Bu çalışma Konsept hibe UC Discovery Proof tarafından ve NIH (R00 EB007241) tarafından desteklenmiştir. Yazarlar ayrıca bu deneyde ona yardım için Jacqueline Hubbard teşekkür etmek istiyorum.

1. Cerrahi Hazırlık <ol><li> 6-8 hafta yaşları arasında Bayan CD 1 farelerde bizim deneylerde kullanıldı. </li><li> Bir ketamin ve ksilazin kombinasyonu (80 mg / kg ketamine/10 mg / kg ksilazin) bir intraperitoneal enjeksiyon ile fare anestetize. ~ 37 ° C&#39;de optimum vücut ısısını sağlamak için sıcakkanlı pad üzerinde fare yerleştirin Sürekli hayvanın refleksler (örneğin, künt forseps ile ayak pinching) takarak anestezi seviyesini izlemek ve gerekli olduğunda daha fazla anestezi enjekte. </li><li> Yapay gözyaşı merhemi ile iki gözü yağlayın. Bir jilet kullanarak kafa derisi üzerinde tüyleri çıkarın ve% 70 alkol hazırlık yastıkları kullanarak kalan tüyleri. Kafa derisi üzerinde Nair epilasyon kremi ince bir tabaka uygulayın ve etkili olması için 2 dakika bekleyin. Yavaşça salin kullanarak Nair ve kalan saç pamuklu ve alkol hazırlık yastıkları nemlendirilmiş siliyorum. Derisi artık tamamen tüysüz olmalıdır. </li><li> Bir betadin çubukla sopa kullanarak derisi Dezenfekte ve 7 ile temizleyin0% etanol hazırlık pedleri. </li><li> ~ 37 arasında optimum vücut ısısı sağlamak için cerrahi örtüler içinde dikkatli bir şekilde sarmak hayvan ° C ve kafatası immobilize etmek için bir stereotaksik çerçeve üzerine monte hayvan. Hafif istikrarı sağlamak için kafatası dokunun. Ikinci malzeme listesi Tablo 1 &#39;de verilmiştir. </li></ol> 2. İnceltilmiş-kafatası Kortikal Pencere Hazırlama <ol><li> Gözler arasındaki orta hat noktada kesi başlayın. Kulakları arasındaki orta hat noktasına kaudal devam edin. Forseps ile Parça deri. </li><li> Diseksiyon mikroskop altında inceltilmesi alanı bulun ve hafifçe cımbız kullanarak fasya çıkarın. Inceltilmiş kortikal pencere oluşturmadan önce steril pamuklu çubukla kafatası kurulayın. Deneylerde, biz 4 yarattı × 4 mm kraniyal pencere ~ bregma posterior ve lateral 1 mm inceltilmiş. </li><li> Hafif süpürme hareketi onl kullanarak cerrahi el matkap matkap boyutu 0,75 mm yuvarlak karbit frez kullanarak kafatası incelme Başlayany. Kafatası üzerine doğrudan baskı uygulamayın. Steril serum fizyolojik ve pamuklu çubuk kullanılarak kemik tozları ve kafatası aşırı ısınmasını önlemek için her 20-30 sn sondaj durdurun. Salin de kafatası boyunca ısı gondererek yardımcı olacaktır. </li><li> Kompakt kemik dış tabakası tamamen çıkarıldıktan sonra orta süngerimsi kemik tabakası şimdi görünür olmalıdır. Kan damarları süngerimsi kemik tabakası daha belirgin olarak bazı hafif kanama olabilir. Yeşil bir taş bur geçin ve süngerimsi tabaka daha hassas olduğu için ekstra dikkatli kullanarak sondaj devam ediyor. Kraniyal pencere boyunca düzgünlüğü oluştururken yeşil taş bur az kemik materyali kaldıracaktır. Kemik tozu ve kafatası soğutmak için arada sondaj durdurun. </li><li> Sonunda, kafatası daha şeffaf ve beyin üzerinde damar haline gelmiştir artık görünür bir parlatma frezle kafatası parlatma başlar. Bu sağlayacak kafatası aşağı iken pürüzsüzleştirici incelme daha hassas. Sku inceliği Girişyavaşça forseps ile dokunarak ll. Kafatası biraz esnek olduğunda parlatma durdurun. </li><li> Inceltilmiş kranial penceresi şimdi tamamen düz ve yansıtıcı ve görüntüleme (Şekil 1) için hazır olmalıdır. Beynin çok saçılma dokuların doğası nedeniyle, kafatası optimum nüfuz derinliği için en az 55 um ile inceltilerek olmalıdır. Ikinci malzeme listesi Tablo 1 &#39;de verilmiştir. </li></ol> 3. Optik Koherens Tomografi Görüntüleme <ol><li> Ameliyat tamamlandıktan sonra anestezi uygun düzeyde sağlamak ve gerekirse ek anestezi yönetmek için hayvanın solunum hızı ve refleksler kontrol edin. Stereotaksik çerçeveden hayvan çıkarın, hayvan görüntüleme istasyonuna cerrahi örtüler sarılmış ve ulaşım hayvan tutun. </li><li> Görüntüleme reflekslerin işaretleri kontrol edin ve gerekirse ek yapay gözyaşı uygulamadan önce. Kafatası sabitlemek için stereotaksik çerçeve için hayvan monte edin. </li><li> Altında yer hayvanEkim kamera ve optik ışın (Şekil 2) &#39;de pozisyon TSCW. Kafatası ve beyin bir kesit görünümü artık (Şekil 3) görüntülenebilir. </li><li> Ilgi alanı yer almaktadır kez Veri toplama başlayabilirsiniz. Görüntüleme amaçları için, 4.0 mm kadar bir genişliğe sahip bir görüntüleme penceresi sağlamak için galvo aynalar kullanılır. 2 mm&#39;lik bir görüntü derinliği 6 olay mW güç ve bir odak noktası inceltilmiş kafatası altında 1 mm ile elde edilmiştir. Her kesit alanı görüntü başına 0.14 sn bir kazanım oranı ile 2.048 eksenel taramaları oluşuyordu. </li><li> Beynin hacimsel taramaları de xy sagital yönde ışın tarama birinci galvo ayna ve koronal tarama ikinci galvo ayna ile tarama için galvo ayna iki kümesi kullanılarak 2D kesitsel görüntü dizisi toplanması ile elde edilebilir yönü. </li></ol>

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Çıkar çatışması ilan etti.

OCT ve bir inceltilmiş-kafatası ile Görüntüleme ancak son 15, 16 araştırılmıştır yeni nöro-görüntüleme tekniğidir. Deneylerde, in vivo olarak fare modelinde bir TSCW aracılığıyla SD-OCT görüntüleme fizibilitesi. Sonuçlardan, kafatası yaklaşık 55 um ile inceltilir ve nüfuz derinliği, sırasıyla eksenel ve yanal yönde 8 mikron ve 20 mikron arasında resim çözünürlüğü ile yaklaşık olarak 1 mm elde edilir. Normal bir kafatası (Şekil 4) ile karş?...

Erken 1990 yılında tanıtılmasından bu yana, OKT doku yapısı ve fonksiyonu 2 biyolojik görüntüleme için yaygın olarak kullanılır olmuştur. OCT, fiber-optik Michelson interferometresi 2,4 ile düşük koherens ışık kaynağı uygulayarak geri saçılmış ışık 4 eko gecikme zamanı ölçülerek kesitsel görüntüler oluşturur. Ayrıca Fourier etki OCT (FD-OCT) olarak bilinen SD-OCT, ilk kez 1995 5&#39;ten ve geleneksel zaman alanı OCT (TD-OCT) ile karşılaştırıldığında üstün bir görüntüleme yöntemidir sunmaktadır. SD-OCT, referans kol yüksek hızlı ve ultra yüksek çözünürlüklü görüntü elde etme 6-9 sonuçlanan sabit tutulur. Halen, TSCW modelleri, geleneksel bir kranyotomi yerine iki-fotonlu mikroskopi beyin in vivo görüntüleme uygulamaları için büyük ölçüde kullanılmaktadır. Bu TSCW ek ima sağlamak için özel bir kafatası plaka veya cam bir kapak kayma 10-13 ile eş zamanlı olarak kullanılmıştıristikrar ging. Bizim çalışmalarımızda, biz TSCW kullanıldığında bu gibi aksesuarlar OCT görüntüleme için gerekli olmadığını gözlemledik. Bunların optik huzme ile karışabilen ve OCT görüntüleri değiştirebilir Bu nedenle, bir kafatası levha ya da cam kapak slipi eksikliği görüntüleme penceresinin boyutu, daha geniş bir hareket alanı sağlar. A inceltilmiş-kafatası hazırlık iki foton mikroskopi 10-13 kullanarak beyin görüntüleme çalışmalarında avantajlı olduğu kanıtlanmıştır. Deneylerde, biz TSCW yoluyla in vivo görüntü kortekse bir SD-OCT sistemi kullanmaktadır. Bizim özel SD-OCT görüntüleme kurulumu sırasıyla 8 mikron ve 20 mikron bir aksiyel ve lateral çözünürlük sonuçlanan 97 nm bant genişliği ile 1295 nm de iki superluminescent diyotlar (SLD), 14 oluşan bir geniş bant, düşük koherens ışık kaynağı içerir . Bizim optik görüntüleme cihazı ile, bir TSCW aracılığıyla görüntüleme o yapılar ve fonksiyonları tanımlama ve görselleştirme büyük bir potansiyele sahip olduğunu tasavvurptically yoğun beyin dokusu.

<table border="1"><tbody><tr><td> Malzemeler </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Ketamin </td><td> Phoenix Pharmaceuticals </td><td> 57319-542-02 </td><td></td></tr><tr><td> Ksilazin </td><td> Akorn, Inc </td><td> 139-236 </td><td></td></tr><tr><td> Yapay Tears merhem </td><td> Rugby </td><td> 0536-6550-91 </td><td></td></tr><tr><td> Nair </td><td> Church &amp; Dwight A.Ş. </td><td> 4010130 </td><td></td></tr><tr><td> Steril Alkol Hazırlık Pad </td><td> Kendall Sağlık </td><td> 6818 </td><td></td></tr><tr><td> Pamuk Aplikatörler Yatırılan </td><td> Fisherbrand </td><td> 23-400-115 </td><td></td></tr><tr><td> Betadine Çözüm Swabstick </td><td> Purdue Ürünler &lt;/ Td&gt; <td> 67618-153-01 </td><td></td></tr><tr><td> Tuzlu Çözüm, .9% </td><td> Phoenix Pharmaceuticals </td><td> 57319-555-08 </td><td></td></tr><tr><td> Stereotaktik Çerçeve </td><td> Stoelting </td><td></td><td></td></tr><tr><td> Yüksek Hızlı Cerrahi El Matkap </td><td> Foredom </td><td></td><td> 38,000 rpm </td></tr><tr><td> Carbide Yuvarlak Bur </td><td> Stoelting </td><td></td><td> 0,75 mm </td></tr><tr><td> Dura-Yeşil Taşlar </td><td> Shofu </td><td></td><td> Sap: HP Şekil: BA1 </td></tr><tr><td> CompoMaster Kaba &amp; CompoMaster Cilalayıcı </td><td> Shofu </td><td></td><td> Şekil: Mini-Pt. </td></tr><tr><td> SpaceDrapes </td><td> Braintree Scientific, Inc </td><td></td><td></td></tr></tbody></table>

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

Optik koherens tomografi (OCT) yüksek uzaysal-zamansal çözünürlüğe sahip biyomedikal görüntüleme tekniğidir. Onun minimal invaziv yaklaşım ile Ekim dermatoloji oftalmoloji, ve gastroenteroloji 1-3 yaygın olarak kullanılır olmuştur. Bir inceltilmiş-kafatası kortikal pencere (TSCW) kullanarak, in vivo görüntü korteks için bir araç olarak spectral-domain OCT (SD-OCT) yöntemi kullanır. Daha çok yönlülük sağlar gibi yaygın, açık bir kafatası nöro-görüntüleme için kullanılır olmuştur, ancak, bir TSCW yaklaşım daha az invaziv ve nöropatoloji çalışmalarda uzun süreli görüntüleme için etkili bir araçtır. Burada, serebral korteksin vivo OCT görüntüleme içinde bir fare modelinde bir TSCW yaratma yöntemi sunuyoruz.

Serebral korteks üzerinde inceltilmiş pencere oluşturduktan sonra damar artık görsel olarak daha belirgin olmalıdır (Şekil 1) ve (1 mm&#39;ye) daha derin bir görüntüleme derinliği için izin verecektir. 140 mikron (Şekil 1) ölçülen ve daha büyük optik netlik sağlar Normal bir kafatası ile karşılaştırıldığında sağ korteks yaklaşık 55 mikron kadar inceltilir. Daha 10-15 um ile inceltme 11 ancak cam kapak slipi ve kafatası levhaların kullanılmas...

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

Biz bir fare modelinde bir inceltilmiş-kafatası kortikal pencere (TSCW) oluşturma bir yöntem mevcut<em&gt; In vivo</emSerebral korteksin&gt; OCT görüntüleme.

Için inceltilmiş-kafatası Kortikal Pencere Tekniği<em&gt; In Vivo</em&gt; Optik Koherens Tomografi Görüntüleme

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

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

15.0K Görüntüleme.  University of California, Riverside. Biz bir fare modelinde bir inceltilmiş-kafatası kortikal pencere (TSCW) oluşturma bir yöntem mevcut In vivo OCT görüntüleme.

Video: 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.

Kronik İki foton için İnce kafatası Pencere Tekniği In vivo</emNöro-inflamasyon Modelleri murin mikroglia> Görüntüleme

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

Nörobilim

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.

Fare Visual Cortex İnceltilen kafatası Hazırlık Kronik Görüntüleme

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.

Fare Beyin Uzun vadeli Görüntüleme için Parlak ve Takviyeli inceltilmiş-kafatası Pencere

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.

Kortikal Nöronlar In Vivo İki foton Of Görüntüleme Deneyimi bağımlı Moleküler Değişiklikler

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.

İki-Foton<em&gt; In vivo</em&gt; Bir inceltilmiş-kafatası Hazırlık kullanarak Fare Cortex dendritik dikenler Görüntüleme

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.

Optik Koherens tomografi: Fare retina Ganglion hücreleri Vivo içinde görüntüleme

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.

Vivo Yenidoğan farelerde kortikal nöronların iki fotonlu görüntüleme

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.

Optik koherens tomografi ve Optokinetic yanıt yapısal ve işlevsel görsel sistemin çıktıları fareler ve sıçanlar olarak kullanma

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.

Optik Koherens Tomografi Anjiyografi ile Değerlendirilen Retinal Vasküler Reaktivite

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.

Uyanık Farelerde Tekrarlanan In Vivo Görüntüleme için Kraniyal Pencerenin İmplantasyonu

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