该出版物中报告的研究得到国家牙科和颅面研究研究所 (NIDCR) 和国立卫生研究院全国癌症研究所 (R56 DE025098、UG3 DE027695 和 F30 CA206296) 的支持。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。这项工作也得到了 NSF DMR 1206219 和 IADR 口腔护理奖 (2016) 创新的支持。
我们要感谢 Eri 圆山博士和安德鲁 Hollomon 为他们提供的唾液收集帮助。我们要感谢培伦翁协助进行腺体解剖。我们要感谢马修·格尔斯在图准备方面的协助。我们要感谢伊莲 Smolock 博士和艾米莉. 吴对这篇手稿的批判性阅读。

下文概述的所有体内程序均经罗切斯特罗切斯特大学动物资源大学委员会批准。纽约。
1. 准备
<ol><li>使用分析天平, 重20毫克的匹。将其溶于2毫升的无菌盐水中, 离心管。 注意: 由于匹属轻敏感, 并且随着时间的推移而失去活动, 这个解决方案应该在注射之日准备好, 并在被管理之前保护光线。</li>
	<li>使用分析平衡, 权衡和识别所有收集管和玻璃毛细血管。收集管和玻璃毛细血管的数量应与小鼠数量相匹配。</li>
</ol>2. 气管切开术
<ol><li>用分析天平称 C57/BL6 小鼠。</li>
	<li>使用0.5 毫升注射器与 29G x ½ "针, 麻醉小鼠与腹腔注射无菌盐水溶液100毫克/千克氯胺酮和10毫克/千克甲苯噻嗪。当鼠标不再响应刺激 (即没有踏板退出反射) 时, 继续执行以下步骤, 通常在注射后5到10分钟内发生。</li>
	<li>在用棉签涂抹润滑剂后, 轻轻地涂抹在眼睛上, 把鼠标放在舞台上的仰卧位置。麻醉动物必须保持在温暖的表面上。 注意: 所有步骤都在室温下执行。</li>
	<li>用手术胶带把鼻子、四肢和尾巴固定在舞台上。</li>
	<li>用酒精擦干净和湿脖子。 注意: 这将有助于防止毛皮进入随后的切口。</li>
	<li>用镊子提高腹中线颈部皮肤, 用解剖剪刀做一个小而浅的切口。引导剪刀进入开口, 慢慢地打开皮下的刀片, 分开组织平面。</li>
	<li>保持皮肤抬起, 小心地切到1厘米以下的嘴。</li>
	<li>在第一切口的下等和上级方面做两个侧向切口。使用镊子, 轻轻地反射离开皮肤, 使进入头部和颈部结构。</li>
	<li>使用解剖显微镜在8X 放大, 可视化颌下腺 (中线:图 1)。</li>
	<li>使用细钳, 轻轻地抬起腺体, 以暴露四舌骨下 (带) 肌肉在气管上方。避免撕裂或扰乱周围的血管或排泄管道。</li>
	<li>如果从多个鼠标收集唾液, 请在继续之前对每只鼠标重复步骤 2.1-2.10。 注: 本程序可同时安全执行多达5只鼠标。</li>
	<li>用小解剖剪刀, 移除带肌内侧部分, 同时尽量靠近中线。在手术过程中, 尽量减少气管的可视性, 保持带肌。避免偷任何附近的船只, 因为如果有严重出血继发量耗尽, 在诱导分泌物时, 匹比不会有效。</li>
	<li>从气管中反射出带肌。</li>
	<li>可视化喉、气管和甲状腺。确保气管的上部组织不明显。</li>
	<li>用小解剖剪刀在气管的下/后向甲状腺进行水平切口。确保气道是专利的, 并清除液体。</li>
</ol>3. 唾液收集
<ol><li>将胶带移近口部和角部解剖阶段向下 (45°, cranially), 以协助唾液流动。</li>
	<li>使用0.5 毫升注射器与 29G x ½ "针, 执行腹腔注射10µL/克体重的匹凡总剂量100毫克/千克。</li>
	<li>开始计时器。如果多剂量的老鼠, 通过快速移动和由助手同时注射其余的老鼠缩短提前时间。</li>
	<li>使用标准钳, 打开口管进入毛细管。</li>
	<li>一旦唾液中的一颗珠子被观察到, 将毛细管的近端放到液体中, 远端端放到位于解剖阶段以下的收集管 (两管预称重)。 注: 在 C57/BL6 雌性小鼠 6-10 周的年龄, 总流涎发生在2分钟的匹比注射。</li>
	<li>如果唾液是在口中生成的, 而不是流过管子, 重试上一步。确保管不直接放置在粘膜表面, 这会阻碍入口。</li>
	<li>收集唾液, 总共12分钟后, 匹凡刺激。确保气孔保持清晰。 注: 在这段时间内, 增加的潮汐量和分泌物 (唾液、泌尿、粪便) 是正常的。</li>
	<li>如果老鼠有压抑的唾液功能 (g., 由于伤害或干预), 使用 P200 微将剩余的液体从口腔转移到收集管。如果从多个鼠标收集, 请确保提示被更改。</li>
	<li>记录12分钟后收集唾液加管的重量。</li>
</ol>4. 腺体解剖
<ol><li>弄死小鼠由 CO2安乐死 (如 AVMA 指南为动物安乐死: 2013 编辑) 然后切开, 注意保存头和脖子的结构。因此, 避免颈椎脱位是安乐死的一种方法。</li>
	<li>将鼠标放在舞台上的仰卧位置。将原始站点中的腺体重新定位到气管 (图 1)。</li>
	<li>使用解剖显微镜在8X 放大, 可视化主要腺体: PG, SMG 和 SLG。为了区分解剖过程中的腺体, 确定 PG 是弥漫的, 躺在与 SMG 和 SLG 侧。 注: 如果解剖 PG, 这应该首先做, 因为它是最不明确的, 最有可能被撕裂。</li>
	<li>用镊子抓住 pg 的尾部, 将其从底层结构中拉出, 在用解剖剪刀切开 pg 头前轻轻地施加张力。</li>
	<li>用镊子将 SMGs 周围的胶囊破坏。轻轻地分开左, 右的 SMG。</li>
	<li>用镊子从粘附的 nonglandular 组织中释放 SMG 的背侧。</li>
	<li>用镊子把沃顿商学院的导管拉紧, 用解剖剪刀切开导管, 从颈部取出 SMG。</li>
	<li>用镊子轻轻地将 SLG 与 SMG 分开。 注: SLG 在 SMG 的 superolateral 方面。</li>
	<li>使用分析天平记录 SMG 重量。</li>
</ol>

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作者没有什么可透露的。

本文提出一种评价涎腺功能的多步法方法, 可用于研究腺损伤和治疗。我们的程序包括气管切开, 唾液收集和腺体解剖, 每个实验应用都能支持涎腺生物学的综合研究。例如, 小鼠气管切开术在阻塞口腔的过程中被用于一般的气道管理。
正确的解剖和气管切开是需要的, 以刺激唾液分泌100毫克/千克。另一种方法是, 减少的匹比, 可以避免对气管切开的需要, 因此用于纵向评估唾液...

涎腺疾病包括失调或分泌障碍的症状, 导致生产过剩 (sialorrhea) 或减产 (口干症和 hyposalivation) 的唾液1。在这两种情况下, 都有兴趣提高我们对涎腺生物学的认识, 迈向治疗发展的最终目标2。
涎腺是高度敏感器官, 并且经常被损坏在头颈癌放疗期间, 导致永久干嘴 (口干症)3, 4.与其他敏感组织不同, 涎腺的更替率相对较低, 分泌性损失的机制不太了解5,6。在这种独特的损伤设置, 组织再生和防护策略需要唾液功能评估。实验中, 小鼠唾液收集是评估辐射和治疗药物的腺体反应的一个特别有价值的工具。
在这里, 我们提出了一个方法, 以执行和量化刺激唾液分泌使用的匹毒蕈, 一个强有力的7激动剂。匹凡刺激自主神经系统诱发腺体分泌8,9。为了适当地完成这项试验, 需要进行气管切开, 以确保老鼠在整个过程中保持一个专利气道, 并减少口腔内的分泌物窒息和吸入的风险10。
这是一个终端程序, 最终清除三大涎腺: 腮腺 (PG), 颌下腺 (SMG) 和舌下 (SLG)。在功能性研究中, 腺体重量被记录下来, 通常用来规范化唾液测量11,12,13。这些数据在辐射研究中尤为重要, 其中腺体萎缩是预期结果14,15
在文献中有可变性的关于如何刺激唾液分泌被执行和报告的16。例如, 在文学范围内的匹比剂量至少三级17,18,19,20,21,22,23。在这里, 我们提出了一个优化的高剂量匹凡协议, 目的是提高方法执行的重现性, 以及提供一个模块化的技术平台 (气管切开, 唾液收集和腺体解剖), 可以适应需要。
除了协议演示外, 我们还包括有代表性的2周内唾液流动的功能数据 (4 剂量 6.85) 到 SMG 地区。

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			<td height="21" style="height:21px;width:219px;">Pilocarpine hydrochloride</td>
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			<td style="width:119px;">P6503</td>
			<td style="width:225px;">Pilocarpine</td>
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			<td>Spring Scissors for Tracheostomy</td>
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			<td height="21" style="height:21px;width:219px;">Lubricant Eye Ointment</td>
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			<td>Refresh Lacri-Lube</td>
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			<td height="42" style="height:42px;width:219px;">Goat polyclonal anti-Nkcc1</td>
			<td style="width:128px;">Santa Cruz Biotech</td>
			<td style="width:119px;">SC-21545</td>
			<td>Nkcc1 Antibody</td>
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			<td height="42" style="height:42px;width:219px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>DAPI</td>
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			<td height="21" style="height:21px;width:219px;">GraphPad Prism</td>
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			<td>Applicator for eye ointment</td>
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			<td style="width:128px;">BD</td>
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			<td>Syringe for intraperitoneal injection</td>
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murine salivary functional assessment via pilocarpine stimulation following fractionated radiation

Hyposalivation 通常观察到 Sjögren 综合征的自身免疫反应或继放射损伤的主要涎腺。在这些情况下, 有关疾病发病机制和有效干预的问题仍然存在。一种优化的技术, 允许对涎腺的功能性评估是非常宝贵的研究外分泌腺生物学, 功能障碍和治疗。在这里, 我们提出一步一步的方法来执行的匹凡刺激唾液分泌, 包括气管切开和解剖三大鼠涎腺。我们还详细介绍了适当的小鼠头部和颈部解剖学在这些技术的访问。这种方法是可伸缩的, 允许多个鼠标同时处理, 从而提高了工作流的效率。我们的目的是提高这些方法的重现性, 每一个都有进一步的应用领域内。除了唾液收集, 我们还讨论了量化和规范化这些组织的功能能力的指标。有代表性的数据包括从颌下腺功能下降2周后, 分光辐射 (4 剂量 6.85)。

当执行高剂量的匹比刺激唾液收集, 重要的是保持呼吸道, 以防止吸入或窒息的分泌物在口腔。提供了气管切开的示意图 (图 1)。气管切开后, 气孔必须保持清晰的组织和液体。
为了加强唾液收集过程中的毛细血管作用, 应将小鼠的头部置于45°角度向下定位。这些步骤可以在 > 1 鼠标上执行, 但不建议一次?...

Watch this Scientific Journal Video about Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation at JoVE.com

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

小鼠涎腺照射后通过匹属刺激进行唾液功能评估

我们提出了一个详细的方法来执行唾液收集, 包括小鼠气管切开和隔离三大涎腺。

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

murine-salivary-functional-assessment-via-pilocarpine-stimulation

Research

JoVE Journal

Medicine

11.0K 次观看.  University of Rochester. 我们提出了一个详细的方法来执行唾液收集, 包括小鼠气管切开和隔离三大涎腺。

视频: Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

沃顿商学院的导管插管通过小鼠颌下腺唾液腺

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

科研

医学

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

一个小鼠模型，通过神经肌肉电刺激肌肉训练

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

心肌梗死和功能预后评估的猪

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

控制帕金森氏症与自适应深部脑刺激

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

弥漫性光学光谱治疗急性电离辐射引起的皮肤毒性的定量评价使用小鼠模型

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Retroductal颌下腺灌输和本地化分割照射在涎腺功能减退症大鼠模型

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

高分辨率光学制图对小鼠心房电生理的评价

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

小鼠致耐受性树突状细胞的发育及功能表征

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

下颌骨和腮腺表型模型对组织型培养的放射治疗是机体活力特征的关键

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...