Name: near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium
Uploaded: 2015-03-14T14:00:00.000+00:00
Duration: 6 min 56 s
Description: 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

著者は、mRNA抽出とPCR、およびサポートのためのガーネットパッシーとロドニー·ウィリアムズ記念財団との援助のためにポール博士分かっていると氏ジュヌヴィエーヴ·フォンを認識したい。

倫理声明：以下のとおり、すべての手順は、シドニーの動物倫理委員会の大学によって承認された。 1.動物注：1と8から9カ月齢のマウス（C57 / BL6）は、動物資源センター（パース、オーストラリア）から入手した。マウスは、シドニー大学のボッシュ齧歯類の施設に収容した。 <ol><li>食料と水を自由にアクセスできる12/12時間の明/暗サイクル上の標準的なマウスケージの住宅マウス。 </li><li>近赤外線に各年齢群で除算マウス（NIR）処理された、または偽の比較のために（対照）処置群。 </li></ol> 2.近赤外（NIR）照射とシャム治療<ol><li>治療レジメンの完了前に髪の迅速な再成長を防ぐために可能な限り厳密に電気かみそりでマウスの頭頸部領域に毛皮を剃る。マウスの病原体を含まない状態を維持するために、70％エタノールを使用してシェービング動物や別々のケージから動物との間にF10獣医消毒剤の間に楽器をきれいにする。動物が過剰に処理されないので、治療前の先頭に3日間 - この2か。 </li><li>尾の近位端を保持することによってマウスを拘束し、誤った結果をもたらし得る応力を最小限にするように、一方またはベンチトップの手のひらにリラックスできるようにする。 </li><li> LED NIrを（670 nm）を（発光ダイオード）1デバイスホールド- 2センチ離し暴露（坊主頭）地域からの90秒（ 図1A）のための装置のスイッチを入れる。 注：90秒の曝露の結果として温度変化を100mlの水に0.2°C &lt;として測定された。 </li><li>動物の偽処置群について2.3が、デバイスがオフになった（ 図1B）を残す-繰り返して、2.2を繰り返します。 </li><li>動物の近赤外ブロックされて治療群2.3が、アルミ箔でデバイスをカバーする - 繰り返して、2.2を繰り返します。 </li><li> approximat毎日2.5 - 繰り返して、2.2ステップ5日間連続してE 24時間間隔。 </li><li>五日目、治療後に両耳から前庭感覚上皮（3クリステと1卵形黄斑）を抽出（下記のセクション3を参照してください）。 </li></ol> 3.組織抽出20 <ol><li> 26のNaHCO 3、11グルコース、250グリセロール、2.5のKCl、1.2のNaH 2 PO 4、1.2のMgCl 2、および2.5のCaCl 2：（mm）をからなるグリセロールベースの人工脳脊髄液（ACSF）の300ミリリットルを準備します。 7.4のpHを確立し、カルシウム沈殿（曇り）を避けるためにカルボゲン溶液（95％O 2および5％CO 2）、CaCl 2を添加する前にガス。氷スラリーが形成されるように45分間、-80℃の冷凍庫に溶液を冷却する。 </li><li>標識されたスクリュートップマイクロチューブ内のRNA単離、溶解緩衝液を調製する（液体窒素中で凍結および融解の間時の管の圧力変化蓋のポッピングを停止する）に従って製造業者の説明書または通常の実験室の実践に。アルミデュワーフラスコ内の液体窒素をご用意ください。 注：液体窒素を取り扱う際、防護服とeyewareを使用してください。その気体の形態のボリュームが液体の形の約700倍であると窒息の原因となりますので、液体窒素は風通しのよい部屋で使用されていることを確認。 </li><li>深く腹腔内注射を介してケタミン（400 mg / kgを）をマウスに麻酔し。後肢反射が完全にマウスが完全に麻酔をしていることを示すものとして沈静化することを許可する。 </li><li>鋭いステンレス製のはさみでマウスを斬首し、かみそりの刃（丸みを帯びた＃22）を使用して、頭蓋骨の矢状皮膚に沿って切開する。この時点で、全体を通して3.5のステップ- 3.9、組織上の氷冷ACSFの通常のアプリケーションによって、できるだけ涼しい頭蓋冠、脳、および基礎となる前庭装置を保つ。 </li><li> Smalのを作る標準パターンハサミの尖った腕の使い方Lラムダで頭蓋骨を切開し、矢状縫合に沿って切断。 </li><li>静かに脳をドラッグすることなく、骨の下面にできるだけ近いブレードを保ち頭頂骨の下の浅い骨鉗子の一方のアームをスライドさせます。安全で脳が露出するまで後方に横方向に頭頂骨と後頭骨を引き離す。 </li><li>小さなステンレス製のへらを使用して、内耳神経（CN VIII）を露出するために離れて、前と中頭蓋窩から脳を持ち上げる。直接前庭有毛細胞を神経支配する一次求心性軸索上の不必要な緊張を防ぐために神経を横断する。 </li><li> CN VIIIの切断後TOTOで脳を削除します。 </li><li>中頭蓋窩における蝸牛及び末梢前庭器官を含む骨迷路を観察します。各骨迷路の横に二つの小さな切開部を作成し、前方の三半規管を保持し、後で引いて、構造全体を切除同盟国。 </li><li>継続的にカルボゲンで灌流しながら、すぐに（ ステップ3.1で説明したように）の氷冷ACSF溶液を含む解剖皿に切除された迷路を浸す。 </li><li>実体顕微鏡下では、蝸牛によって迷路を押しながら、ピンセットで皿の底に固定します。 <ol><li>前方半規管（SSC）膨大部上記の骨に小さな開口部に傷を付けまっすぐ細かい鉗子を使用してください。 </li><li>静かにすぐに骨の下に到達し、外側に離れて膨大部からフリックすることで、この開口部を拡大する。開口部に鉗子を押し、以下の脆弱な膜迷路を損傷しないためにここに注意が必要です。卵形、前方および横方向の膨大部がすべて露出するまで、このように続けます。可能であれば、また事後膨大部を取り外します。 </li></ol></li><li>細かい鉗子を使用して、静かに卵形嚢を持ち上げ、彼らが完全に切り離されるまで、骨迷路から離れて膨大部。可能な場合には、それらを保持するそれらに関連する半規管によって感覚上皮の損傷を避けるために。いくつかのケースでは、半円形膜状管の近位部は、骨から膨大部を解放するために虹彩鋏で切断する必要があるかもしれない。 </li><li>確実にステップ3.2で先に作成した溶解バッファに鉗子の先端と場所との間に前庭器官を把握する。静かに前庭器官が鉗子から切り離されていることにバッファ内の周りに鉗子を旋回。これは実体顕微鏡下でピンセットを持って来ることでケースです確認してください。 <ol><li>微小管の蓋のネジと、すぐに液体窒素でサンプルを凍結する。 </li></ol></li></ol> 4. RNA抽出およびRT-PCR <ol><li>メーカーの指示又は好ましい実験室プロトコルに従ってメッセンジャーRNA（mRNA）の抽出の標準的な方法に従ってください。 注：mRNAの小さな収量を特定するためにキャリアRNAを利用し、市販のキットが、この中で使用されたプロトコル。低い溶出量は、mRNAの最終濃度を増加させることができる。 注：負の「酵素なし」のコントロール（NECS）と「無DNAテンプレート」コントロール（NTCの）も観察された影響の妥当性を確保するために完了する必要があります。 </li><li>標的遺伝子21-23の相補的DNA（cDNA）の増幅のmRNAの逆転写の標準的な方法を適用する。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないと宣言。

ここで説明する代表的な結果は、NIR光の短い頭蓋送達（5日間90秒/日）は、偽処置マウスと比較して老齢マウスにおける抗酸化物質の発現のレベルを上昇させるのに十分であることを示している。放出されながら熱は、ラットの前庭求心24について報告されているよう、ミトコンドリアおよび/ ​​またはニューロンの活性化の源を表すことができる- NIrをすることによって放出される?...

定率性能およびその後滝一般的であり、残念ながら、多くの場合、自然老化1の特徴を定義する。この減少の影響は、物理的および社会的の両方である、と大幅に高齢者の生活の質を低減することができます。応答では、物理療法やリハビリテーションは、滝の研究の焦点となっているが、繰り返し滝の有病率の一貫した減少と関連していない。同時に、作業は、末梢または中枢前庭系（バランスを維持する責任システム）乏しく、これらのシステムを標的とする潜在的な治療戦略の変更や制限された不均衡の根本的な原因を調査する。 加齢性黄斑変性症2-4、アルツハイマー病モデル5-8、およびパーキンソン病9-12を含む加齢関連神経変性疾患に関する最近の研究は、済州の神経保護効果を示した近赤外（NIR）光のmple非侵襲的アプリケーション。さらに、前庭系に、NIrをインビトロ13前庭一次求心性ニューロンの活性を増大させるために使用されてきた。 NIR光のメカニズムはよく理解されていないが、NIrをを使用してほとんどの研究は、NIRは、細胞代謝を促進するために、ミトコンドリア複合体IV（シトクロムcオキシダーゼ）14-17を刺激することを示唆している。前庭感覚上皮ではI型有毛細胞の表皮下プレートは、ミトコンドリア18で緻密であり、そのように治療のNIR治療のための作用部位を表すことができる。 ここで、頭蓋の簡単な非侵襲的処置レジメンは、細胞の代謝を測定するために使用することができる（および含意、ミトコンドリア機能によって）マウス前庭感覚上皮に記載されているNIrを適用した。また議論前庭感覚上皮の調製物であり、それは、NIRはubiquitoの発現を増加させることが示されている感覚上皮における我々の抗酸化剤（スーパーオキシドジスムターゼ1） -以前に蝸牛の有毛細胞の生存19にとって重要であることが示される。

<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

加齢とのバランス機能の低下を減衰させるための戦略は、主にバランスの作業や運動などの物理的な治療法に焦点を当てている。しかし、これらのアプローチは、バランスの衰退の根本的な原因に対処していない。マウスを用いて、前庭感覚上皮における細胞の代謝に近赤外光（NIR）の影響を評価した。収集されたデータは、このシンプルで安全な介入は自然老化の有害な影響からこれらの脆弱な細胞を保護することができることを示している。 mRNAは、孤立した末梢前庭感覚上皮（膨大部稜と卵形黄斑）から抽出し、続いてcDNAライブラリーに転写した。このライブラリは、その後ユビキタス酸化防止剤（SOD-1）の発現についてプローブした。抗酸化遺伝子の発現は、その後、細胞代謝を定量した。若い（4週間）でのNIR経頭蓋配信を使用し、古い（8から9ヶ月）マウス、および5日間の簡単な治療レジメン（90秒/日AYS）、この作業は、単独NIrを前庭感覚上皮におけるミトコンドリアの機能を改善するのに十分であり得ることを示唆する。前庭有毛細胞の機能を改善するための治療に使用可能な、手頃な価格の、非侵襲的な方法は現在存在しないので、外部NIrを放射線の適用は、前庭感覚上皮inthe細胞代謝の老化の影響に対抗する潜在的な戦略を提供する。

若い（4週間）と古いでNIrを治療の影響を比較する（8から9ヶ月）マウスを、我々は若いた（n = 16）に、酸化防止剤、スーパーオキシドジスムターゼ1（SOD-1）の発現を測定し、古いた（n = 20）偽処理、または近赤外線遮断され、近赤外処理したマウス。 図2は、若い偽処置動物と比較して若いNIrを処理動物で2倍以上のβアクチン正規化されたSOD-1発現の有意な増加を示しているた（p...

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

ミトコンドリア機能障害は、細胞老化の特徴である。本論文では、高齢化マウス前庭感覚上皮におけるミトコンドリア機能を改善するために、非侵襲的近赤外（NIR）処理を使用しています。

近赤外（NIR）の光は、マウス前庭感覚上皮におけるミトコンドリア機能のマーカーの発現を増加させる

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

Research

JoVE Journal

Biology

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

9.9K ビュー.  University of Sydney. ミトコンドリア機能障害は、細胞老化の特徴である。本論文では、高齢化マウス前庭感覚上皮におけるミトコンドリア機能を改善するために、非侵襲的近赤外（NIR）処理を使用しています。 

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

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

高齢マウス前庭系の行動評価

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

行動学

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

加齢黄斑変性症マウスモデルにおける網膜色素性上皮病変を評価および定量化するためのプロトコル

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

遺伝学

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.

電気生理学および高分解能二光子顕微鏡用マウス前庭感覚上皮の切り抜き半無傷準備

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

神経科学

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).

マウスにおける造影MRIによる視神経線維の整合性in vivoイメージング

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

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

マウスの光嫌悪を用いた片頭痛様行動の調査

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

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.

高解像度呼吸法による坐骨神経のミトコンドリア機能の評価

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

生化学

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.

マウスにおける経頭蓋 Photobiomodulation 療法のプロトコル

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

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Ex Vivoのレティナル組織サンプルにおけるミトコンドリア呼吸と解糖の測定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active 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

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.

酸化ストレスに赤/近赤外光療法の波長および強度の範囲の効果の評価のための手法<em&gt;インビトロ</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

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.

携帯マイトファジーのマーカーとしてEYFP-パーキン集計の評価のためのタイムラプスビデオ顕微鏡

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 ...