这项工作是由美国国立卫生研究院的研究经费R01AR062207，R01AR061484，R56AI100901，K01AR053210，并且针对的疾病研究资助由风湿病研究基金会（以CJ刘）部分支持。

所有与动物的外科手术应该由本地机构的动物护理和伦理委员会，以尽可能避免疼痛而造成手术的不适努力的批准。 1。准备<ol><li>选择8-12周龄雄性C57BL / 6小鼠体重手术约25g。 </li><li>通过腹膜内注射含有甲苯噻嗪（5毫克/千克）和氯胺酮（40毫克/千克）鸡尾酒的麻醉动物。 </li><li>剃须膝盖用剃刀，然后手术悬垂的动物和消毒手术部位用优碘和酒精（3次）并覆盖鼠标眼睛软膏。 </li></ol> 2。手术过程<ol><li>做1厘米的纵向段内侧髌切口，显露膝关节。 </li><li>通过髌骨和髌韧带外侧脱位轻轻打开膝关节。 <ol><li>切开膝关节囊纵向通过段内侧髌切口在步骤2.1。 </li><li>解剖膝关节囊用钳子。 </li><li>抓住后爪与左手的远端部分，并执行髌骨和髌韧带与钳的横向错位。按住后爪轻轻的，并确保避免在爪子创伤。关节囊被解剖越好，越少力将被要求作出的错位。 </li></ol></li><li>操作过程中滴落的关节软骨的表面上的无菌盐水中，以避免干燥出软骨表面。 </li><li>切入内侧meniscotibial韧带其中锚内侧半月板的胫骨平台。避免伤及内侧半月板下方的软骨。 <ol><li>确定内侧半月板股骨内侧髁和内侧胫骨平台之间的定位。 </li><li>确定meniscotibial韧带是连接内侧半月板外侧与胫骨髁间嵴。 </li><li>抱喜ND爪子轻轻的手，划破内侧meniscotibial韧带仔细用10号手术刀片。确保不造成损伤关节软骨和韧带等。在许多情况下，覆盖脂肪垫需要被拉到一边而不是为了识别韧带被删除。 </li></ol></li><li>关闭膝关节囊用6-0可吸收缝线。 </li><li>关闭用6-0丝线缝合皮肤。 </li></ol> 3。术后护理<ol><li>申请一滴0.25％布比卡因在每只小鼠以减少手术后疼痛的外科手术部位。 </li><li>离开手术小鼠自由地去水和食物。 </li></ol> 4。手术DMM模型组织学评分<ol><li>牺牲小鼠在指定的时间点（ 例如，4周，8周，12周，在这里，我们表明8周的结果作为代表），在手术后。 </li><li>整个膝关节固定，脱钙，EMBE石蜡dded，然后在5微米的间隔连续切片。 <ol><li>全切后肢与第10号刀片。 </li><li>解剖后肢的皮肤和肌肉仔细，固定的试样，用4％PFA中于室温3天。 </li><li>取出PFA和清洁水样品。此后，脱钙在EDTA中的样品在4℃下2周。 </li><li>脱水的样品中的乙醇梯度。详细地说，保持样品在70％乙醇中1小时，然后改变它的乙醇，并保持在样品中一组新的70％乙醇的O / N。之后，把样品在80％和90％乙醇因此，并保持1小时后，分别后，再用100％乙醇中的O / N。 </li><li>除去乙醇。保持样品中邻二甲苯1小时，并切换到一组新的邻二甲苯。重复此步骤3次。 </li><li>嵌入样品到使用徕卡包埋中心石蜡模具。之后，将样品用旋转切片机（Leica RM225切片在5微米5，德国），然后收集在玻璃载玻片上。 </li><li>串行矢状切片切成每个样品从外侧髁的内髁的中心的中心跨越的区域。 </li></ol></li><li>番红O染色进行，然后通过OARSI评分系统计分和统计分析，如先前25所述。 <ol><li>首先，deparaffinize的幻灯片中邻二甲苯，和滋润他们在乙醇梯度蒸馏水。此后，幻灯片通过韦格特的铁苏木素液，0.05％快速绿色（FCF）溶液，1％醋酸溶液和0.1％番红O解决方案染色。使用树脂介质安装的幻灯片。 </li><li>评分基于蛋白多糖（红色）的软骨和破坏的软骨结构比例的损失番红Ø染色切片，做统计分析的组织学评分。 </li></ol></li></ol>

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我们在此声明，我们没有利益冲突。

据报道，小鼠的品系是非常重要的DMM模型诱导，小鼠作为不同菌株有不同的OA严重性DMM后，用在129/SvEv应变水平最高，其次是C57BL6，129/SvInJ然后FVB / Ñ26。转基因的一大部分是建立在C57BL6小鼠，如PGRN-/ - 小鼠，我们在本研究中使用，这是相对容易的DMM。然而，如果转基因是基于不敏感的菌株如FVB / N小鼠，它是必要的回交这些小鼠与DMM诱导前敏感菌株。用于数字万用表诱导小鼠的性别也是非?...

骨关节炎（OA），也称为退行性关节炎，影响到世界人口的15％，超过46万人在美国，并且其特征在于滑膜炎，软骨变性和骨赘形成1。它可以是遗传，代谢，生物力学和生化因素之间复杂的相互作用的结果。 OA的基本机制继续回避科学界。目前有许多动物模型中，可以模仿OA 2,3的发病机制。它建立各种转基因小鼠的两个可用性和实验的成本效益，因为动物模型小鼠中是很重要的。其中各种不同的实验OA模型中，手术引起的内侧半月板（DMM）模型的不稳定是由于其良好的重现性和OA的发展过程中一个相对进展较慢一个广为接受的OA模型。这两个属性有是关键的不同处理或转基因3-8 OA进展的评估。然而，外科手术的OA模型的一致性在外科手术，其结果是受多种因素影响，手术小鼠模型中的应用是有限的。 的progranulin（PGRN）是表示在各种细胞多功能生长因子。它是已知的PGRN起着各种生理和疾病过程，如伤口愈合9，肿瘤10和炎症11-15的关键作用。研究还表明，PGRN的不足可引起神经系统的退化性疾病在人类和小鼠16-18。它是已知的PGRN表达在人关节软骨，其水平是在患者的骨关节炎和类风湿关节炎的19软骨显著升高。此外，PGRN也起着软骨细胞增殖20，分化和软骨内至关重要的作用生长板的发育21,22中骨化。最近，我们报道了PGRN通过结合TNF受体拮抗TNF-α和表现出抗炎作用在炎性关节炎模型13,14,23,24。然而，PGRN的在OA中的作用，特别是在体内 ，仍然是一个谜。在此，我们提出的方法诱导手术DMM模型，并通过在WT和PGRN-/建立数字万用表模型研究PGRN的在OA发展中的作用 - 小鼠。

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			<td height="21" style="height:21px;width:216px;">No. 10 Surgical blades</td>
			<td style="width:135px;">FEATHER</td>
			<td style="width:119px;">25-2976#10</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;">6-0 suture</td>
			<td>APPLIED DENTAL</td>
			<td>WG-N53133</td>
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establishment of a surgically-induced model in mice to investigate the protective role of progranulin in osteoarthritis

内侧半月板不稳定（DMM）模型是研究在骨关节炎（OA） 在体内的致病众多的关节炎相关分子的病理生理作用的一个重要工具。然而，详细的，尤其是可视化协议，用于在小鼠体内建立这种复杂的模型，无法使用。在此我们利用野生型和progranulin的（PGRN）的 - / - 小鼠为例子来介绍一种协议，用于诱导DMM模型小鼠，与OA相比下成立了这个手术引起的模型的发作。在老鼠身上进行的操作要么假手术，这只是开了关节囊，或数字万用表操作，这将削减MENISCO，胫骨韧带，造成内侧半月板不稳定。用组织学检测（ 如番红O染色），办公自动化相关基因的表达，降解软骨细胞外基质分子和骨赘骨关节炎形式严重性进行评估通报BULLETIN。 DMM操作成功诱导的OA发生和发展中都野生型和PGRN-/ - 小鼠，并导致了在此手术诱导模型更严重的表型OA PGNR生长因子损失。

成功建立了小鼠DMM模型，PGRN的缺陷夸大手术引起的OA发展。 老鼠-深水和数字万用表操作（ 图1）WT和PGRN-/执行了。操作8周后，处死小鼠，并番红O染色对从膝关节的部分来进行，随后关节炎得分基于组织学的统计分析。 如图2A所示 ，有软骨的基于番红O染色，这暗示了类似的基线情况（上图），没有明显的退化在两个基因型在假手术组。 ...

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Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

我们描述了一个协议内侧半月板（DMM）模型小鼠中，一种有效的工具，骨关节炎（OA）研究的不稳定。此外，我们已经证明progranulincan夸大OA的发生和发展利用该模型的不足，表明progranulin的发挥在OA的发病机制中起保护作用。

建立了外科手术引起的小鼠模型，以调查的progranulin在骨性关节炎的保护作用

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

establishment-surgically-induced-model-mice-to-investigate-protective

Research

JoVE Journal

Bioengineering

27.1K 次观看.  NYU Hospital for Joint Diseases. 我们描述了一个协议内侧半月板（DMM）模型小鼠中，一种有效的工具，骨关节炎（OA）研究的不稳定。此外，我们已经证明progranulincan夸大OA的发生和发展利用该模型的不足，表明progranulin的发挥在OA的发病机制中起保护作用。 

视频: Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

建立一种结构逼真的心肌细胞的有限元几何模型研究蜂窝结构在心肌系统生物学中的作用

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

一种三肽稳定的油酸纳米乳液

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...