This study was supported by the NIH grant NS074559 (to C. K.). We thank all collaborators who contributed to our research articles that the present methodology report is based upon.

Questo protocollo è approvato dalla cura e l&#39;uso Comitato Istituzionale Animal (IACUC) della Emory University e segue il National Institutes of Health Linee guida per la cura e l&#39;uso di animali da laboratorio. 1. Setup <ol><li> Preparare il letto chirurgico su pad riscaldamento collegata con pompa di calore a 37 ° C per almeno 15 minuti prima della chirurgia. Posizionare un paranuca con il barile di 3 ml della siringa sul letto chirurgico. Preparare il gas anestesia con 2% isoflurano in aria medicale. </li><li> Preparare pinze autoclavato, forbici, porta micro aghi, emostatico, tamponi di cotone e suture. Preparare colla tissutale e unguento oculare. </li><li> Impostare il sistema ipossia e termoregolatori con lampada di riscaldamento e sonda rettale. Preparare gas ipossia con 2% isoflurano nel 7,5% O 2 equilibrato per il 92,5% di N 2. </li><li> Un&#39;ora prima dell&#39;intervento chirurgico, i topi sono analgesized mediante iniezione sottocutanea di un lento rilascio Meloxicam (4.0 mg / kg). </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 2. Transient cerebrale ipossia-ischemia (Figura 1B) <ol><li> Anestetizzare 10-13 settimane di età C57BL maschio / 6 topi di peso 22-30 g nella camera di induzione dell&#39;anestesia con il 3% isoflurano finché l&#39;animale non risponde ai piedi stretta, e quindi rimuovere i peli sul collo a destra con un rasoio elettronico. </li><li> Posizionare i mouse sul letto chirurgico legato 2% isoflurano in aria medicale a portata di 2 l / min. Arti anteriori Sicuro stese lungo paranuca ai lati con del nastro medico. </li><li> Pulire il sito chirurgico per incisione con betadine seguito da alcol e poi tamponi di cotone. </li><li> Sotto il microscopio dissezione, fare un&#39;incisione a 0,5 centimetri destro del collo dell&#39;utero con una pinza rette e un micro forbici circa 0,2 cm lateralmente dalla pelle linea mediana. </li><li> Utilizzare un paio di belle pinze dentellate per separare la fascia e il tessuto per esporre la destra carotide comune (RCCA). Separare con cura il RCCA dal nervo vagale usando un paio di pinza sottile liscia. </li><li> Live nodo due pretagliati 5-0 sutura di seta (rilasciabile) sul RCCA, e poi cucire la pelle utilizzando 4-0 Nylon monofilamento di sutura (Figura 1C). </li><li> Applicare una pomata occhio su entrambi gli occhi per prevenire la secchezza. </li><li> Trasferire rapidamente i topi al sistema ipossia e mettere il naso e la bocca in maschera facciale con 2% isoflurano in 7,5% O 2 a portata di 0.5-1 L / min per 30 min. <ol><li> Durante ipossia, utilizzare termoregolatori con lampade riscaldanti per controllare la temperatura rettale a 37,5 ± 0,5 ° C. Monitorare la frequenza respiratoria a 80-120 respiri / min. Il mantenimento della temperatura corporea superiore a 37 ° C durante l&#39;ipossia è importante creare infarto cerebrale coerente. Bassa frequenza respiratoria di solito accade dopo 20 min ipossia. Rimuovere la maschera facciale e consentire il normale alimentazione di aria se la frequenza respiratoria scende al di sotto di 40. Questo richiede 1-2 minuti e non conta nella durata ipossia 30 min. </li></ol></li><li> Dopo ipossia, trasferiretopi per un letto chirurgica e rilasciare i due punti di sutura da RCCA. Chiudere la ferita utilizzando colla tissutale e quindi tornare topi per gabbia. Escludere gli animali se entrambi due nodi vivi sono inaspettatamente rilasciati dopo ipossia. </li><li> Monitorare i topi per 5-10 minuti per recuperare da ipossia e anestesia. Posizionare il cibo bagnata nella gabbia e restituirlo alla struttura di cura degli animali. Nota: Gli animali che presentano lievi a comportamenti circuitazione grave a 24 ore dopo Thi sono correlati con infrazione del cervello. La maggior parte degli animali con sintomi di sequestro muoiono prima della timepoint 24 ore dopo Thị. </li></ol> 3. Laser Speckle Contrast Imaging Nota: Anche se questa non è una procedura essenziale del modello THI, un sistema di imaging contrasto bidimensionale speckle laser 16 può essere utilizzato per caratterizzare i cambiamenti del flusso sanguigno cerebrale (CBF) durante o dopo ipossia-ischemia transitoria. Per documentare le alterazioni del CBF sotto THI, registrare subito dopo la step 2.6. In alternativa, per confrontare recupero CBF dopo THI insulto, queste procedure possono essere eseguite dopo la fase 2.10. <ol><li> Inserire un topo anestetizzato in posizione prona ed eseguire da 1 cm a lunga incisione mediana sul cuoio capelluto con il cranio esposto, ma non aperto. </li><li> Monitorare CBF in entrambi gli emisferi cerebrali sotto un imager flusso di sangue, secondo il protocollo del produttore e avviare la registrazione del flusso ematico cerebrale subito dopo l&#39;intervento chirurgico CCAO (passo 2,6). Proseguire per 50 min. </li><li> Mostra immagine CBF con unità arbitrarie in una tavolozza di 16 colori e analizzare in tempo reale le regioni selezionate utilizzando il software MoorFLPI seguendo le istruzioni del produttore (Figura 2). </li><li> Dopo aver registrato l&#39;immagine CBF, chiudere il cuoio capelluto con colla tissutale e rispedire l&#39;animale verso la gabbia. </li></ol> Amministrazione 4. tPA <ol><li> Iniettare animali durante la vena della coda con il solvente o 10 mg / kg ricombinante tPA (220-300 &amp;# 956; l di 1 mg / m tPA) a 0.5, 1 o 4 ore dopo tCCAo più ipossia (Figura 4). </li></ol> 5. Cervello rilevamento dei danni con diverse opzioni Nota: Per raccogliere i campioni di cervello, eutanasia i topi a 1, 4 o 24 ore dopo Thị. <ol><li> Metodo Eseguire quantificazione del volume di infarto in vivo cloruro di 2,3,5-trifeniltetrazolio (TTC) a 24 ore dopo THI insulto come descritto precedente. 17 <ol><li> Intraperitoneale iniettare animali con 1,4 M soluzione di mannitolo (~ 0,1 ml / g di peso corporeo) 30 minuti prima di perfusione transcardial. Topi profumato transcardial con PBS seguita da 10 ml di 2% TTC. </li><li> Rimuovere il cervello degli animali con strumenti chirurgici dopo 10 min e riporre in paraformaldeide 4% per la fissazione durante la notte e la sezione in spessore di 1 mm con un vibratome. </li><li> Scatta una serie di quattro diapositive cervello sezionati al microscopio digitale e quantficare il volume dell&#39;infarto come il rapporto tra l&#39;area infartuata (area bianca sul lato destro) alla zona della integro, nell&#39;emisfero controlaterale utilizzando il software ImageJ. </li></ol></li><li> In alternativa, effettuare la formazione di trombosi mediante immunofluorescenza a 1 ora dopo Thi insulto. <ol><li> Congelare il cervello fisso in OCT e la sezione del cervello a 12 micron di spessore con un criostato. </li><li> Incubare il vetrino di cervello di coniglio con anticorpo anti-fibrinogeno (1: 100) in seguito da capra anti-coniglio Alexa fluo 488 dye (1: 200) per osservare la fluorescenza su un microscopio a fluorescenza. </li></ol></li><li> In alternativa, eseguire nave ostruzione da iniezione vena della coda di 100 ml 2% Evans colorante blu a 4 ore dopo Thị insulto. <ol><li> Euthanize i topi e rapidamente tagliare la testa per rimuovere cervelli in paraformaldeide al 4% dopo l&#39;iniezione blu Evans. Nota: Ci vogliono 5-10 min per Evans blu circolazione con il colore blu sia anteriori e hind arti. </li><li> Seziosu cervelli fissi a 100 micron di spessore con un microtomo scorrimento e osservare la fluorescenza usando un filtro di emissione 680 nm su un microscopio a fluorescenza. </li></ol></li></ol>

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L&#39;ictus è un problema di salute importante di crescente importanza per ogni società con un invecchiamento della popolazione. A livello globale, l&#39;ictus è la seconda causa di morte, con una stima di 5,9 milioni di eventi fatali nel 2010, pari al 11,1% di tutti i decessi 18. L&#39;ictus è anche la terza principale causa di anni di vita disability-adjusted (DALY) hanno perso a livello mondiale nel 2010, passando dalla quinta posizione nel 1990 19. Questi dati epidemiologici evidenziano la ...

La trombolisi e ricanalizzazione è la più efficace terapia dell&#39;ictus ischemico acuto nella pratica clinica 1. Tuttavia, la maggior parte delle ricerche neuroprotezione preclinica è stata eseguita in un modello transitorio meccanico ostruzione (endoluminale sutura occlusione dell&#39;arteria cerebrale media) che produce un rapido recupero del flusso sanguigno cerebrale dopo la rimozione della occlusione vascolare e mostra poco o nessun benefici di tPA trombolisi. È stato suggerito che la scelta dubbia di modelli ictus contribuito, almeno in parte, alla difficoltà nel tradurre terapia neuroprotettiva ai pazienti 2,3. Quindi, vi è una crescente richiesta per l&#39;impiego di modelli di ictus tromboembolico tPA-reattiva nella ricerca preclinica, ma tali modelli hanno anche problemi tecnici (vedi Discussione) 4-7. Qui si descrive un nuovo modello di ictus trombotico in base transitorio ipossico-ischemica (THI) insulto unilaterale e le sue risposte alla terapia endovenosa tPA 8. Il modello di ictus THI è stato sviluppato sulla base della procedura di Levine (legatura permanente della carotide comune unilaterale seguita da esposizione ad ipossia transitoria in una camera), che è stato inventato per esperimenti con ratti adulti nel 1960 9. La procedura originale Levine caduta nell&#39;oscurità perché ha prodotto solo danni al cervello variabile, ma lo stesso insulto causato neuropatologia coerente in cuccioli di roditori quando fu reintrodotta da Robert Vannucci e dai suoi colleghi come un modello di neonatale ipossico-ischemica encefalopatia (HIE) nel 1981 10. Negli ultimi anni, un po &#39; investigatori ri-adattato il modello Levine-Vannucci di topi adulti regolando la temperatura della camera ipossica 11. E &#39;plausibile che le lesioni cerebrali incoerenti nella procedura originale Levine possono derivare da sbalzi di temperatura del corpo dei roditori adulti nella camera ipossica. Per verificare questa ipotesi, abbiamo modificato la procedura di Levine con la somministrazione di gas ipossicoattraverso una maschera, mantenendo la temperatura al cuore roditore a 37 ° C sul tavolo operatorio 12. Come previsto, il controllo della temperatura corporea severi notevolmente aumentato la riproducibilità della patologia cerebrale HI-indotta. L&#39;insulto HI innesca anche la coagulazione, l&#39;autofagia, e grigi e lesioni 13 bianco-materia. Altri ricercatori hanno usato anche il modello HI a indagare post-ictus risposte infiammatorie 14. Una caratteristica unica del modello ictus HI è che ricalca triade di Virchow di formazione di trombi, compresa la stasi del flusso di sangue, danno endoteliale (ad esempio a causa di stress ossidativo HI-indotta), e ipercoagulabilità (attivazione piastrinica HI-indotta) ( Figura 1A) 15. In quanto tale, il modello HI può catturare alcuni meccanismi fisiopatologici rilevanti per ictus ischemico del mondo reale. Con questa idea in mente, abbiamo perfezionato ulteriormente il modello HI con legatura reversibile delle Nazioni Uniteilateral carotide comune (quindi per creare un transitorio HI insulto), e testato le sue risposte a tPA trombolisi con o senza edaravone. Edaravone è un scavenger di radicali liberi già approvato in Giappone per il trattamento di ictus ischemico entro 24 ore di insorgenza 9. I nostri esperimenti hanno dimostrato che il più breve 30 minuti transitorio HI innesca infarto trombotico, e che il trattamento combinato tPA-edaravone conferisce benefici sinergici 8. Qui si descrivono le procedure chirurgiche dettagliate e considerazioni metodologiche del modello THI, che possono essere utilizzati per ottimizzare i trattamenti riperfusione di ictus ischemico acuto.

<table><tbody><tr><td>adult male mice</td><td>Charles River</td><td>C57BL/6&amp;nbsp;</td><td>10-13 weeks old (22-30 g)</td></tr><tr><td>Mobile Laboratory Animal Anesthesia System</td><td>VetEquip</td><td>901807</td><td>anesthesia</td></tr><tr><td>Medical air (Compressed) air tank</td><td>Airgas</td><td>UN1002</td><td>anesthesia</td></tr><tr><td>Isoflurane</td><td>Piramal Healthcare</td><td>NDC 66794-013-25</td><td>anesthesia</td></tr><tr><td>Multi-Station Lab Animal AnesthesiaSystem</td><td>Surgivet</td><td>V703501</td><td>hypoxia system</td></tr><tr><td>7.5% O&lt;sub&gt;2&lt;/sub&gt; balanced by 92.5% N&lt;sub&gt;2&lt;/sub&gt; tank</td><td>Airgas</td><td>UN1956</td><td>hypoxia system</td></tr><tr><td>Temperature Controller with heating lamp&amp;nbsp;</td><td>Cole Parmer&amp;nbsp;</td><td>EW-89000-10</td><td>temperature controllers</td></tr><tr><td>Rectal probe</td><td>Cole Parmer&amp;nbsp;</td><td>NCI-00141PG</td><td>temperature controllers</td></tr><tr><td>Dissecting microscope&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td>SZ40</td><td>surgical setup</td></tr><tr><td>Heat pump with warming pad</td><td>Gaymar&amp;nbsp;</td><td>TP700</td><td>surgical setup</td></tr><tr><td>Fine curved forceps (serrated)</td><td>FST</td><td>11370-31</td><td>surgical instrument</td></tr><tr><td>Fine curved forceps (smooth)</td><td>FST</td><td>11373-12</td><td>surgical instrument</td></tr><tr><td>micro scissors</td><td>FST</td><td>15000-03</td><td>surgical instrument</td></tr><tr><td>micro needle holders</td><td>FST</td><td>12060-01</td><td>surgical instrument</td></tr><tr><td>Halsted-Mosquito hemostats</td><td>FST</td><td>13008-12</td><td>surgical instrument</td></tr><tr><td>5-0 silk suture&amp;nbsp;</td><td>Harvard Apparatus</td><td>624143</td><td>surgical supplies</td></tr><tr><td>4-0 Nylon monofilament suture</td><td>LOOK</td><td>766B</td><td>surgical supplies</td></tr><tr><td>Tissue glue</td><td>Abbott Laboratories</td><td>NC9855218</td><td>surgical supplies</td></tr><tr><td>Puralube Vet ointment</td><td>Fisher</td><td>NC0138063&amp;nbsp;</td><td>eye dryness prevention&amp;nbsp;</td></tr><tr><td>MoorFLPI-2 blood flow imager</td><td>Moor</td><td>780-nm laser source</td><td>Laser Speckle Contrast Imaging</td></tr><tr><td>Mannitol</td><td>Sigma</td><td>M4125</td><td>&lt;i&gt;in vivo&lt;/i&gt; TTC</td></tr><tr><td>2,3,5-triphenyltetrazolium chloride (TTC)&amp;nbsp;</td><td>Sigma</td><td>T8877</td><td>&lt;i&gt;in vivo&lt;/i&gt; TTC</td></tr><tr><td>Vibratome</td><td>Stoelting</td><td>51425</td><td>brain section for &lt;i&gt;in vivo&lt;/i&gt; TTC&amp;nbsp;</td></tr><tr><td>Digital microscope</td><td>Dino-Lite</td><td>AM2111</td><td>whole-brain imaging</td></tr><tr><td>O.C.T compound</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>goat anti-rabbit Alexa Fluro 488</td><td>Invitrogen</td><td>A11008</td><td>Immunohistochemistry</td></tr><tr><td>Cryostat</td><td>Vibratome</td><td>ultrapro 5000</td><td>brain section for IHC</td></tr><tr><td>Evans blue</td><td>Sigma</td><td>E2129</td><td>Detecting vascular perfusion</td></tr><tr><td>Microtome</td><td>Electron Microscopy Sciences</td><td>5000</td><td>brain section for histology</td></tr><tr><td>Avertin (2, 2, 2-Tribromoethanol)</td><td>Sigma</td><td>T48402</td><td>euthanasia</td></tr><tr><td>Fluorescent microscope</td><td>Olympus</td><td>DP73</td><td></td></tr><tr><td>Meloxicam SR</td><td>ZooPharm</td><td></td><td>NSAID analgesia</td></tr></tbody></table>

a thrombotic stroke model based on transient cerebral hypoxia-ischemia

Ricerca Stroke ha sopportato molte battute d&#39;arresto nel tradurre terapie neuroprotettive nella pratica clinica. Al contrario, la terapia del mondo reale (tPA trombolisi) raramente produce benefici in modelli sperimentali meccanici basati occlusione-, che dominano la ricerca preclinica ictus. Questa scissione tra la panchina e comodini suggerisce la necessità di impiegare modelli tPA-reattiva nella ricerca preclinica ictus. A tal fine, un modello semplice e tPA-reattiva ictus trombotico è inventato e descritto qui. Questo modello si compone di occlusione transitoria dell&#39;arteria carotide comune unilaterale e consegna del 7,5% di ossigeno attraverso una maschera in topi adulti per 30 minuti, mantenendo la temperatura rettale animale a 37,5 ± 0,5 ° C. Sebbene legatura reversibile della carotide unilaterale o ipossia ciascun soppressa flusso sanguigno cerebrale solo transitoriamente, la combinazione di entrambi insulti causato durata deficit riperfusione, fibrina e piastrine deposizione e grande Infarct nel territorio cerebrale mezzo dell&#39;arteria fornito. Importante, coda vena iniezione di tPA ricombinante a 0,5, 1, o 4 ore post-THI (10 mg / kg) disponibile riduzione tempo-dipendente delle dimensioni mortalità e infarto. Questo nuovo modello di ictus è semplice e può essere standardizzata attraverso laboratori per confrontare i risultati sperimentali. Inoltre, induce trombosi senza craniectomy o introdurre emboli preformati. Alla luce di questi meriti unici, il modello THI è un&#39;utile aggiunta al repertorio della ricerca preclinica ictus.

Bidimensionale imaging a contrasto di speckle laser (LSCI) 16 è stato utilizzato per confrontare le alterazioni del flusso ematico cerebrale (CBF) per 30 min transitoria occlusione carotidea unilaterale (tCCAO), 30 minuti di esposizione ad ipossia (7,5% di ossigeno), e 30 min unilaterale carotidea legatura sotto ipossia (THI). Questo esperimento ha rivelato che tCCAO sotto normossia soppressa la CBF sulla carotide legatura emisfero ~ 50% del valore basale, che recuperato rapidamente al di sopra dell&#39;85% ...

Watch this Scientific Journal Video about A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia at JoVE.com

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Un trombotica Stroke modello basato sulla transitoria cerebrale ipossia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

a-thrombotic-stroke-model-based-on-transient-cerebral-hypoxia-ischemia

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

14.7K Views.  The Emory University School of Medicine. Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients. 

Video: A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Il modello di encefalopatia ipossico ischemica di ischemia perinatale

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Ricerca

Biologia

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Ischemia Photothrombotic: un modello fotochimico lesione corticale minimamente invasiva e riproducibile per mouse Studi Stroke

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Medicina

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Corsa di modellazione in Mouse: Coagulazione permanente dell&#39;arteria cerebrale media distale

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Transient Medio occlusione dell&#39;arteria cerebrale Modello di Stroke neonatale nei ratti P10

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

Un modello di ictus fototrombotico arricchito di fibrina e sensibile al tPA

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Neuroscienze

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Modellazione dell'ictus nei topi: lesioni corticali focali da fototrombosi

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Modellazione dell'ictus nei topi: occlusione transitoria dell'arteria cerebrale media attraverso l'arteria carotide esterna

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Un modello di ischemia cerebrale transitoria mediante occlusione dell'arteria cerebrale media

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Autore in primo piano: Valutazione del danno da ictus ischemico attraverso il modello di occlusione dell'arteria cerebrale media 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Modello transitorio di occlusione dell'arteria cerebrale media dell'ictus

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

Un trombotica Stroke modello basato sulla transitoria cerebrale ipossia-ischemia

Riepilogo

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Corsa di modellazione in Mouse: Coagulazione permanente dell'arteria cerebrale media distale

Transient Medio occlusione dell'arteria cerebrale Modello di Stroke neonatale nei ratti P10

Un modello di ictus fototrombotico arricchito di fibrina e sensibile al tPA

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Modello transitorio di occlusione dell'arteria cerebrale media dell'ictus

Modello transitorio di occlusione dell'arteria cerebrale media dell'ictus