Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.

Remarque: Toutes les procédures sur les animaux ont été approuvés par le soin et l&#39;utilisation Commission institutionnelle animale de l&#39;Université du Texas Health Science Center de San Antonio et étaient conformes aux directives arrivent. 1. L&#39;anesthésie pour corticale Préparation <ol><li> Passer la souris dans une chambre d&#39;induction avec 2-3% d&#39;isofluorane mélangé avec l&#39;oxygène pour induire une anesthésie. Observez la diminution du taux de respiration que la souris est induite. Pincez la patte de la souris pour déterminer si la souris est prête à passer à l&#39;ogive. Remarque: le niveau d&#39;anesthésie est une étape critique dans une préparation in vivo et il faut veiller à ne pas induire un niveau qui va provoquer une ischémie globale. </li><li> Une fois que la souris est suffisamment anesthésié, transférer l&#39;animal à la chirurgie / plate-forme d&#39;imagerie et placer le nez de la souris dans le cône de nez et d&#39;appliquer 1-1,2% isofluorane de maintenir un état anesthésié. Assurez-vous que la souris est allongée sur un co de températureCoussin chauffant ntrolled pour maintenir la température corporelle (37 ° C +/- 0,5 ° C) tout au long des procédures restantes. Placez vétérinaire pommade sur les yeux pour prévenir la sécheresse tandis que sous anesthésie. </li><li> Surveiller la physiologie de la souris en utilisant un système d&#39;oxymétrie de pouls à l&#39;aide du pied ou de la queue de clip fourni avec le système. Vérifiez que la fréquence respiratoire est maintenue entre 50-65 respirations / min. Vérifiez que la fréquence cardiaque reste entre 300 à 450 bpm et la saturation en oxygène est maintenue entre 97-98% pour assurer la survie à long terme de l&#39;animal. </li><li> Lorsque la souris est correctement anesthésié, de se raser les cheveux sur le crâne à l&#39;aide des tondeuses électriques, enlever les poils résiduelle et propre avec de la bétadine, suivi par un tampon imbibé d&#39;éthanol. Répétez cette procédure jusqu&#39;à trois fois pour assurer un environnement chirurgical stérile. </li></ol> 2. Procédure chirurgicale <ol><li> Avec le cuir chevelu entièrement nettoyée et rasée, faire une incision de 5 mm dans le cuir chevelu de la souris pour révéler le fissu crânienneres et de localiser bregma. </li><li> Utilisez un applicateur de coton stérile pour éliminer toute fascia restants recouvrant le crâne. </li><li> Collez une bague sur mesure en acier inoxydable (figure 1) avec de la colle de tissu à l&#39;os recouvrant le cortex pariétal en utilisant les coordonnées stéréotaxiques de Bregma: -1 à -3 mm et latérale: 2-4 mm. Remarque: La colle définit généralement dans les 2 minutes après le placement de l&#39;anneau sur l&#39;os. </li><li> Fixez la bague à un titulaire stéréotaxique (figure 2) pour stabiliser la souris et empêcher tout mouvement pendant l&#39;imagerie. </li><li> En vertu d&#39;une dissection chirurgicale de qualité microscope, percer lentement une section de 1-2 mm dans le crâne à l&#39;aide d&#39;un outil de dremel-comme la vitesse contrôlée (Meisinger 3,9 mm foret) en veillant à maintenir le niveau de la zone comme il est percé. Atteindre cet objectif en utilisant un motif en zig-zag. Pour éviter l&#39;accumulation de chaleur, régler la vitesse de forage à bas et prendre des pauses fréquentes. </li><li> Lorsque le crâne crânienne devient brillant en apparence, continuer l&#39;amincissementdu crâne en utilisant une lame de scalpel en utilisant le même motif en zig-zag afin de maintenir le niveau de surface amincie pour faciliter le retrait en douceur de couches minces de crâne crânienne. Avec la pointe de la lame de bistouri faire de petits coups de linéaires avec une légère pression pour enlever des couches minces d&#39;os à la fois. Continuer jusqu&#39;à ce que le système vasculaire est clairement visible à travers le microscope de dissection. </li><li> Si l&#39;expérimentateur perce à travers ou briser le crâne pendant le processus d&#39;amincissement, euthanasier l&#39;animal en raison de dommages susceptibles de le cortex sous-jacent. Remarque: le crâne de la souris est d&#39;environ 300 um d&#39;épaisseur et est constituée de deux couches minces d&#39;os compact (une externe et une couche interne) et une couche d&#39;os spongieux pris en sandwich entre les deux couches de l&#39;os compact. La couche externe de l&#39;os compact et plus de l&#39;os spongieux sont éliminés dans la zone de perçage 5 mm pour résultat une couche d&#39;environ 50 um d&#39;os compact restant (voir la figure 2B). Visualization de la vascularisation fera en sorte que le crâne amincie intact finale est d&#39;environ 50 um d&#39;épaisseur. Le crâne est donc toujours présent lorsque amincie à cette épaisseur. </li></ol> 3. Microscope Set-up <ol><li> Utiliser un système de microscope inversé (classique, à foyer commun ou système à deux photons) qui a un onduleur objectif. Remarque: Il est également possible d&#39;utiliser un microscope droit standard. Le facteur limitant sera l&#39;espace entre la scène et les objectifs. Les modifications apportées à la scène peuvent être nécessaires pour accomplir cette configuration. </li><li> Fixez la plate-forme chirurgicale / imagerie à un stade sur mesure qui se trouve côté de la base du microscope. Remarque: La plate-forme est faite en utilisant un vérin de laboratoire pour permettre un mouvement vertical de la plate-forme chirurgicale / imagerie sous l&#39;objectif. Le vérin de laboratoire est monté sur une plaque fixée à quatre pôles cylindriques. (Voir Figure 2). </li><li> Positionner l&#39;onduleur objectif contenant un 20Xobjectif sur la fenêtre crânienne. Utilisez une source de lumière externe pour trouver la fenêtre crânienne en regardant à travers les oculaires du microscope et de positionner l&#39;objectif dans le domaine de l&#39;imagerie. Remarque: La zone de formation d&#39;image sera noté par la présence de la vasculature. </li><li> Pour les objectifs à base d&#39;eau, en utilisant le fluide spinal cérébral artificiel (aCSF) (NaCl 130 mM; KCl 30 mM; 12 mM de KH 2 PO 4; 200 mM de NaHCO 3; 30 mM de HEPES et 100 mM de glucose) en tant que milieu en raison du risque fuite dans la cavité crânienne pendant l&#39;imagerie (Figure 3). </li></ol> 4. Rose Bengale Dye préparation, l&#39;administration et l&#39;induction de l&#39;AVC <ol><li> Préparer une solution / ml frais de 20 mg de Rose Bengale artificielle liquide céphalo-rachidien (aCSF); filtrer et stériliser avant l&#39;administration. Ne pas réutiliser ou stocker le Bengale Rose une fois qu&#39;il a été mitigée. Faire une solution fraîche pour chaque expérience. </li><li> Donner une injection dans la veine de la queue 0,1 ml de Rose Bengale tandis que sla mise en conserve fenêtre crânienne avec un laser 561 nm pour assurer l&#39;injection adéquate de la solution. Remarque: Rose Bengale sera visualisée dans les 5 secondes après l&#39;injection dans le système vasculaire du cerveau. Le navire entier doit être rempli avec Rose Bengale. </li><li> Après une injection suffisante de colorant rose Bengale choisir un récipient approprié pour une thrombose sur la base de récipient de diamètre (40 à 80 um) pour assurer la reproductibilité d&#39;un volume de la lésion donnée. Différencier les artères et les veines en regardant la direction du flux sanguin: artères vont se déplacer de plus grand diamètre à des navires de plus petit diamètre, les veines se déplacent de plus petits navires de plus grand diamètre. Ceci est facilement accompli par la visualisation une fois Rose Bengale est injecté. </li><li> Changez le réglage de microscope comme suit: <ol><li> Augmenter le temps de séjour. Remarque: Ce variera en fonction du système de microscope est utilisée. </li><li> Augmenter la puissance du laser à 100%. </li><li> Recueillir des images de séquence de temps à 1 image / s en utilisant lavitesse de balayage maximale. </li></ol></li><li> Balayer la souris jusqu&#39;à un caillot stable est formé à l&#39;intérieur de la cuve. Note: Ceci est généralement réalisé dans les 5 min de balayage continu (Voir Figure 4). </li><li> Suite à l&#39;induction de la formation de caillot avec Rose Bengale, retirer la souris à partir de la zone d&#39;imagerie revenir à la loupe binoculaire. Retirez délicatement l&#39;anneau en acier inoxydable du crâne crânienne. Examinez la fenêtre crânienne pour des saignements. Si le saignement se produit, mettre fin à l&#39;expérience. </li><li> Utilisez 6.0 monofilament suture pour fermer l&#39;incision sur le crâne. Passer onguent antibiotique sur la ligne de suture pour prévenir l&#39;infection. Injecter Buprenex (0,05 mg / kg) sous-cutanée toutes les 12 heures pendant trois jours pour la gestion de la douleur. </li><li> Retourner la souris pour une chambre de récupération après le retrait de l&#39;anesthésie jusqu&#39;à pleinement éveillé et se déplacer librement. </li><li> Retour de la souris dans une cage propre pour complément d&#39;enquête à une date ultérieure. </li></ol> 5. Imagerie longitudinale les jours suivants <ol><li> Employer les méthodes suivantes pour effectuer l&#39;imagerie longitudinale les jours suivants post-photothrombosis. <ol><li> Anesthésier la souris comme décrit dans la section 1 des méthodes. </li><li> Après une anesthésie adéquate ré-ouvrir le cuir chevelu en supprimant toutes les sutures résiduelles de rouvrir la peau recouvrant le domaine de l&#39;imagerie percé précédemment. </li><li> Utilisez un applicateur de coton stérile pour éliminer toute fascia restants recouvrant le crâne. </li><li> Collez une bague sur mesure en acier inoxydable (figure 1) avec de la colle de tissu à l&#39;os recouvrant le domaine de l&#39;imagerie précédente. </li><li> Fixez la bague à un titulaire stéréotaxique (figure 2) pour stabiliser la souris et empêcher tout mouvement pendant l&#39;imagerie. </li><li> Localisez le système vasculaire sous-jacente du crâne préalablement amincie. Utilisation injection dans la veine de la queue de FITC-dextrane pour vérifier la présence du caillot précédemment induit. </li><li> Utilisez 6.0 monofilament suture pour fermer la danscision sur le crâne. Passer onguent antibiotique sur la ligne de suture pour prévenir l&#39;infection. Injecter Buprenex (0,05 mg / kg) sous-cutanée toutes les 12 heures pendant trois jours pour la gestion de la douleur. </li><li> Retourner la souris pour une chambre de récupération après le retrait de l&#39;anesthésie jusqu&#39;à pleinement éveillé et se déplacer librement. </li></ol></li></ol> 6. Vérification de l&#39;induction de l&#39;AVC (post-mortem) <ol><li> À la conclusion d&#39;une étude, vérifier que le volume de la course en utilisant 2,3,5-triphényltétrazolium chlorure (TTC) coloration comme indiqué sur la Figure 5. La méthode complète peut être trouvée dans 12. </li></ol>

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Les auteurs déclarent qu&#39;ils ont aucun intérêt financier concurrentes.

La capacité à traduire expérimentale course physiopathologie de l&#39;animal à l&#39;application humaine a été en proie à l&#39;échec. Cependant, l&#39;utilisation de modèles animaux, tels que le modèle de photothrombosis, permet d&#39;améliorer la compréhension de la physiopathologie de la course et l&#39;exploration de nouvelles approches thérapeutiques pour fournir une neuroprotection après un AVC. Petits coups et microinfarctions produites par le modèle photothrombotique corticales sont cliniquement p...

La technique décrite permet la visualisation de la réponse cellulaire in vivo, immédiatement après l&#39;induction de Rose Bengale photothrombosis dans une souris intactes. Rose Bengale (4,5,6,7-tétrachloro-2 &#39;, 4&#39;, 5 &#39;, 7&#39;-tétraiodofluorescéine) est un colorant photosensible utilisée pour induire un AVC ischémique dans des modèles animaux (souris et le rat). Suite à une injection en bolus de RB par la veine de la queue et l&#39;illumination ultérieure par un crâne amincie avec une lumière laser de 564 nm, un thrombus est induit provoque un accident vasculaire cérébral physiologique 1. La méthode a été initialement décrit par Rosenblum et El-Sabban, en 1977, et a ensuite été adapté par Watson dans les milieu des années 1980 1,2. En bref, le rose Bengale est irradié avec de la lumière verte d&#39;excitation (laser 561 nm dans notre cas), ce qui génère la production d&#39;espèces réactives de l&#39;oxygène, qui active ensuite le facteur tissulaire, un initiateur de la cascade de coagulation. L&#39;induction de la cascade de coagulation produit une ischémie lesion qui est pathologiquement pertinentes pour AVC clinique 3. AVC a une physiopathologie complexe en raison de l&#39;interaction de nombreux types de cellules différents, y compris les neurones, les cellules gliales, l&#39;endothélium et le système immunitaire. Le choix de la meilleure technique pour étudier un processus cellulaire particulier nécessite plusieurs considérations. Techniques expérimentales retrouvent globalement dans une des trois catégories: in vitro, in vivo et in silico chacun ayant des avantages et des inconvénients Des études in vitro ont l&#39;inconvénient principal de l&#39;élimination des cellules de leur milieu naturel et peuvent donc ne pas reproduire les effets observés dans une intacte,. animal vivant. Dans les techniques in vivo Pour renforcer la réplication expérimentale d&#39;états pathologiques avec une importance accrue de translation. Dans silico se réfère généralement à la modélisation informatique d&#39;une maladie ou d&#39;un processus cellulaire, et alors que de plus en plus utilisé pour étudier les interactions médicamenteuses potentielles pour examenple, toute information recueillie doit encore être testée dans des cellules ou des tissus vivants. Le modèle idéal de la course dans le cadre d&#39;un laboratoire doit démontrer les caractéristiques pathologiques similaires à ceux observés dans la population humaine. Bien qu&#39;il existe des caractéristiques physiologiques communes de l&#39;AVC dans la population humaine, il ya aussi beaucoup de différences selon le type de dommage subi. AVC dans la population humaine se produit que de petites ou grandes occlusions des vaisseaux, des lésions hémorragiques, et l&#39;artère à artère ou cardio-embolies qui se traduisent par des volumes d&#39;infarctus variées ainsi que des différences dans les mécanismes liés à chaque pathologie. L&#39;avantage de l&#39;utilisation de modèles de course des animaux est la génération des infarctus reproductibles qui imitent les caractéristiques d&#39;un AVC humaine. Les modèles les plus courants de course d&#39;origine animale comprennent occlusion de l&#39;artère en utilisant: occlusion de l&#39;artère cérébrale moyenne (méthodes de filaments ou emboliques endovasculaires) qui distale MCAO modèles et le modèle de photothrombosis. Les avantages d&#39;uninconvénients de chaque modèle d ont été examinés ailleurs (voir 4 et 5). Les modèles globaux ischémiques (MCAO), tout en étant relativement facile à réaliser sont moins pertinents pour la course humaine que sont les modèles de course focaux. En outre, ces procédés sont très variables dans l&#39;induction de lésions d&#39;infarctus cérébral reproductibles. Le modèle de photothrombosis est hautement reproductible aussi longtemps que l&#39;expérimentateur contrôle leurs expériences bien, offrant un net avantage sur les modèles MCAO. Toutefois, en raison de la microvascularisation insulte le modèle a été décrit pour afficher une pénombre ischémique minimale, la zone où les cellules sont pensés pour être récupérable 6,7. En outre, l&#39;oedème vasogénique et la formation de l&#39;oedème cytotoxique peut également être induites après irradiation de la zone d&#39;imagerie. Malgré ces limites de la technique a fourni un nouvel aperçu de nombreux processus physiologiques suivants course 8, 9, 10, 11.

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rose bengal photothrombosis by confocal optical imaging in vivo: a model of single vessel stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Le but de cette méthode est d&#39;induire un AVC ischémique dans des modèles animaux (souris et rats) après une injection en bolus de RB par la veine de la queue et l&#39;illumination subséquente d&#39;un crâne amincie avec une lumière laser de 561 nm. Les images de la figure 4 montrent l&#39;évolution de la formation de caillots à l&#39;intérieur d&#39;une seule cuve après irradiation de la zone à 0, 1, 1,5 et 2 min. Avant la formation de caillots tout le navire est blanche due à écouleme...

Watch this Scientific Journal Video about Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke at JoVE.com

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rose Bengale Photothrombosis par imagerie optique confocal<em&gt; In Vivo</em&gt;: Un modèle de l&#39;unité des maladies du navire

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

rose-bengal-photothrombosis-confocal-optical-imaging-vivo-model

Research

JoVE Journal

Medicine

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

14.9K vues.  The University of Texas Health Science Center San Antonio. Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection. 

Vidéo: Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Non-invasive de mesure optique du métabolisme cérébral et hémodynamique chez les nourrissons

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Recherche

Médecine

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Dans l&#39;imagerie in vivo de angiogenèse tumorale en utilisant la fluorescence confocale vidéomicroscopie

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Un modèle murin d&#39;hémorragie sous-arachnoïdienne

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo L&#39;imagerie optique de tumeurs cérébrales et l&#39;arthrite Utilisation fluorescent SAPC-DOPS nanovésicules

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combiné<em&gt; In vivo</em&gt; Optique et μCT imagerie pour surveiller l&#39;infection, l&#39;inflammation, et Bone Anatomy dans une infection orthopédique Implant souris

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Induits par la chimiothérapie vasculaire Toxicité - en temps réel<em&gt; In vivo</em&gt; Imagerie de déficience navire

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Photothrombosis induite par ischémie focale comme un modèle de lésion médullaire chez la souris

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualisation quantitative de l&#39;infiltration de leucocytes dans un modèle murin de myocardite fulminante par microscopie à feuilles lumineuses

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

L&#39;approche d&#39;échantillonnage à 4 navires pour les études intégratives de la physiologie du plaidoyer humain<em&gt; In vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...