笔者要感谢保罗·维悌希博士和吉纳维芙方女士的mRNA的提取和PCR援助，以及加内特凋谢和罗德尼·威廉斯纪念基金会的支持。

伦理学声明:下面列出的所有程序批准了悉尼动物伦理委员会的大学。 1.动物注:1和8 - 9个月大的小鼠（C57 / BL6）是从动物资源中心（珀斯，澳大利亚）中获得。老鼠被安置在博世灭鼠设施，在悉尼大学。 <ol><li>家鼠在上一个12/12小时亮/暗循环以获得食物和水随意标准鼠标笼中。 </li><li>鸿沟小鼠各年龄组为近红外（NIR）治疗，或假处理（对照）组进行比较。 </li></ol> 2.近红外（NIR）照射及深水治疗<ol><li>剃鼠标的头部和颈部区域的毛皮用电动剃刀尽可能防止头发快速再生完成治疗方案的前。为了保持小鼠的无病原体状态，使用70％的乙醇剃须动物，并从不同的笼子里的动物之间的F10兽用消毒剂清洗之间的乐器。这样做2 - 3天前开始治疗等动物不是过度处理。 </li><li>抑制小鼠握住尾部的近端，并允许它在用一只手或台式手掌放松，以便尽量减少应力，这可能会导致错误的结果。 </li><li>持有NIR（670 nm）的LED（发光二极管）器件1 - 2厘米的距离暴露（光头）区和开关设备上的90秒（ 图1A）。 注意:温度变化的90秒曝光结果测定为&lt;0.2℃下在100毫升的水中。 </li><li>对于假治疗组动物的2.3，但离开该装置关闭（ 图1B） -重复步骤2.2。 </li><li>重复步骤2.2 - 2.3对NIR阻断治疗组的动物，但覆盖器件有铝箔。 </li><li>每天approximat 2.5 - 重复步骤2.2E 24小时的间隔，连续5天。 </li><li>提取两个耳朵上的第五天后处理前庭感觉上皮（3嵴和1个椭圆囊斑）（见下文第3节 ）。 </li></ol> 3.组织提取20 <ol><li>制备300毫升基于甘油的人工脑脊液（ACSF）的组成（以mM计）:26的NaHCO 3，11葡萄糖，250甘油，2.5氯化钾，1.2的NaH 2 PO 4，1.2的MgCl 2，和2.5 氯化钙 。在加入氯化钙 ，燃气与卡波金（95％O 2和5％CO 2）的溶液来建立的pH为7.4，并避免钙沉淀（混浊）。寒意在-80℃冷冻该溶液45分钟，以使冰浆料形成。 </li><li>准备在标螺杆顶端微管的RNA分离裂解缓冲液（停止盖子的爆裂时冻结和解冻在液氮之间管压力的变化），根据制造商或通常的做法实验室的指示。有液氮准备在铝杜瓦瓶中。 注:液氮操作时，使用的防护服和eyeware。确保使用液氮在一个通风良好的房间作为其气体形式的体积大约是700倍，它的液体形式，并可能导致窒息。 </li><li>通过腹膜内注射深深地麻醉小鼠用氯胺酮（400毫克/千克）。允许后肢反射完全消退作为指示小鼠完全麻醉。 </li><li>杀头的小鼠用锋利的不锈钢剪刀，并沿着用刀片头骨的矢状皮肤切口（四舍五入＃22）。在这一点上和整个步骤3.5 - 3.9，保持颅，脑和潜在的前庭器官尽可能凉爽通过在组织常规应用冰冷学联的。 </li><li>使用标准模式剪刀尖手臂做出SMaL公司升切口头骨的Lambda和沿矢状缝切割。 </li><li>轻轻滑动的浅表骨钳一个臂顶骨保持刀片尽可能接近到骨头的下表面，而不拖动大脑下方。安全和扯远了顶骨横向和枕骨后部，直到大脑暴露。 </li><li>用一个不锈钢小铲子和解除大脑远离前部和中颅窝暴露前庭耳蜗神经（CN八）。横切神经以防止在该初级传入轴突直接支配前庭毛细胞不必要的紧张。 </li><li> CN八横断后取出大脑全盘 。 </li><li>观察含耳蜗和中颅窝周围前庭器官的骨迷路。让两个小切口，每个骨迷路旁边，握住前半规管和后拉切除整个结构盟友。 </li><li>立即浸入切除迷宫中含有冰冷ACSF溶液（如在步骤3.1中所述），同时连续地卡波金灌注解剖盘中。 </li><li>在立体显微镜下，按住迷宫由耳蜗并将其固定钳盘的底部。 <ol><li>直接使用细镊子划伤小开在上面的前半规管（SSC）壶腹骨。 </li><li>轻轻达到立即骨下方，向外弹离壶腹放大此开幕。谨慎行事这里没有推入钳开放和破坏脆弱的膜迷路下方。继续以这种方式，直到椭圆囊，前部和侧壶腹都暴露出来。如果可能的话删除后壶腹也。 </li></ol></li><li>用细镊子，轻轻抬起囊，并从骨迷路壶腹距离，直到他们完全不沾边。如果可能的话，他们持有由它们相关联的半规管，以避免损坏感觉上皮。在一些情况下，半圆形膜质导管的近端部分可能需要与虹膜剪切以从骨释放壶腹。 </li><li>牢固地抓住镊子的顶端和地点之间的前庭器官到先前在步骤3.2中制备的裂解缓冲液。轻轻绕摇动镊子在缓冲，以确保前庭器官已经从钳子脱离。检查，这是通过使钳子的立体显微镜下的情况。 <ol><li>拧上微管盖，并立即冷冻在液氮中的样品。 </li></ol></li></ol> 4. RNA提取和RT-PCR <ol><li>根据制造商的说明或优选的实验方案遵循信使RNA（mRNA）的提取的标准方法。 注:利用载体分子RNA，以帮助隔离mRNA的小产量商品化试剂盒在此使用协议。低洗脱体积可用于增加最终浓度的mRNA。 注:负"无酶"的控制（NEC的）和"无DNA模板"对照（NTC却）也必须被完成，以确保观察到的效果的有效性。 </li><li>适用的mRNA逆转录，以互补的DNA的靶基因的21-23（cDNA中）和扩增的标准方法。 </li></ol>

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作者宣称，他们没有竞争的财务权益。

这里所描述的代表性的结果表明，当与假治疗的小鼠相比较简短颅递送NIR光的（90秒/天5天）足以提高抗氧化剂的表达在老年小鼠的水平。而发射的热可以代表线粒体和/或神经元激活的一个来源，据报道为大鼠前庭传入神经​​24 -我们的热量由NIR发射测量LED器件是&lt;0.2℃，90秒，因此不太可能导致这里描述的变化。此外，与报告所强调以上，这里使用的NIR治疗不直接向感觉上皮或前庭传?...

余额递减性能和后续的瀑布是常见的，而自然老化1的不幸往往定义功能。这种下降的影响，既可以是自然和社会，和显著降低生活质量的老年人。作为回应，物理治疗和康复一直是研究的重点为瀑布，但尚未有一致的减少重复跌倒的发生率有关。与此同时，工作调查在周边或中央前庭系统（负责保持平衡的系统）是稀少，并且针对这些系统的潜在的治疗策略的变化和不平衡有限的根本原因。 在年龄相关的神经变性病症，包括年龄相关性黄斑变性2-4，阿耳茨海默氏病模型5-8，和帕金森氏病9-12最近的工作已经表明SI的神经保护作用mple非侵入性的应用的近红外（NIR）光。另外，在前庭系统，NIR已被用于提高在体外 13前庭初级传入神经 ​​元的活性。而没有很好理解NIR光的机构，利用NIR大多数的研究表明，NIR刺激线粒体复合物IV（细胞色素C氧化酶）14-17，以促进细胞代谢。在前庭感觉上皮I型毛细胞的表皮下板是致密的线粒体18，因此可以代表用于治疗NIR治疗作用的部位。 这里，经颅的简要的，非侵入性的治疗方案应用于NIR可用于测量细胞代谢（和暗示线粒体功能）在小鼠前庭感觉上皮进行说明。还讨论了为制备的前庭感觉上皮和它表明NIR增加一个ubiquito的表达我们的抗氧化剂（超氧化物歧化酶1）中的感觉上皮-先前证明是对耳蜗毛细胞存活19重要。

<table><tbody><tr><td>Quantum WARP 10</td><td>Quantum Devices</td><td>2070N030-A</td><td></td></tr><tr><td>Screw top microtubules</td><td>Quality Scientific Plastics</td><td>520-GRD-Q</td><td></td></tr><tr><td>Ketamine</td><td>Parnell, Alexandria Australia</td><td></td><td></td></tr><tr><td>Standard pattern scissors</td><td>FST</td><td>14001-12</td><td></td></tr><tr><td>Carbon steel surgical blades #22</td><td>Livingstone</td><td>SBLDCL 22</td><td></td></tr><tr><td>Friedman-Pearson rongeurs</td><td>FST</td><td>16221-14</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>A60S</td><td></td></tr><tr><td>Dumont #5 SF forceps</td><td>FST</td><td>11252-00</td><td></td></tr><tr><td>Isolate II RNA Micro Kit</td><td>Bioline</td><td>BIO-52075</td><td></td></tr></tbody></table>

near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

策略随着年龄的增加衰减下降平衡功能主要是专注于物理疗法，包括平衡工作和锻炼。然而，这些方法没有解决平衡下降的根本原因。使用小鼠，近红外光（NIR）的细胞上，在前庭感觉上皮的代谢的影响进行了评估。收集的数据表明，这种简单和安全的干预可能保护这些脆弱的细胞从自然老化的有害影响。的mRNA从分离的前庭外周感觉上皮（壶腹嵴和椭圆囊斑）萃取，随后转录成cDNA文库。这个库然后探查无处不抗氧化剂（SOD-1）的表达。抗氧化剂的基因表达，然后用于量化细胞代谢。用年轻的经颅交付NIR（4周）及以上（8 - 9个月）小鼠，并简要治疗方案（90秒/天5天AYS），这项工作表明NIR单独可足以改善线粒体功能的前庭感觉上皮。由于目前没有可用的，负担得起的，非侵入性的治疗方法，提高前庭毛细胞功能，外部NIR辐射的应用程序提供，以抵消对细胞在矿井前庭感觉上皮代谢老化的影响，潜在的战略。

比较近红外治疗的年轻年长的影响（4周）和（8 - 9个月）小鼠，我们测得的抗氧化剂超氧化物歧化酶1（SOD-1）的表达年轻的（N = 16）和老年人（N = 20）小鼠是NIR治疗，假治疗，或NIR封; 图2示出了相比于年轻假处理的动物一个显著增加β肌动蛋白标准化的SOD-1的表达超过2倍于年轻NIR处理的动物（P &lt;0.01），年轻的NIR阻断动物（P &lt;0.01）。较旧的NIR处理的动物也显示出超过一个时与旧NI...

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

线粒体功能障碍是细胞衰老的标志。本文使用的非侵入性的近红外（NIR）处理，以改善在老化小鼠前庭感觉上皮线粒体功能。

近红外（NIR）光增加线粒体功能的标志物的表达，在鼠标前庭上皮细胞

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

near-infrared-nir-light-increases-expression-marker-mitochondrial

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.9K 次观看.  University of Sydney. 线粒体功能障碍是细胞衰老的标志。本文使用的非侵入性的近红外（NIR）处理，以改善在老化小鼠前庭感觉上皮线粒体功能。 

视频: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

科研

神经科学

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

生物化学

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

医学

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

小鼠经颅光生物调节治疗方案

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

生物学

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

可筛选的<em&gt;在体内</em&gt;测定线粒体调节剂使用转基因生物发光<em&gt;秀丽隐杆线虫</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

时间推移视频显微镜对EYFP-Parkin的聚集性评估作为蜂窝Mitophagy一个标记

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

用于分析神经科学中线粒体网络的 MATLAB 优化

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

作者聚焦:研究神经元线粒体中视黄酸受体的优化自动化方法

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

通过亚型特异性视黄酸受体对活神经元线粒体稳态调节进行优化的自动分析

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...

近红外（NIR）光增加线粒体功能的标志物的表达，在鼠标前庭上皮细胞

摘要

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此视频中的章节

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

小鼠经颅光生物调节治疗方案

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法

可筛选的

时间推移视频显微镜对EYFP-Parkin的聚集性评估作为蜂窝Mitophagy一个标记

通过亚型特异性视黄酸受体对活神经元线粒体稳态调节进行优化的自动分析

通过亚型特异性视黄酸受体对活神经元线粒体稳态调节进行优化的自动分析