Ce travail a été soutenu par la preuve UC Découverte de la subvention Concept et par le NIH (R00 EB007241). Les auteurs tiennent également à remercier Jacqueline Hubbard pour son aide dans cette expérience.

1. Préparation chirurgicale <ol><li> CD 1 souris femelles âgées de 6-8 semaines ont été utilisés dans nos expériences. </li><li> Anesthésier la souris avec une injection intrapéritonéale d&#39;une combinaison de kétamine et de xylazine (80 mg / kg ketamine/10 mg / kg de xylazine). Placez la souris sur un tapis homéothermes afin d&#39;assurer une température corporelle optimale à ~ 37 ° C Surveiller en permanence le niveau de l&#39;anesthésie en testant les réflexes animal (par exemple, pincer pied avec le forceps émoussé) et injecter plus de l&#39;anesthésie si nécessaire. </li><li> Lubrifiez les deux yeux avec une pommade larmes artificielles. Enlever les poils sur le cuir chevelu à l&#39;aide d&#39;un rasoir et enlever les poils résiduel en utilisant 70% de tampons imbibés d&#39;alcool. Appliquer une fine couche de crème Nair épilation sur le cuir chevelu et attendre 2 minutes pour qu&#39;elle prenne effet. Essuyer doucement les cheveux Nair et restant en utilisant une solution saline imbibé des cotons-tiges et tampons de préparation d&#39;alcool. Scalp devrait maintenant être complètement chauve. </li><li> Désinfecter le cuir chevelu à l&#39;aide d&#39;un bâton bétadine tige et nettoyer avec 70% tampons imbibés d&#39;éthanol. </li><li> Soigneusement envelopper l&#39;animal dans les draps chirurgicaux pour assurer la température du corps optimale de ~ 37 ° C et monter l&#39;animal sur un cadre stéréotaxique pour immobiliser le crâne. Appuyez légèrement sur le crâne pour assurer sa stabilité. Une liste des matériaux utilisés est fournie dans le tableau 1. </li></ol> 2. Diluée-crâne Préparation fenêtre corticale <ol><li> Démarrez l&#39;incision au point médian entre les deux yeux. Continuer caudalement au point médian entre les oreilles. Partie de la peau avec une pince. </li><li> Localisez la zone à éclaircir sous un microscope à dissection et retirez délicatement la façade à l&#39;aide de pinces. Sécher le crâne avec des cotons-tiges stériles avant de créer la fenêtre amincie corticale. Dans nos expériences, nous avons créé un 4 × 4 mm aminci fenêtre crânienne ~ 1 mm postérieure et latérale au bregma. </li><li> Commencez amincissement du crâne à l&#39;aide d&#39;une fraise en carbure de tour de taille foret de 0,75 mm dans une perceuse à main chirurgicale en utilisant la lumière ONL mouvement de balayagey. Ne pas appliquer une pression directe sur le crâne. Arrêtez le forage toutes les 20-30 secondes pour enlever la poussière d&#39;os en utilisant une solution saline stérile et tampons de coton et d&#39;éviter la surchauffe du crâne. La solution saline peut aussi aider à dissiper la chaleur à travers le crâne. </li><li> Une fois que la couche externe de l&#39;os compact est complètement enlevé la couche d&#39;os spongieux milieu devrait maintenant être visible. Il peut y avoir un léger saignement que les vaisseaux sanguins sont plus apparents dans la couche d&#39;os spongieux. Passez à une fraise pierre verte et continuer le forage en utilisant des précautions supplémentaires que la couche spongieuse est plus délicate. La fraise pierre verte sera enlever de la matière osseuse moindre tout en créant régularité tout au long de la fenêtre crânienne. Arrêtez le forage de temps en temps pour enlever la poussière d&#39;os et de refroidir le crâne. </li><li> Enfin, lorsque le crâne est devenu plus transparent et le système vasculaire dans le cerveau est maintenant visible, commencez à polir le crâne à l&#39;aide d&#39;une fraise de polissage. Cela permettra une évaluation plus précise amincissement tout en lissant le crâne. Vérifiez la minceur de l&#39;skull en tapotant doucement sur elle avec une pince. Arrêtez polissage lorsque le crâne est légèrement flexible. </li><li> La fenêtre amincie crânienne devrait maintenant être parfaitement lisse et réfléchissante et prêt pour l&#39;imagerie (figure 1). En raison de la nature des tissus fortement diffusantes du cerveau, du crâne doit être dilué à au moins 55 um pour la profondeur de pénétration optimale. Une liste des matériaux utilisés est fournie dans le tableau 1. </li></ol> 3. Tomographie par cohérence optique d&#39;imagerie <ol><li> Après la chirurgie est terminée, vérifier le taux de respiration des animaux et les réflexes pour assurer un niveau adéquat d&#39;anesthésie et d&#39;administrer une anesthésie supplémentaire si nécessaire. Supprimer animal du cadre stéréotaxique, garder des animaux enveloppé dans les draps chirurgicaux, et le transport des animaux à la station d&#39;imagerie. </li><li> Avant d&#39;imagerie vérifier les signes de réflexes et d&#39;appliquer des larmes artificielles supplémentaires si nécessaire. Monter sur des animaux pour la cadre stéréotaxique pour fixer le crâne. </li><li> Placer sous animauxOctobre caméra et la position de l&#39;TSCW sous le faisceau optique (figure 2). Une vue en coupe transversale du crâne et le cerveau peuvent maintenant être visualisées (figure 3). </li><li> L&#39;acquisition des données peut commencer une fois domaine d&#39;intérêt est située. À des fins d&#39;imagerie, on utilise des miroirs galvanométrique pour réaliser une fenêtre de formation d&#39;image avec une largeur de 4,0 mm. Une profondeur d&#39;imagerie de 2 mm a été obtenue avec 6 mW de puissance incidente et un point focal 1 mm au-dessous du crâne amincie. Chaque surface de section transversale se composait de 2.048 balayages axiaux avec un taux d&#39;acquisition de 0,14 secondes par image. </li><li> Balayages volumétriques du cerveau peut également être obtenue en recueillant une série de 2D des images en coupe à l&#39;aide de deux jeux de miroirs de balayage galvanométrique pour xy par le miroir galvanométrique premier balayage du faisceau dans la direction sagittale et le deuxième miroir de galvanomètre de balayage dans la coronaire direction. </li></ol>

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Aucun conflit d&#39;intérêt déclaré.

Imagerie par OCT et amincie crâne est un roman de neuro-imagerie technique qui n&#39;a été que récemment enquêté sur 15, 16. Dans nos expériences, nous avons démontré la faisabilité de SD-OCT par un TSCW dans un modèle de souris in vivo. À partir de ces résultats, le crâne est amincie à environ 55 um et la profondeur de pénétration est obtenue à environ 1 mm avec une résolution d&#39;image de 8 um et 20 um dans la direction axiale et latérale, respectivement. Dans le profil d&#39;...

Depuis son introduction dans le début des années 1990, l&#39;OCT a été largement utilisé pour l&#39;imagerie biologique de la structure des tissus et la fonction 2. Octobre génère des images en coupe en mesurant le temps de retard d&#39;écho de 4 lumière rétrodiffusée par la mise en oeuvre à faible cohérence source de lumière avec un interféromètre de Michelson à fibres optiques 2,4. SD-OCT, également connu sous le nom de domaine de Fourier PTOM (FD-OCT), a été introduit en 1995 5 et offre une modalité d&#39;imagerie de qualité supérieure par rapport aux traditionnels dans le domaine temporel OCT (TD-OCT). Dans l&#39;affaire SD-OCT, le bras de référence est maintenue stationnaire résultant en une vitesse élevée et ultra acquisition d&#39;image à haute résolution 6-9. À l&#39;heure actuelle, les modèles TSCW ont été largement utilisés pour des applications in vivo d&#39;imagerie cérébrale de microscopie à deux photons au lieu d&#39;une craniotomie traditionnelle. Ces TSCW ont été utilisés simultanément avec une plaque de crâne personnalisée ou une lamelle de verre 10-13 à fournir d&#39;autres imaGing stabilité. Dans nos études, nous avons constaté que des accessoires tels que ceux-ci ne sont pas nécessaires pour imagerie OCT quand un TSCW est utilisé. Par conséquent, l&#39;absence d&#39;une plaque de crâne ou des lamelles de verre permet une plus large gamme de taille de la fenêtre d&#39;imagerie, car ils peuvent interférer avec le faisceau optique et modifier des images octobre Une préparation amincie crâne s&#39;est avérée avantageuse dans les études d&#39;imagerie du cerveau en utilisant la microscopie à deux photons 10-13. Dans nos expériences, nous utilisons un système SD-OCT à l&#39;image du cortex in vivo par un TSCW. La coutume SD-OCT installation d&#39;imagerie contient une large bande, la source de lumière à faible cohérence constitué par deux diodes superluminescentes (SLD) centrés à 1295 nm avec une largeur de bande de 97 nm résultant en une résolution axiale et latérale de 8 um et 20 um, respectivement 14 . Avec notre appareil d&#39;imagerie optique, nous prévoyons que l&#39;imagerie à travers un TSCW a un grand potentiel dans l&#39;identification et la visualisation des structures et des fonctions dans l&#39;otissu cérébral ptically dense.

<table border="1"><tbody><tr><td> Matériels </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> La kétamine </td><td> Phoenix Pharma </td><td> 57319-542-02 </td><td></td></tr><tr><td> Xylazine </td><td> Akorn, Inc </td><td> 139-236 </td><td></td></tr><tr><td> Pommade larmes artificielles </td><td> Rugby </td><td> 0536-6550-91 </td><td></td></tr><tr><td> Nair </td><td> Church &amp; Dwight Co., Inc </td><td> 4010130 </td><td></td></tr><tr><td> Stérile alcool Prep Pad </td><td> Kendall Healthcare </td><td> 6818 </td><td></td></tr><tr><td> Cotton Tipped Applicators </td><td> Fisherbrand </td><td> 23-400-115 </td><td></td></tr><tr><td> Tige montée Solution de Bétadine </td><td> Produits Purdue &lt;/ Td&gt; <td> 67618-153-01 </td><td></td></tr><tr><td> Solution Saline, .9% </td><td> Phoenix Pharma </td><td> 57319-555-08 </td><td></td></tr><tr><td> Cadre stéréotaxique </td><td> Stoelting </td><td></td><td></td></tr><tr><td> Haute Vitesse Perceuse à main chirurgicale </td><td> Foredom </td><td></td><td> 38000 rpm </td></tr><tr><td> Carbide Bur ronde </td><td> Stoelting </td><td></td><td> 0,75 mm </td></tr><tr><td> Dura-Green Pierres </td><td> Shofu </td><td></td><td> Shank: HP Forme: BA1 </td></tr><tr><td> CompoMaster grossier et CompoMaster Polisher </td><td> Shofu </td><td></td><td> Forme: 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

Tomographie par cohérence optique (OCT) est une technique d&#39;imagerie biomédicale de haute résolution spatio-temporelle. Grâce à son approche minimalement invasive OCT a été largement utilisé en ophtalmologie, dermatologie, gastro-entérologie et 1-3. En utilisant une fenêtre corticale amincie crâne (TSCW), nous employons domaine spectral OCT (SD-OCT) modalité comme un outil pour l&#39;image du cortex in vivo. Communément, un crâne ouvert, a été utilisé pour la neuro-imagerie car il offre une plus grande polyvalence, cependant, une approche TSCW est moins invasive et est un moyen efficace pour l&#39;imagerie à long terme dans les études de neuropathologie. Ici, nous présentons une méthode de création d&#39;un TSCW dans un modèle de souris in vivo pour imagerie OCT du cortex cérébral.

Après avoir créé une fenêtre amincie sur le cortex cérébral du système vasculaire devrait maintenant être visuellement plus importante (Figure 1) et permettra une profondeur plus grande image (jusqu&#39;à 1 mm). Le cortex droit est amincie à environ 55 um par rapport à un crâne normal mesuré à 140 um (figure 1) et fournit une plus grande clarté optique. Dilution supplémentaire à 10-15 um est possible 11 cependant pas nécessaire que l&#39;utilisation de lamel...

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

Nous présentons une méthode de création d&#39;une fenêtre corticale amincie crâne (TSCW) dans un modèle de souris pour<em&gt; In vivo</em&gt; Imagerie OCT du cortex cérébral.

Diluée-skull Technique fenêtre corticale pour<em&gt; In Vivo</em&gt; Tomographie par cohérence optique d&#39;imagerie

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 vues.  University of California, Riverside. Nous présentons une méthode de création d'une fenêtre corticale amincie crâne (TSCW) dans un modèle de souris pour In vivo Imagerie OCT du cortex cérébral.

Vidéo: 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.

Une technique de la fenêtre mince crâne pour chronique à deux photons In vivo</emImagerie> de Murine microglies dans les modèles de la neuro-inflammation

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

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Neurosciences

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.

Imagerie chronique des souris cortex visuel en utilisant une préparation Diluée-crâne

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.

Une poli et renforcé Diluée-crâne fenêtre for Long-term imagerie du cerveau de souris

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.

In Vivo Imagerie biphotonique d&#39;expérience qui dépendent des changements moléculaires dans les neurones corticaux

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.

Deux-Photon<em&gt; In vivo</em&gt; Imagerie des épines dendritiques dans le cortex de souris utilisant un crâne-Diluée Préparation

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.

Tomographie par cohérence optique : Imagerie souris ganglionnaires rétiniennes cellules In Vivo

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.

In Vivo Two-photon Imaging de neurones corticaux en souris néonatales

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.

Utilisant la tomographie par cohérence optique et réponse optocinétique comme lectures structurale et fonctionnelle de système visuel chez les souris et les Rats

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.

Réactivité vasculaire rétinienne évaluée par l’angiographie de la tomographie de la cohérence optique

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.

Implantation d’une fenêtre crânienne pour l’imagerie in vivo répétée chez des souris éveillées

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