We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1。组织收获和肌肉组织的冷冻<ol><li>安乐死的小鼠用异氟烷（2-氯-2-（二氟甲氧基）-1,1,1-三氟乙烷）的过量。执行此步骤在通风橱中并利用一第二容器的方法，如干燥器或钟罩含有棉签浸泡在异氟醚。 </li><li>用力挤压脚垫确认死亡。使用安乐死的辅助方法，如颈椎脱位。 </li><li>湿用70％酒精的皮毛，以减少对肌肉松弛头发缕粘着。用细镊子，如＃5剥离皮肤脱落的肢体。 </li><li>小心地从骨骼中分离的兴趣（）在这种情况下，胫骨前（TA）的肌肉的下面，从肌腱开始并移除。 </li><li>切除的肌肉，它覆盖在华侨城（最佳切削温度化合物），并把它平放在一个标有一次性冻结模具。确保肌肉在其正常的生理方向（头至大l）在不拉伸。可替代地，冻结小发泡胶正方形肌肉和利用昆虫针以确保肌肉的正确长度针它。 </li><li>寒意2-甲基丁烷（异戊烷）中采用液氮钢制烧杯中。这平均需要3 - 5分钟。不断搅拌间歇直至白色沉淀开始形成在烧杯的底部和壁上。 注意:当这种情况发生时，异戊烷已达到最佳冷冻温度（-150℃） </li><li>浸含在冷冻2-甲基丁烷肌肉的模具。冷冻小肌肉，如隔膜和趾长伸肌（EDL）6 - 12秒，较大的肌肉如腓肠肌比目鱼（GS）和肱三头肌15 - 20秒。不要冷冻肌肉太久，因为这可能会导致开裂。 </li><li>转让的冷冻组织干冰，让异戊烷蒸发（注意，此过程平均需要大约15 - 20分钟）。包裹组织中铝箔并将其存储在üLTRA低温（-70℃〜-80℃），直到切片。 </li></ol> 2.嵌入组织在OCT准备组织块<ol><li>干冰运输组织，以cyrostat防止解冻。转移组织到低温恒温室（设定为-20至-24℃，以避免组织的充分解冻），并让他们平衡30分钟。 </li><li>打开珀耳帖冷却海湾上。如果低温恒温器不具备这种冷却，使用喷雾冷却喷雾快速冻结十月不要让肌肉解冻的任何一点的嵌入，因为这可能会导致冻害期间。 </li><li>添加均匀的薄层华侨城在试样上盘，让它冷却。当大多数的OCT被冻结在底部，同时仍然液体在顶部，插入在正确的方向的组织（垂直华侨城为横截面和平行华侨城为纵向部分）。使用喷雾冷却喷雾迅速冷冻十月触摸组织unembeded部分热转ractor并等待45秒。 </li><li>添加另一个薄层OCT的周围的肌肉，喷洒气溶胶冷却喷雾和提取的热量作为在上一步。 </li><li>在保持小幅度增加OCT和快速使用气溶胶喷雾冷却和加热萃取的结合冻结华侨城，直到整个肌肉被覆盖。 </li><li>放的热量提取的组织块的顶部，等待5分钟。 </li></ol> 3.准备部分和肌肉半山区肚识别章节<ol><li>安装试样到样品头。拧紧旋钮固定盘。确保在试样圆盘与所述样品头良好的接触。 </li><li>调节室内温度°之间-21和-24℃，等到设定温度达到。 </li><li>其厚度调节设置（7微米）;修剪通过推进或使用粗，细设置缩回块的块。 </li><li>收集部分通过按滑动的顶部上的部分中，使用相反电荷之间的吸引力，以方便切割部分以滑动的粘附预热带正电的显微镜载玻片。 </li><li>通过测定各部分的横截面面积（CSA）识别中期腹部分。连接到显微镜的计算机上执行此操作使用图像处理软件。 注:相衬图像的拍摄和整个肌肉的圆周经由上的软件程序跟踪功能测定。这将产生两个CSA以及最小鼬直径（测量纤维横截面尺寸的被表示为对一个粒子相反两侧的两个平行切线之间的最小距离）的测量。注意:当CSA和鼬最小直径测量开始高原，肚皮中旬已经达到。 </li><li>库存和归档的幻灯片在-80℃的未来染色。 注意:OCT不需要之前被除去以stainin摹程序​​。一旦载玻片盖上盖玻片，他们准备立即染色。 </li></ol> 4.染色注:虽然详细的一步一步的程序，可以在产品数据表中找到，并应遵循染色程序，个人优化可能需要以获得最佳的染色。 <ol><li>因为安装介质（见表）是不与水混溶的，通过分级醇增加脱水载玻片（每次2分钟，70％，95％和100％的乙醇），并清除它们用二甲苯（100％，5分钟），以确保幻灯片不会成为不透明随着时间的推移。 </li><li>盖玻片部分，并让他们干。图片显微镜配备图像处理软件。 </li></ol>

<ol>
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A., Kostek, C. A., Miller, 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=Inhibition+of+apoptosis+improves+outcome+in+a+model+of+congenital+muscular+dystrophy.">Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.</a> The Journal of clinical investigation. 114, 1635-1639 (2004).</li><li>Kumar, A., Yamauchi, J., Girgenrath, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle-specific+expression+of+insulin-like+growth+factor+1+improves+outcome+in+Lama2Dy-w+mice,+a+model+for+congenital+muscular+dystrophy+type+1A.">Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.</a> Human molecular genetics. 20, 2333-2343 (2011).</li><li>Mehuron, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+matricellular+proteins+is+an+early+signature+of+pathology+in+laminin-deficient+muscular+dystrophy.">Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.</a> Skeletal muscle. 4, 14 (2014).</li><li>Yamauchi, J., Kumar, A., Duarte, L., Mehuron, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggering+regeneration+and+tackling+apoptosis:+a+combinatorial+approach+to+treating+congenital+muscular+dystrophy+type+1+A.">Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.</a> Human molecular genetics. 22, 4306-4317 (2013).</li><li>Sheriffs, I. 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The authors have nothing to disclose.

组织学和免疫组化已被用作主要的工具在理解表现和病理的进展在各种神经肌肉疾病。经典地，肌肉活检第一固定在甲醛，然后用石蜡包埋蜡。虽然甲醛固定保持组织结构，并允许组织存储和运输，在RT，这也灭活酶和蛋白质交联迫使进一步措施，以实现精确的免疫染色。这个问题与使用冰冻切片消除，但这种方法提出了获得最佳的染色效果和准确的诠释14,15独特的技术挑战。 <p class="j...

检查肌肉组织学中起着不同类型神经肌肉疾病1-4中的认识至关重要的作用。当与其他技术相结合，它使研究人员能够更好地了解病理的表现和进展的，也可以是必需评估治疗干预5-10的功效。然而，要准确，组织需要以适当的方式被保留以便切除之前立即保持生理状态。这种去除，保存，切片，和染色过程中需要非常小心。 组织可以以两种不同的方式进行光学显微镜研究来制备。第一种方法，福尔马林固定的石蜡包埋（FFPE）的部分，需要组织到被嵌入石蜡11,12之前被固定在10％的天然缓冲甲醛。固定步骤，有助于更好地保存形态，而且交联内源性PRoteins因而需要复杂的抗原修复程序用于免疫染色和消除酶染色对这些部分12的可能性。冰冻切片，另一方面，不需要进行预固定或处理;因此，蛋白质能保留原生生物构13,14。此外，冷冻切片也允许快速的周转时间，这有时是必要的，例如在肉瘤和其他外科活组织检查15的术中诊断。但是，如果没有正确完成，该方法是容易冻结损坏，它引入了改变肌肉结构使得部分不适宜用于组织学检查。本文将概述准备肌肉活检，以达到正确检查最佳染色最有效的方法。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica CM 1850&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td>Buffalo Grove, IL 60089 United States, office phone 1-800 248 0123 
			Richard Allen Scientific</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse 50i&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Olympus</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Nikon Imaging Software Basic Research</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Image J, Metamorph</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>14043-220-05</td>
			<td>JD Medical Dist. Co., Inc.</td>
			<td>JD Medical Distrubuting Co., Inc 1923 West Peoria Avenue Phoenix, Arizona 85029</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>4583</td>
			<td>Sakura Inc.</td>
			<td>Sakrua Fintek, USA, Torrence, CA 90501, USA 
			Leica Microsystem</td>
		</tr>
		<tr>
			<td>Freeze It</td>
			<td>23-022524</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Disposable tissue mold</td>
			<td>22-363-554</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Colorfrost plus slides</td>
			<td>9991001</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>650501-4L</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>HC-1300-1GL</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>M 32631-SL</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>HC-7001GA</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Cytoseal 280</td>
			<td>8311-4</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Hematoxylin Gill #3</td>
			<td>CS402-1D</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>6766007</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
	</tbody>
</table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

肌肉活检组织学评价曾担任在神经肌肉疾病的病理发展过程的理解不可或缺的工具。然而，为了这样做，适当的照料需要切除并保存组织，以达到最佳的染色时，必须考虑。组织保存的一种方法包括固定在甲醛组织，然后用石蜡包埋它们。此方法保留形态良好，并且允许长期储存于RT但是麻烦的，并且需要处理的有毒化学品。此外，甲醛固定导致抗原的交联，这需要抗原修复协议有效免疫染色。与此相反，冷冻切片不需要固定，如此保留生物抗原的构象。此方法还提供了一个明显的优势在周围时间快转，使得它在情况需要像intraopera快速组织学评估特别有用略去手术活检。这里，我们描述制备肌肉活检用于可视化具有不同的组织学和免疫染色的最有效的方法。

该组向上收获和组织冷冻可以看出，在图1中 。切下的组织应存储在浸泡在PBS中，以避免在干燥（ 图1A）纱布。当组织被选择以用于组织学分析，他们应该被浸入OCT和布置在低温模具的正确方向，并尽可能接近生理长度尽可能以确保准确的形态（ 图1B）。异戊烷适于组织冷冻的浆液可以通过冷却的异戊烷在液氮中，并搅拌，直到小白色沉淀出现在底部来实现?...

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do&#039;s and Don&#039;ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

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JoVE Journal

Medicine

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

31.6K 次观看.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

视频: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

科研

医学

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

主动脉钙化和炎症血管平滑肌细胞和成像的钙化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

解剖的小鼠胰腺的组织学分析与代谢谱

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

超声心动图与小鼠心脏形态学的组织学检查

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

大鼠舌的机械和粘弹性的体内评估

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

代谢组学和利皮多米分析脑肿瘤的现场采样与提取

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

心血管疾病大型动物模型中心脏的解剖技术和组织学取样

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

长期培养用人心肌组织的制备

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...