この作品は、コンセプト助成のUCディスカバリープルーフによっておよびNIH（R00 EB007241）によってサポートされていました。著者らはまた、この実験では彼女の援助のためのジャクリーン·ハバードに感謝したいと思います。

1。手術の準備<ol><li> 6-8週間の年齢の間に雌性CD-1マウスは、私たちの実験で使用した。 </li><li>ケタミンおよびキシラジンの組み合わせ（80 mg / kgのketamine/10 mg / kgをキシラジン）の腹腔内注射でマウスを麻酔。 〜37℃で最適な体温を確保するための恒温パッドの上にマウスを置き継続的に動物の反射を（ 例えば 、鉗子で鈍足をつまんで）テストすることによって、麻酔のレベルを監視し、必要に応じてそれ以上の麻酔を注入します。 </li><li>人工涙液軟膏で両眼を潤滑します。カミソリを使って頭皮に毛を削除し、70％アルコールの準備パッドを用いて残留毛を取り除く。頭皮上Nairさんの脱毛クリームの薄い層を適用し、それを有効にするには、2分間待ちます。穏やかに生理食塩水を使用してNairさん、残りの髪は綿棒とアルコールの準備パッドを湿らせ拭き取ってください。頭皮は完全に無毛である必要があります。 </li><li>ベタジンスワブスティックを使用して頭皮を消毒すると7をクリーン0％エタノール準備パッド。 </li><li> 〜37の最適な体温を確保するための手術用ドレープで動物を注意深く包む℃、頭蓋骨を固定する定位フレームに動物をマウントします。軽くて安定性を確保するために頭蓋骨をタップします。使用される材料のリストを表1に提供されています。 </li></ol> 2。間引き頭蓋骨皮質ウィンドウ準備<ol><li>目の間の正中線の時点で切開を開始します。耳の間の正中線点まで尾を続行します。鉗子との部分は皮膚。 </li><li>解剖顕微鏡下で薄くする領域を見つけ、ゆっくりとピンセットを用いて筋膜を除去します。間伐皮質のウィンドウを作成する前に、滅菌綿棒で頭蓋骨を乾かします。私たちの実験では、4×4mmの間伐頭蓋窓〜後部とブレグマ横1ミリメートルを作成しました。 </li><li>光抜本的な動きのONLを使用して外科ハンドドリルにドリルビットサイズ0.75ミリメートルの丸カーバイドバーを用いて頭蓋骨を薄く開始yである。頭蓋骨の上に直接圧力をかけないでください。滅菌生理食塩水と綿棒を用いて骨ほこりを取り除くために、頭蓋骨の過熱を防ぐため、毎年20〜30秒の掘削を停止します。生理食塩水はまた、頭蓋骨全体に熱を放散させるのに役立ちます。 </li><li>緻密骨の外側の層が完全に除去されたら真ん中の海綿骨の層が表示されるようにする必要があります。血管が海綿骨層でより明らかになりますようにいくつかのわずかな出血があるかもしれません。緑石のいがに切り替えて、海綿層がより繊細であるため特別な注意を用いた掘削を続ける。頭蓋窓全体に均一の作成中に緑色の石のいがが少ない骨材を除去します。骨塵を除去し、頭蓋骨を冷却するために時折ドリルを停止します。 </li><li>最後に、頭蓋骨の透明性と脳への血管系になったときに表示されるようになりました、研磨バールを使って頭蓋骨を研磨開始。頭蓋骨を下にスムージングしつつ、間伐より正確にできるようになります。 SKUの薄さをチェック静かにピンセットでそれをタップして、LL。頭蓋骨が少し柔軟になったときに研磨を停止します。 </li><li>間伐頭蓋窓は完全に滑らかで反射およびイメージング（ 図1）のために準備ができているはずです。非常に脳の組織の散乱の性質上、頭蓋骨は、最適な深さの浸透のために少なくとも55ミクロンまで薄くする必要があります。使用される材料のリストを表1に提供されています。 </li></ol> 3。光コヒーレンストモグラフィーイメージング<ol><li>手術が完了した後、麻酔の適切な水準を確保し、必要に応じて追加の麻酔を投与する動物の呼吸速度と反射神経をチェックします。 、定位フレームから動物を削除する手術用ドレープに包まれた動物を維持し、イメージングステーションに動物を輸送する。 </li><li>イメージングは​​、反射の標識を確認し、必要に応じて追加の人工涙液を適用する前に。頭蓋骨を確保するための定位フレームに代わって動物をマウントします。 </li><li>下に動物を置き10月のカメラと位置光ビーム（ 図2）に基づくTSCW。頭蓋骨と脳の断面図になりました（ 図3）可視化することができる。 </li><li>関心領域が配置された後、データ取得を開始することができます。イメージング目的のために、私たちは4.0ミリメートルの幅で撮像窓を達成するためにガルボミラーを使用しています。 2mmの撮像深度は6入射電力は、mWと焦点間伐頭蓋骨1mm以下で得られた。それぞれの断面積は、画像1枚あたり0.14秒の取得率は2048軸方向走査で構成されていた。 </li><li>脳の体積スキャンも、xyはサジタル方向にビームを走査する第一ガルバノミラーとコロナでスキャン第二ガルバノミラーで走査するためのガルバノミラーの2セットを使用して2D断面一連の画像を収集することによって得ることができる方向。 </li></ol>

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特別な利害関係は宣言されません。

10月と間引き頭蓋骨によるイメージングはごく最近検討されている小説のニューロイメージング技術15、16です。私たちの実験では、in vivoでのマウスモデルにおけるTSCWを通して、SD-OCTイメージングの実現可能性を実証した。我々の結果から、頭蓋骨が約55μmまで薄くされると侵入深さは、それぞれ、軸方向および横方向に8μmと20μmの画像解像度で約1ミリメートルで得られる。?...

1990年代初頭の登場以来、10月は、組織の構造と機能2の生体イメージングのために広く使用されてきました。 10月は、光ファイバ·マイケルソン干渉計2,4低コヒーレンス光源を実装することにより、後方散乱光4のエコー遅延時間を測定することにより、断面画像を生成します。また、フーリエドメインOCT（FD-OCT）として知られている、SD-OCTは、1995年に初めて5年に導入され、従来のタイムドメインOCT（TD-OCT）と比較して優れた画像診断法を提供しています。 SD-OCTでは、参照アームが高速·超高解像度の画像取得6-9で得られた静止状態に保たれています。 現在、TSCWモデルは従来の開頭術の代わりに、2光子顕微鏡の in vivoでの脳の画像アプリケーションでのために主に使用されている。これらTSCWは追加IMAを提供するために、カスタム頭蓋骨板またはガラスカバースリップ10月13日と同時に使用されてきた安定性を変更する。我々の研究では、TSCWが使用されている場合これらのような付属品はOCTイメージングのために必要ではないことを観察した。彼らは光ビームと干渉し、OCT画像を変更することがありますしたがって、頭蓋骨板またはガラスカバースリップの欠如は、イメージング·ウィンドウサイズの広い範囲を可能にします。 間引き頭蓋骨の準備は、二光子顕微鏡10-13を用いて脳のイメージング研究に有利で ​​あることが判明した。私たちの実験では、TSCWを通じて体内画像皮質に、SD-OCTシステムを利用しています。当社のカスタムSD-OCTイメージングセットアップは、それぞれ8μmと20μmの軸方向および横方向の解像度が得97 nmの帯域幅で1295 nmの中心とする2つのスーパールミネッセントダイオード（SLD）14からなる広帯域、低コヒーレンス光源が含まれています。弊社の光学撮像素子では、我々はTSCW介して撮像がoで構造と機能を特定し、可視化に大きな可能性を持っていることを想像するptically密脳組織。

<table border="1"><tbody><tr><td> 材料 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>ケタミン</td><td>フェニックス医薬品</td><td> 57319-542-02 </td><td></td></tr><tr><td>キシラジン</td><td> Akorn株式会社</td><td> 139から236 </td><td></td></tr><tr><td>人工涙液軟膏</td><td>ラグビー</td><td> 0536-6550-91 </td><td></td></tr><tr><td>ナイル</td><td>チャーチ＆ドワイト株式会社</td><td> 4010130 </td><td></td></tr><tr><td>滅菌アルコール準備パッド</td><td>ケンドール·ヘルスケア</td><td> 6818 </td><td></td></tr><tr><td>綿はアプリケーターをひっくり返した</td><td> Fisherbrand </td><td> 23-400-115 </td><td></td></tr><tr><td>ベタジンソリューションSwabstick </td><td>パデュー製品&lt;/ TD&gt; <td> 67618-153-01 </td><td></td></tr><tr><td>食塩水、0.9パーセント</td><td>フェニックス医薬品</td><td> 57319-555-08 </td><td></td></tr><tr><td>定位フレーム</td><td> Stoelting </td><td></td><td></td></tr><tr><td>高速外科ハンドドリル</td><td> Foredom </td><td></td><td> 38000回転</td></tr><tr><td>超硬丸バール</td><td> Stoelting </td><td></td><td> 0.75ミリメートル</td></tr><tr><td>のDura-グリーンストーンズ</td><td>松風</td><td></td><td>シャンク：HP 形状：BA1 </td></tr><tr><td> CompoMaster粗＆CompoMasterポリッシャー</td><td>松風</td><td></td><td>形状：ミニ白金。 </td></tr><tr><td> SpaceDrapes </td><td>トリーティフィック株式会社</td><td></td><td></td></tr></tbody></table>

thinned-skull cortical window technique for in vivo optical coherence tomography imaging

光コヒーレンストモグラフィ（OCT）は、高時空間分解能で生物医学イメージング技術です。その低侵襲アプローチと10月は皮膚科、眼科、消化器1-3に広く使用されています。間引き頭蓋骨皮質ウィンドウ（TSCW）を使用して、我々は、in vivoでの画像の皮質をするためのツールとしてスペクトラルドメインOCT（SD-OCT）モダリティを採用しています。一般的に、それは、より汎用性を提供するように開かれ、頭蓋骨がニューロイメージングのために使用されてきた、しかし、TSCWアプローチは、低侵襲であり、神経病理学的研究における長期的イメージングのための効果的な平均である。ここでは、大脳皮質の in vivoイメージングの 10月のマウスモデルにおけるTSCWを作成する方法を紹介します。

大脳皮質上間伐ウィンドウを作成した後血管系は、より視覚的に顕著でなければなりません（ 図1）と深い撮像深度（1mmまで）が可能になります。右皮質は140μmで（ 図1）で測定した正常な頭蓋骨に比べ、より大きな光学的透明度を提供するので、約55ミクロンまで薄くされる。さらに10から15μmまで薄くするガラスカバースリップと頭蓋骨プレートの使用など、しか?...

Watch this Scientific Journal Video about Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging at JoVE.com

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

私達はのためのマウスモデルにおける間引き頭蓋骨皮質ウィンドウ（TSCW）を作成する方法を紹介<em生体内で&gt;</em大脳皮質の&gt; OCTイメージング。

ための間引き頭蓋骨皮質ウィンドウ·テクニック<emインビボで&gt;</em&gt;光干渉断層イメージング

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

thinned-skull-cortical-window-technique-for-vivo-optical-coherence

Research

JoVE Journal

Neuroscience

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

15.0K ビュー.  University of California, Riverside. 私達はのためのマウスモデルにおける間引き頭蓋骨皮質ウィンドウ（TSCW）を作成する方法を紹介 OCTイメージング。

ビデオ: Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

慢性二光子用薄型頭蓋骨のウィンドウテクニック in vivoで</em神経炎症のモデルのマウスミクログリアの>イメージング

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

神経科学

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

間伐-頭蓋骨の準備を使用してマウス視覚野の慢性的イメージング

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

マウス脳の長期イメージングのためのポリッシュと強化間引き頭蓋骨ウィンドウ

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

二光子<em&gt; in vivoで</em&gt;間引き頭蓋骨の準備を使用して、マウス皮質における樹状突起棘のイメージング

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光干渉断層計: マウス網膜神経節細胞In Vivoイメージング

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

生体内で新生仔マウスの大脳皮質ニューロンの 2 光子イメージング

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

マウスおよびラットの視覚系の構造と機能読み出しとして光干渉断層計と動視反応を使用してください。

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

光コヘレンス断層撮影解剖学によるレチナル血管反応性

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

覚醒マウスにおける 反復生体内 イメージングのための頭蓋窓の移植

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...