞瘤肿瘤细胞的起源细胞类型，具体的治疗方法可以开发基于。为此，颅内原位基因标记的肿瘤细胞类型肿瘤的发展中，收件人的后续分析allografting将使测定肿瘤细胞的细胞来源。在这里，我们描述了颅内原位allografting髓母细胞瘤细胞从原发肿瘤组织的实验协议，这个过程也可以用于移植建立细胞株的细胞。

Watch this Scientific Journal Video about Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice at JoVE.com

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

这个协议描述鼠标髓母细胞瘤组织的隔离和分裂，并随后allografting到免疫功能低下的受体小鼠的肿瘤细胞，以开展二次髓母细胞瘤。

在免疫功能低下小鼠颅内髓母细胞瘤细胞原位Allografting

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

intracranial-orthotopic-allografting-medulloblastoma-cells

Research

JoVE Journal

Neuroscience

12.1K 次观看.  Vanderbilt University. 这个协议描述鼠标髓母细胞瘤组织的隔离和分裂，并随后allografting到免疫功能低下的受体小鼠的肿瘤细胞，以开展二次髓母细胞瘤。

视频: Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

髓母细胞瘤干细胞的分离，浓缩，和维修

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

科研

神经科学

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

颅内注射腺相关病毒载体

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

颅内药物治疗和啮齿动物疼痛检测

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

小鼠体内合成蛋白的定量测量

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular pressure (IOP) which&#160;can identify various pathogenic paradigms related to the glaucoma pathogenesis. Ex vivo human anterior segment perfusion organ culture models have previously been successfully utilized and applied as effective technologies for the discovery of glaucoma pathogenesis and testing of therapeutics. Preclinical drug screening and research performed on ex vivo human organ systems can be more translatable to clinical research. This article describes in detail the generation and operation of a novel ex vivo human translaminar pressure model called the translaminar autonomous system (TAS). The TAS model can independently regulate ICP and IOP using human donor posterior segments. The model allows for studying pathogenesis in a preclinical manner. It can reduce the use of living animals in ophthalmic research. In contrast to in vitro experimental models, optic nerve head (ONH) tissue structure, complexity, and integrity can also be maintained within the ex vivo TAS model.

We describe and detail the use of the translaminar autonomous system. This system utilizes the human posterior segment to independently regulate the pressure inside the segment (intraocular) and surrounding the optic nerve (intracranial) to generate a translaminar pressure gradient that mimics features of glaucomatous optic neuropathy.

用于调节人类供体后段眼内压和颅内压的跨层自主系统模型

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

研究钝力损伤对成年斑马鱼影响的可扩展模型

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...