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.

Nota: Tutte le procedure sugli animali sono state approvate dalla cura e l&#39;uso degli animali Comitato Istituzionale della University of Texas Health Science Center di San Antonio e sono stati coerenti con le linee guida arrivano. 1. anestetizzante per corticale Preparazione <ol><li> Posizionare il mouse in una camera di induzione con 2-3% isofluorano mescolato con l&#39;ossigeno per indurre anestesia. Osservare il calo tasso di respirazione come il mouse è indotta. Stringere la zampa del mouse per determinare se il mouse è pronto per passare al cono. Nota: il livello di anestesia è un passo fondamentale in qualsiasi preparazione in vivo e si deve prestare attenzione a non indurre un livello tale da provocare ischemia globale. </li><li> Una volta che il mouse sia sufficientemente anestetizzato, trasferire l&#39;animale alla chirurgia piattaforma / imaging e mettere il naso del mouse nella cono e applicare 1-1,2% isofluorane a mantenere uno stato anestetizzato. Assicurarsi che il mouse è sdraiata su una temperatura di coriscaldamento pad ntrolled per mantenere la temperatura corporea (37 ° C +/- 0.5 ° C) durante le procedure rimanenti. Posizionare veterinario pomata sopra gli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Monitorare la fisiologia del mouse utilizzando un sistema pulsossimetria utilizzando la clip coda o piede fornito con il sistema. Controllare che la frequenza respiratoria è mantenuta tra 50-65 atti / min. Verificare che la frequenza cardiaca rimane tra 300 e 450 bpm e la saturazione di ossigeno viene mantenuta tra 97-98% per garantire la sopravvivenza a lungo termine dell&#39;animale. </li><li> Quando il mouse è adeguatamente anestetizzato, radersi i capelli sul cranio con cesoie elettriche, rimuovere i capelli residui e pulita con betadine, seguito da un tampone di etanolo. Ripetere questa procedura fino a tre volte per garantire un ambiente sterile chirurgico. </li></ol> 2. Procedura chirurgica <ol><li> Con il cuoio capelluto completamente pulito e rasato, fare una incisione di 5 mm del cuoio capelluto del mouse per rivelare il fissu cranicores e per individuare bregma. </li><li> Usare un applicatore di cotone sterile per rimuovere eventuali residui fascia sovrastante il cranio. </li><li> Colla un anello misura in acciaio inox (Figura 1) con tessuto adesivo all&#39;osso sovrastante corteccia parietale utilizzando le coordinate stereotassica di Bregma: -1 a -3 mm e laterale: 2-4 mm. Nota: La colla definisce tipicamente entro 2 minuti dopo il posizionamento dell&#39;anello sull&#39;osso. </li><li> Fissare l&#39;anello al titolare stereotassico (Figura 2) per stabilizzare il mouse e per evitare movimenti durante l&#39;imaging. </li><li> Sotto un microscopio da dissezione chirurgica grado, lentamente praticare un tratto di 1-2 mm nel cranio con un Dremel simile velocità controllata (Meisinger 3,9 millimetri punta) facendo attenzione a mantenere il livello di zona, è forato. Raggiungere questo obiettivo con un percorso a zig-zag. Per evitare l&#39;accumulo di calore, impostare la velocità di punta al minimo e fate pause frequenti. </li><li> Quando il teschio cranica diventa shinny in apparenza, continua il diradamentodel cranio con una lama di bisturi utilizzando lo stesso andamento a zig-zag per mantenere il livello della superficie assottigliata per facilitare la rimozione regolare di strati sottili del cranio cranica. Utilizzando la punta della lama di bisturi fare piccoli tratti lineari con una leggera pressione per rimuovere gli strati sottili di osso per volta. Continuare questo fino a quando il sistema vascolare è chiaramente visibile attraverso il microscopio da dissezione. </li><li> Se lo sperimentatore perfora attraverso o rompere il cranio durante il processo di assottigliamento, l&#39;eutanasia degli animali a causa di probabili danni alla corteccia sottostante. Nota: Il cranio mouse è di circa 300 micron di spessore ed è composto da due strati sottili di osso compatto (uno esterno e uno strato interno) e uno strato di osso spugnoso a sandwich tra i due strati di osso compatto. Lo strato esterno dell&#39;osso compatto e più dell&#39;osso spugnoso vengono rimossi nella zona di foratura 5 millimetri conseguente approssimativa strato 50 micron dell&#39;osso compatto rimanente (vedere la Figura 2B). Visualizzione del sistema vascolare farà in modo che la finale intatto cranio diluito è di circa 50 micron di spessore. Il cranio è quindi ancora presente quando assottigliato a questo spessore. </li></ol> 3. Microscopio Set-up <ol><li> Utilizzare un sistema di microscopio invertito (convenzionale, confocale o sistemi a due fotoni) che ha un inverter obiettivo. Nota: È anche possibile utilizzare un microscopio verticale standard. Il fattore limitante sarà lo spazio tra la fase e gli obiettivi. Le modifiche al palco può essere necessario per realizzare questa configurazione. </li><li> Fissare la piattaforma chirurgica / imaging per una fase su misura che si trova oltre la base del microscopio. Nota: La piattaforma è realizzata con un martinetto di laboratorio per consentire il movimento verticale della piattaforma chirurgica / immagini oggetto dell&#39;obiettivo. La presa di laboratorio è montato su una targhetta fissata a quattro pali cilindrici. (Vedi figura 2). </li><li> Posizionare l&#39;inverter obiettivo contenente un 20Xobiettivo nel finestra del cranio. Utilizzare una fonte di luce esterna per trovare la finestra del cranio, cercando attraverso gli oculari del microscopio e posizionare l&#39;obiettivo nell&#39;area imaging. Nota: L&#39;area di esposizione verrà indicata dalla presenza del sistema vascolare. </li><li> Per obiettivi a base d&#39;acqua, usare fluido cerebrale spinale artificiale (aCSF) (130 mM NaCl; 30 mM KCl, 12 mM KH 2 PO 4; 200 mM NaHCO 3; HEPES 30 mM e 100 mM di glucosio) come mezzo a causa della potenziale perdite nella cavità cranica durante l&#39;imaging (Figura 3). </li></ol> 4. Rosa Bengala Dye Preparazione, gestione e induzione di corsa <ol><li> Preparare una soluzione / ml fresco 20 mg di Rosa Bengala in artificiale cerebrali liquido spinale (aCSF); filtrare e sterilizzare prima della somministrazione. Non riutilizzare o conservare la Rose del Bengala, una volta che è stato mescolato. Fare una soluzione fresca per ogni esperimento. </li><li> Dare un iniezione vena 0,1 ml coda della Rosa Bengala mentre sinscatolare finestra cranica con un laser 561 nm a garantire un&#39;adeguata iniezione della soluzione. Nota: Rosa Bengala verrà visualizzato entro 5 secondi dopo l&#39;iniezione nella vascolatura del cervello. L&#39;intero vaso deve essere riempito con Rosa Bengala. </li><li> Dopo l&#39;iniezione adeguato di Rosa Bengala colorante scegliere una nave appositamente per la trombosi base di diametro del vaso (40-80 micron) per garantire la riproducibilità di un determinato volume della lesione. Distinguere tra arterie e vene guardando la direzione del flusso sanguigno: arterie si muoveranno da diametro maggiore di vasi di diametro minore, vene spostano da piccolo a vasi di diametro maggiore. Questo è facilmente realizzabile da visualizzazione una volta Rosa Bengala viene iniettato. </li><li> Modificare l&#39;impostazione del microscopio come segue: <ol><li> Aumentare il tempo di sosta. Nota: Questo può variare a seconda del sistema di microscopio essendo utilizzato. </li><li> Aumentare la potenza del laser al 100%. </li><li> Raccogliere le immagini di sequenza temporale a 1 frame / sec con ilvelocità di scansione massima. </li></ol></li><li> Acquisire il mouse fino un coagulo stabile si forma all&#39;interno del vaso. Nota: Questo si ottiene in genere entro 5 minuti di scansione continua (vedi figura 4). </li><li> Dopo l&#39;induzione di formazione di coaguli con Rose del Bengala, togliere il mouse dalla area di esposizione di nuovo al microscopio da dissezione. Rimuovere con attenzione l&#39;anello in acciaio inox dal cranio cranico. Esaminare la finestra del cranio per qualsiasi sanguinamento. In caso di emorragia, interrompere l&#39;esperimento. </li><li> Utilizzare 6.0 monofilamento di sutura per chiudere l&#39;incisione sul cranio. Inserire pomata antibiotica lungo la linea di sutura per prevenire l&#39;infezione. Iniettare Buprenex (0,05 mg / kg) per via sottocutanea ogni 12 ore per tre giorni per la gestione del dolore. </li><li> Ritorna il mouse in una camera di recupero dopo la rimozione dal anestetico fino completamente sveglio e liberamente in movimento. </li><li> Ritorna il mouse in una gabbia pulita per ulteriori indagini in un secondo momento. </li></ol> 5. Imaging longitudinale nei giorni successivi <ol><li> Impiegare i seguenti metodi per eseguire l&#39;imaging longitudinale nei giorni successivi dopo photothrombosis. <ol><li> Anestetizzare il mouse come descritto al punto 1 dei metodi. </li><li> Dopo un&#39;adeguata anestesia riaprire il cuoio capelluto, eliminando eventuali suture residui di riaprire la pelle sovrastante il campo dell&#39;imaging precedentemente realizzato. </li><li> Usare un applicatore di cotone sterile per rimuovere eventuali residui fascia sovrastante il cranio. </li><li> Colla un anello misura in acciaio inox (Figura 1) con tessuto adesivo all&#39;osso sovrastante campo dell&#39;imaging precedente. </li><li> Fissare l&#39;anello al titolare stereotassico (Figura 2) per stabilizzare il mouse e per evitare movimenti durante l&#39;imaging. </li><li> Individuare il sistema vascolare alla base del cranio, in precedenza assottigliata. Utilizzare iniezione vena caudale di FITC-destrano per verificare la presenza del coagulo precedentemente determinati. </li><li> Utilizzare 6.0 monofilamento di sutura per chiudere la acisione sul cranio. Inserire pomata antibiotica lungo la linea di sutura per prevenire l&#39;infezione. Iniettare Buprenex (0,05 mg / kg) per via sottocutanea ogni 12 ore per tre giorni per la gestione del dolore. </li><li> Ritorna il mouse in una camera di recupero dopo la rimozione dal anestetico fino completamente sveglio e liberamente in movimento. </li></ol></li></ol> 6. Verifica della fase di aspirazione (post-mortem) <ol><li> A conclusione di uno studio, verificare stroke volume usando 2,3,5-trifeniltetrazolio cloruro (TTC) colorazione come mostrato in Figura 5. Il metodo completo può essere trovato in 12. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

La capacità di tradurre sperimentale corsa fisiopatologia da animale ad applicazione umana è stata colpita con il fallimento. Tuttavia, l&#39;utilizzo di modelli animali, come il modello photothrombosis, permette una migliore comprensione di ictus fisiopatologia e l&#39;esplorazione di nuovi approcci terapeutici per fornire neuroprotezione a seguito di un ictus. Piccoli colpi corticali e microinfarctions prodotte dal modello photothrombotic sono clinicamente rilevanti per subcliniche o &quot;silenzioso&quot; colpo <su...

La tecnica descritta consente la visualizzazione delle risposte cellulari in vivo subito dopo l&#39;induzione di Rosa Bengala photothrombosis in un mouse intatto. Rosa Bengala (4,5,6,7-tetracloro-2 &#39;, 4&#39;, 5 &#39;, 7&#39;-tetraiodofluorescein) è un colorante fotosensibile usato per indurre ictus ischemico in modelli animali (topo e ratto). A seguito di una iniezione in bolo di RB attraverso la vena della coda e la successiva illuminazione attraverso un cranio diluito con una luce laser 564 nm, un trombo è indotta causando un ictus fisiologica 1. Il metodo è stato originariamente descritto da Rosenblum e El-Sabban nel 1977, ed è stato successivamente adattato da Watson a metà 1980 1,2. In breve, Rosa Bengala viene irradiata con luce verde di eccitazione (laser 561 nm nel nostro caso), che genera la produzione di specie reattive dell&#39;ossigeno, che attiva successivamente fattore tissutale, un iniziatore della cascata coagulativa. L&#39;induzione della cascata coagulativa produce un ischemico lesione che è patologicamente rilevanti per ictus clinica 3. Corsa ha una fisiopatologia complessa a causa della interazione di molti tipi di cellule diverse, tra cui neuroni, glia, endotelio e il sistema immunitario. Scegliere la tecnica migliore per studiare un particolare processo cellulare richiede più considerazioni. Tecniche sperimentali rientrano ampiamente in una delle tre categorie: in vitro, in vivo e in silico con ciascuno che ha vantaggi e svantaggi Gli studi in vitro hanno lo svantaggio principale di rimuovere le cellule dal loro ambiente naturale e quindi non possono riprodurre effetti osservati in un intatto,. animale vivente. Nelle tecniche vivo fornire per la replica sperimentale maggiore di patologie con maggiore rilevanza traslazionale. In silico si riferisce generalmente alla modellazione al computer di una malattia o di processo cellulare, e mentre sempre più utilizzato per studiare potenziali interazioni farmacologiche per esamepio, tutte le informazioni raccolte devono ancora essere testato in cellule o tessuti viventi. Il modello ideale di ictus in ambiente di laboratorio deve dimostrare le caratteristiche patologiche simili a quelli osservati nella popolazione umana. Mentre ci sono caratteristiche fisiologiche comuni di ictus nella popolazione umana, ci sono anche molte differenze a seconda del tipo di lesione sperimentato. Corsa nella popolazione umana si verifica come piccoli o grandi occlusioni dei vasi, lesioni emorragiche, e l&#39;arteria a un&#39;arteria o cardio-embolie che si traducono in vari volumi infarto così come le differenze nei meccanismi relativi a ciascuna patologia. Il vantaggio di utilizzare modelli animali di ictus è la generazione di infarti riproducibili che imitano le caratteristiche di ictus umana. I modelli corsa più comuni animali includono occlusione utilizzando: mezzo occlusione dell&#39;arteria cerebrale (metodi incandescenza embolici o endovascolare), che i modelli distale MCAO e il modello photothrombosis. I vantaggi di und svantaggi di ciascun modello sono stati riesaminati altrove (vedi 4 e 5). Modelli globali ischemici (MCAO), mentre relativamente facile da eseguire sono meno rilevanti per ictus umano che sono modelli ictus focali. Inoltre, questi metodi sono molto variabili nell&#39;indurre riproducibili lesioni infarto cerebrale. Il modello photothrombosis è altamente riproducibile finché sperimentatore controlla loro esperimenti bene, fornendo un chiaro vantaggio rispetto ai modelli MCAO. Tuttavia, a causa microcircolo insultare il modello è stato descritto per visualizzare una penombra ischemica minima, l&#39;area in cui le cellule si pensa siano recuperabili 6,7. Inoltre, vasogenico e formazione edema citotossico possono essere indotte dopo irradiazione dell&#39;area imaging. Nonostante queste limitazioni la tecnica ha fornito una nuova visione molti processi fisiologici seguenti ictus 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.

Lo scopo di questo metodo è di indurre un ictus ischemico in modelli animali (topo e ratto) a seguito di un bolo di RB attraverso la vena della coda e successiva illuminazione di un cranio diluito con una luce laser 561 nm. Le immagini in Figura 4 mostrano la progressione della formazione del coagulo in un unico recipiente dopo irradiazione dell&#39;area a 0, 1, 1,5 e 2 min. Prima di formazione di coaguli l&#39;intero vaso è bianco a causa di flusso libero Rosa Bengala. Dopo l&#39;induzione di irradia...

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.

Rosa Bengala Photothrombosis da confocale Optical Imaging<em&gt; In Vivo</em&gt;: Un modello di singolo vaso Stroke

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 Views.  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. 

Video: 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 invasiva di misurazione ottica del metabolismo cerebrale ed emodinamica nei neonati

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

Ricerca

Medicina

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.

In vivo imaging dell&#39;angiogenesi tumorale mediante fluorescenza confocale Videomicroscopy

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 modello murino di emorragia subaracnoidea

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 Imaging ottico dei tumori cerebrali e artrite Uso fluorescente SAPC-DOPS nanovesicles

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.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

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.

Combinato<em&gt; In vivo</em&gt; Optical e μCT Imaging Monitor per infezione, infiammazione, e Bone Anatomy in una infezione ortopedica impianto in topi

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.

Chemioterapia-indotta vascolare Tossicità - in tempo reale<em&gt; In vivo</em&gt; Imaging di 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 ...

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-indotta ischemia focale come un modello di lesioni del midollo spinale nei topi

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.

Visualizzazione quantitativa di leucociti infiltrati in un modello di murina della miocardite fulminante mediante microscopia a foglio chiaro

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;approccio di campionamento a 4 nave per studi integrativi della fisiologia placentare umana<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 ...