Ringraziamo Annalisa Buffo per penetranti suggerimenti e commenti, e Maurizio Grassano, Marina Boido e Ermira Pajaj per le riprese. Questo lavoro è stato finanziato dal 7 ° PQ-MC-214.003-2 (Marie Curie Initial Training Network AXREGEN) e la Compagnia di San Paolo, progetto gliarep.

1. Pre-chirurgia <ol><li> Pesare Rosa Bengala in una provetta da 1,5 ml e sciogliere in soluzione salina sterile fino a raggiungere una concentrazione finale di 15 mg / ml. Filtro sterilizzare attraverso un filtro 0,2 micron e conservare al buio a temperatura ambiente fino a due mesi. </li><li> Sterilizzare tutti gli strumenti chirurgici in autoclave. L&#39;area chirurgica deve essere sterilizzata meno di un&#39;ora prima di iniziare l&#39;intervento chirurgico. </li><li> Registrare il peso del corpo del mouse per regolare la dose di Rosa Bengala da iniettare. Abbiamo iniettato 10 ml / g di peso animale in topi femmina CD1 12 settimane nel presente protocollo. La quantità di Rosa Bengala necessario per produrre la dimensione desiderata lesione corticale può essere facilmente determinato in una serie separata di esperimenti preliminari testando diversi dosaggi (tipicamente 2 microlitri / g, 5 microlitri / go 10 ml / g di peso corporeo). Si noti che la quantità di Rosa Bengala da iniettare è fortemente dipendente dalle condizioni sperimentali, in particolare il tipo di sorgente luminosautilizzato e la durata di esposizione alla luce. In questo studio, 10 ml / g (150 mcg / g) della dose è risultato essere necessario per indurre photothrombosis con l&#39;esposizione alla luce per 15 minuti, mentre altri gruppi hanno riferito che o 50 mcg / g 6,8 e 100 mcg / g 10 iniezione intraperitoneale era sufficiente per indurre una lesione photothrombotic. </li></ol> 2. Procedura di Anestesia <ol><li> Anestetizzare topi con isoflurano in una camera di induzione trasparente (3,5-4% per l&#39;induzione, 1,5-2% per il mantenimento) in 50% (v / v) oxygen/50% (v / v) diazoto miscela di gas monossido. Anestesia gassosa permette un rapido risveglio degli animali e il livello di gas anestetico può essere regolata facilmente. Alternativamente, i topi possono anche essere anestetizzati con una miscela ketamina-xilazina. </li><li> Quando si raggiunge in profondità dell&#39;anestesia, rimuovere l&#39;animale anestetizzato dalla camera di induzione, posizionarlo nel frame stereotassico e mantenere l&#39;anestesia utilizzando una maschera viso. Regolare la isoflurane della dose per raggiungere un livello adeguato di anestesia. Monitorare la frequenza respiratoria durante tutta la procedura e assicurarsi che sia costante (40-60 respiri al minuto). </li><li> Utilizzare pizzichi punta in modo da garantire che l&#39;animale è profondamente anestetizzati. </li><li> Applicare pomate oftalmiche, al fine di evitare gli occhi si secchi. </li><li> Inserire delicatamente la sonda rettale per monitorare la temperatura durante le procedure chirurgiche. Impostare il pad riscaldamento retroazione controllata associato per il mantenimento della temperatura corporea topo a 37 ± 0,5 ° C. </li></ol> 3. Chirurgia per illuminazione della zona di destinazione <ol><li> Shave il cuoio capelluto del mouse con un rasoio elettrico. </li><li> Fissare saldamente la testa e inserire le barre di auricolari in meato esterno. Siate attenti a non danneggiare i timpani. </li><li> Disinfettare la pelle in superficie con colpi di etanolo al 70% e betadine utilizzando tamponi di cotone alternati. </li><li> Utilizzare un bisturi per fare un&#39;incisione lungo la linea mediana dall&#39;occhio level fino al collo. Applicare divaricatori della pelle per mantenere il cranio esposto. </li><li> Ritrarre delicatamente il periostio ai bordi del cranio con un bisturi e lasciare la superficie del cranio asciugare utilizzando tamponi di cotone sterili. Identificare il bregma e lambda. Posizionare una micropipetta di vetro sul bregma come punto di riferimento, quindi spostarlo le coordinate della regione di interesse. La regione di interesse scelto per questo articolo è approssimativamente roundly sagomata, centrata circa 2 mm lateralmente al bregma, e copre una superficie di circa 30 mm 2, che comprende gran parte della corteccia sensomotoria secondo il topo cervello atlante da Franklin e Paxinos 11. </li><li> Segnare la posizione di interesse con punti di riferimento e di porre in fibra ottica a stretto contatto con la superficie del cranio per evitare dispersione della luce, ma attenzione a non esercitare una pressione su di esso. L&#39;area illuminata può essere limitata applicando sul cranio una maschera con piccola apertura o un tappo sulla punta della fibra ottica guide. </li></ol> 4. Rosa Bengala iniezione e attivazione <ol><li> Caricare la soluzione Rosa Bengala in una siringa da 1 ml e calcolare l&#39;importo da iniettare in base a una dose di 10 ml / g di peso corporeo. </li><li> Procedere ad una iniezione intraperitoneale lenta. </li><li> Lasciate che la diffusa colorante ed entrare nel flusso sanguigno. Dopo 5 minuti, accendere l&#39;illuminatore a luce fredda. Evitare l&#39;illuminazione dell&#39;animale da qualsiasi altra sorgente di luce. Abbiamo usato illuminatore da 150 W intensità in fibra ottica. </li><li> Dopo 15 min di illuminazione, interrompere l&#39;esposizione alla luce e suturare la ferita. Cinque minuti di illuminazione già producono miocardico e 10 min dovrebbe essere sufficiente per ottenere un effetto massimo 12. Per ridurre al minimo la variabilità, abbiamo scelto di illuminare per 15 min. </li></ol> 5. Suturare <ol><li> Rimuovere i divaricatori pelle e applicare la soluzione salina sterile per evitare la disidratazione. </li><li> Chiuda la ferita utilizzando inverso needl taglioE e seta o filo di sutura di nylon. </li><li> Interrompere per anestesia, rimuovere con attenzione il mouse dal apparato stereotassico e metterlo su un rilievo di riscaldamento pre-riscaldato fino a quando non è completamente sveglio, per poi tornare alla sua gabbia. La temperatura corporea deve essere monitorata attentamente tutta la procedura per limitare la variabilità dell&#39;estensione dell&#39;infarto. In funzione della concentrazione e la via di somministrazione, Rosa Bengala può ancora essere rilevato nel flusso sanguigno per diverse ore dopo l&#39;iniezione 13. Preferire la coperta di riscaldamento per il riscaldamento della lampada per evitare potenziali danni secondari (Rosa Bengala assorbimento di lunghezza d&#39;onda è nello spettro verde). </li></ol>

<ol>
	<li>Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., Murray, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+burden+of+disease+and+risk+factors.">Global and regional burden of disease and risk factors.</a> Lancet. 367, 1747-1757 (2001).</li><li>Mathers, C. D., Boerma, T., Ma Fat, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+causes+of+death.">Global and regional causes of death.</a> Br. Med. Bull. 92, 7-32 (2009).</li><li>Rosenblum, W. I., El-Sabban, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+in+the+cerebral+microcirculation:+effect+of+aspirin+and+other+agents.">Platelet aggregation in the cerebral microcirculation: effect of aspirin and other agents.</a> Circ. Res. 40, 320-328 (1977).</li><li>Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+brain+infarction+by+photochemically+initiated+thrombosis.">Induction of reproducible brain infarction by photochemically initiated thrombosis.</a> Ann. Neurol. 17, 497-504 (1985).</li><li>Bergeron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+photochemical+cortical+lesions+in+rat+brain.">Inducing photochemical cortical lesions in rat brain.</a> Curr. Protoc. Neurosci. Chapter 9, Unit 9 16 (2003).</li><li>Lee, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cerebral+ischemia+in+a+mouse+model.">Photochemically induced cerebral ischemia in a mouse model.</a> Surg. Neurol. 67, 620-625 (2007).</li><li>Dietrich, W. D., Watson, B. D., Busto, R., Ginsberg, M. D., Bethea, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cerebral+infarction.+I.+Early+microvascular+alterations.">Photochemically induced cerebral infarction. I. Early microvascular alterations.</a> Acta Neuropathol. 72, 315-325 (1987).</li><li>Schroeter, M., Jander, S., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+induction+of+focal+cerebral+ischemia+in+mice+by+photothrombosis+of+cortical+microvessels:+characterization+of+inflammatory+responses.">Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.</a> J. Neurosci. Methods. 117, 43-49 (2002).</li><li>Kitagawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+ischemia+after+bilateral+carotid+artery+occlusion+and+intraluminal+suture+occlusion+in+mice:+evaluation+of+the+patency+of+the+posterior+communicating+artery.">Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery.</a> J. Cereb. Blood Flow Metab. 18, 570-579 (1998).</li><li>Sigler, A., Goroshkov, A., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hardware+and+methodology+for+targeting+single+brain+arterioles+for+photothrombotic+stroke+on+an+upright+microscope.">Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope.</a> J. Neurosci. Methods. 170, 35-44 (2008).</li><li>Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li><li>Piao, M. S., Lee, J. K., Jang, J. W., Kim, S. H., Kim, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+photochemically+induced+spinal+cord+injury.">A mouse model of photochemically induced spinal cord injury.</a> J. Korean Neurosurg. Soc. 46, 479-483 (2009).</li><li>Silva, V. M., Corson, N., Elder, A., Oberdorster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+ear+vein+model+for+investigating+in+vivo+thrombogenicity+of+ultrafine+particles+(UFP).">The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP).</a> Toxicol. Sci. 85, 983-989 (2005).</li><li>Watson, B. D., Prado, R., Dietrich, W. D., Ginsberg, M. D., Green, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+spinal+cord+injury+in+the+rat.">Photochemically induced spinal cord injury in the rat.</a> Brain Res. 367, 296-300 (1986).</li><li>Van Reempts, J., Van Deuren, B., Van de Ven, M., Cornelissen, F., Borgers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flunarizine+reduces+cerebral+infarct+size+after+photochemically+induced+thrombosis+in+spontaneously+hypertensive+rats.">Flunarizine reduces cerebral infarct size after photochemically induced thrombosis in spontaneously hypertensive rats.</a> Stroke. 18, 1113-1119 (1987).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx. 2, 396-409 (2005).</li><li>Kleinschnitz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+of+platelets+or+intrinsic+coagulation+pathway-driven+thrombosis+does+not+prevent+cerebral+infarctions+induced+by+photothrombosis.">Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis.</a> Stroke. 39, 1262-1268 (2008).</li><li>Porritt, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombosis-induced+infarction+of+the+mouse+cerebral+cortex+is+not+affected+by+the+Nrf2-activator+sulforaphane.">Photothrombosis-induced infarction of the mouse cerebral cortex is not affected by the Nrf2-activator sulforaphane.</a> PLoS One. 7, e41090 (2012).</li><li>Baskin, Y. K., Dietrich, W. D., Green, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+effective+behavioral+tasks+for+evaluating+sensorimotor+dysfunction+following+traumatic+brain+injury+in+mice.">Two effective behavioral tasks for evaluating sensorimotor dysfunction following traumatic brain injury in mice.</a> J. Neurosci Methods. 129, 87-93 (2003).</li><li>Markgraf, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+histopathologic+consequences+of+photothrombotic+occlusion+of+the+distal+middle+cerebral+artery+in+Sprague-Dawley+and+Wistar+rats.">Comparative histopathologic consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague-Dawley and Wistar rats.</a> Stroke. 24, 286-292 (1993).</li><li>Wester, P., Watson, B. D., Prado, R., Dietrich, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photothrombotic+'ring'+model+of+rat+stroke-in-evolution+displaying+putative+penumbral+inversion.">A photothrombotic 'ring' model of rat stroke-in-evolution displaying putative penumbral inversion.</a> Stroke. 26, 444-450 (1995).</li><li>Hu, X., Wester, P., Brannstrom, T., Watson, B. D., Gu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+and+reproducible+focal+cortical+ischemia+with+or+without+late+spontaneous+reperfusion+generated+by+a+ring-shaped,+laser-driven+photothrombotic+lesion+in+rats.">Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats.</a> Brain Res. Brain Res. Protoc. 7, 76-85 (2001).</li><li>Maxwell, K. A., Dyck, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+focal+ischemic+lesions+in+neonatal+mice+by+photothrombosis.">Induction of reproducible focal ischemic lesions in neonatal mice by photothrombosis.</a> Dev. Neurosci. 27, 121-126 (2005).</li><li>Kuroiwa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rat+model+of+photothrombotic+ischemia+and+infarction+within+the+caudoputamen.">Development of a rat model of photothrombotic ischemia and infarction within the caudoputamen.</a> Stroke. 40, 248-253 (2009).</li></ol>

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Le modifiche e sostituzioni A causa del suo picco di assorbimento a 562 nm, un laser a luce verde da una lampada ad arco allo xeno filtrato è stato originariamente scelto per irradiare la fotosensibile Rosa Bengala. Sebbene eccitazione laser-mediata è stato ancora utilizzato recently5, esso può essere sostituito da lampada a luce fredda che anche garantire eccitazione colorante 10,15. Fibre ottiche a luce fredda sono più facili da manipolare e meno costoso di sorgenti laser. Tutt...

Agli inizi del secolo 21 °, ictus ischemico è una malattia devastante che rappresenta la seconda causa di disabilità a lungo termine 1 e la seconda causa di mortalità in tutto il mondo, nei quali ictus rappresentato circa 5,7 milioni di morti nel 2004 2. Nonostante i numerosi sforzi che sono stati messi in, non vi è ancora alcun trattamento efficace disponibile per migliorare il recupero funzionale dopo ictus. I modelli animali di ictus sono ampiamente utilizzati nel campo della ricerca ictus in quanto consentono la modellazione della fisiopatologia del danno ischemico e testare l&#39;efficacia di differenti strategie di neuroprotezione in vivo. La maggior parte di questi modelli mirano a indurre infarto esteso da interrompere (temporaneamente o definitivamente) il flusso di sangue all&#39;interno dell&#39;arteria cerebrale media, mentre altri modelli sono stati sviluppati per studiare le lesioni di piccole dimensioni in settori specifici, in genere il motore e la corteccia somatosensoriale. Tuttavia, diversi fattori possono contribuire a generare corrente alternataalune grado di variabilità in studi sperimentali ictus, compreso il mouse ceppo utilizzato, l&#39;età e il sesso degli animali inclusi nello studio e, soprattutto, la tecnica adottata per indurre il danno ischemico. Con riferimento a quest&#39;ultimo punto, la durata e invasività della chirurgia (cioè la necessità di una craniotomia), così come l&#39;abilità chirurgica richiesta all&#39;operatore di indurre attendibilmente una lesione ischemica sono fattori determinanti per il successo di uno studio imparziale nella corsa in vivo . Il concetto di photothrombosis stato inizialmente proposto da Rosenblum e El-Sabban nel 1977 3 e divenne celebre per la sua applicazione nel cervello di ratto da Watson et al nel 1985, 4 nel quale la tecnica è stata ampiamente migliorata e ha posto le basi del modello attuale 3. - 6. L&#39;approccio photothrombotic mira a indurre un infarto corticale attraverso la foto-attivazione di un colorante fotosensibile precedentemente consegnata nel sistema sanguigno, which provoca trombosi dei vasi locale nelle aree esposte alla luce. Quando il colorante circolante è illuminato alla lunghezza d&#39;onda appropriata da una sorgente di luce fredda, rilascia energia a molecole di ossigeno, che a loro volta generano una grande quantità di prodotti altamente reattive dell&#39;ossigeno singoletto. Questi intermedi dell&#39;ossigeno inducono perossidazione endoteliali membrana cellulare, portando ad adesione e l&#39;aggregazione piastrinica, e infine alla formazione di trombi, che determinano il flusso locale interruzione cerebrale 7. Photothrombosis è un modello ischemico non canonico che non occlude o rompere una sola arteria come accade di solito in tempi umani, ma induce lesioni in vasi più superficiali, che provoca l&#39;interruzione selettiva del flusso sanguigno nelle zone esposte alla luce. Per questo motivo, questo approccio può essere adatto per gli studi cellulari e molecolari della plasticità corticale. Il vantaggio principale di questa tecnica risiede nella sua semplicità di esecuzione.Inoltre, photothrombosis può essere facilmente effettuata in circa 40 minuti per animale, compreso venti minuti di attesa (3 min per anestesia, 1 min a radere il cuoio capelluto, 3 per 5 min per posizionare l&#39;animale sul apparato stereotassico; 2 min per fregare la cuoio capelluto con soluzione antisettica, fare un&#39;incisione e pulire il cranio; 2-4 min per posizionare la fibra di luce fredda; 1 min per iniettare la soluzione di rosa del Bengala; 5 minuti di attesa per la diffusione intraperitoneale; 15 min di illuminazione, e 5 min a pulire la ferita e suturare l&#39;animale). Inoltre, nessuna perizia chirurgica è necessaria per eseguire questa tecnica come la lesione è indotta mediante semplice illuminazione del cranio intatto. A differenza di occlusione arteriosa classica, questo metodo determina occlusione selettiva del microcircolo piale e intraparenchimale all&#39;interno della zona irradiata e riduce la variabilità tra le lesioni come nessuna nave garanzia è lasciato per la fornitura di ossigeno nella zona di mira. Nonostante la sua particolare natura, ilazioni danni photothrombotic meccanismi essenziali che si verificano nel cervello ictus. Similmente a occlusione dell&#39;arteria in corsa umana, l&#39;aggregazione piastrinica e la formazione di coagulo determinano interruzione del flusso sanguigno nella zona irradiata 7. Allo stesso modo, questo modello condivide anche le risposte infiammatorie essenziali come in occlusione dell&#39;arteria cerebrale media 8. Tuttavia, a causa dei limiti ben delimitato, la zona di penombra, che corrisponde ad un campo di metabolismo parzialmente conservata, è molto ridotta o inesistente dopo una lesione photothrombotic. Questo confine chiaro può facilitare lo studio delle risposte cellulari all&#39;interno dell&#39;area corticale ischemica o intatto. Modello di topo Photothrombosis è particolarmente adatto per studi di ictus in una varietà di animali transgenici. Infatti i modelli classici non possono adattarsi a tutti i ceppi e studi lungo periodo in C57BL / 6 ceppo di topi riferito un alto rapporto di mortalità che può causare pregiudizi 9.

<table border="1">
<tbody>
 <tr>
 <td>MATERIAL NAME</td>
 <td>COMPANY </td>
 <td>CATALOGUE NUMBER</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Solutions and chemicals</td>
 </tr>
 <tr>
 <td>Rose Bengal </td>
 <td>Sigma, Italy </td>
 <td>330000 </td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane Vet </td>
 <td>Merial </td>
 <td> 103120022 </td>
 <td></td>
 </tr>
 <tr>
 <td>Betadine </td>
 <td>Asta Medica </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paraformaldehyde </td>
 <td>Sigma-Aldrich </td>
 <td> 158127 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Surgical material and equipment</td>
 </tr>
 <tr>
 <td>Fluosorber Filter </td>
 <td>Havard apparatus </td>
 <td> 340415 </td>
 <td></td>
 </tr>
 <tr>
 <td>150W fiber optic illuminator</td>
 <td>Photonic</td>
 <td>PL3000</td>
 <td></td>
 </tr>
 <tr>
 <td>Temperature Controller for Plate TCAT-2DF </td>
 <td>Havard apparatus</td>
 <td>727561 </td>
 <td></td>
 </tr>
 <tr>
 <td>Stereotaxic Instrument </td>
 <td>Stoelting</td>
 <td> 51950 </td>
 <td></td>
 </tr>
 <tr>
 <td>Operating microscope </td>
 <td>Takagi </td>
 <td> OM8 </td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad </td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Oxygen and nitrogen gas</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Surgery Tools</td>
 <td>World precision instrument </td>
 <td></td>
 <td>Optic fiber taps and mask are custom-made </td>
 </tr>
 </tbody>
</table>

photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies

Il modello di ictus photothrombotic mira a indurre un danno ischemico entro una data area corticale mediante foto-attivazione di un colorante fotosensibile precedentemente iniettato. Seguendo illuminazione, il colorante viene attivato e produce ossigeno singoletto che danneggia i componenti delle membrane cellulari endoteliali, con successiva aggregazione piastrinica e la formazione di trombi, che alla fine determina l&#39;interruzione del flusso sanguigno locale. Questo approccio, inizialmente proposto dal Rosenblum e El-Sabban nel 1977, è stato successivamente migliorato da Watson nel 1985 nel cervello di ratto e ha posto le basi del modello attuale. Inoltre, l&#39;aumento della disponibilità di linee di topi transgenici ha ulteriormente contribuito ad aumentare l&#39;interesse sul modello photothrombosis. Brevemente, un colorante fotosensibile (Rosa Bengala) viene iniettato per via intraperitoneale e entra nel flusso sanguigno. Quando illuminata da una sorgente luminosa fredda, il colorante diventa attivato e induce danno endoteliale con l&#39;attivazione piastrinica e trombosi, causando localeinterruzione del flusso sanguigno. La sorgente luminosa può essere applicato sul cranio intatto senza bisogno di craniotomia, che permette il targeting di qualsiasi area corticale di interesse in modo riproducibile e non invasivo. Il mouse viene poi suturata e ha permesso di svegliarsi. La valutazione del danno ischemico può essere rapidamente realizzato da cloruro trifenil-tetrazolium o cresile colorazione viola. Questa tecnica produce infarto di piccole dimensioni e confini ben delimitati, che è molto vantaggioso per la caratterizzazione delle cellule precise o studi funzionali. Inoltre, è particolarmente adatto per studiare risposte cellulari e molecolari alla base della plasticità cerebrale in topi transgenici.

Questo protocollo produrrà una lesione corticale che è già visibile sulla dissezione della corteccia ad occhio nudo (Figure 1A-1C). La lesione photothrombotic sviluppa in superficiali e profondi strati corticali in cui il tessuto è sufficientemente trasparente da consentire foto-attivazione del Rosa Bengala. Misurare l&#39;entità di infarto cerebrale può essere eseguita rapidamente mediante colorazione istologica con cloruro di trifenil-tetrazolio (TTC) su tessuto fresco o violetto cresolo dopo fi...

Watch this Scientific Journal Video about Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies at JoVE.com

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

Photothrombosis è una tecnica rapida, mini-invasiva per indurre le piccole e ben delimitate del miocardio in aree di interesse in modo altamente riproducibile. È particolarmente adatto per studiare risposte cellulari e molecolari alla base della plasticità cerebrale in topi transgenici.

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 ...

photothrombotic-ischemia-minimally-invasive-reproducible

Research

JoVE Journal

Medicine

34.4K Views.  University of Turin. Photothrombosis è una tecnica rapida, mini-invasiva per indurre le piccole e ben delimitate del miocardio in aree di interesse in modo altamente riproducibile. È particolarmente adatto per studiare risposte cellulari e molecolari alla base della plasticità cerebrale in topi transgenici.

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

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 ...

Ricerca

Medicina

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Lesione Explorer: un video-guida, standardizzato protocollo preciso e affidabile Volumetrics MRI-derivati ​​nella malattia di Alzheimer e normale Anziani

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Minimally Invasive Thumb-sized Pterional Craniotomy for Surgical Clip Ligation of Unruptured Anterior Circulation Aneurysms

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve patient outcomes. We present our experience with a minimally invasive pterional approach for anterior circulation aneurysms performed in a major tertiary cerebrovascular center and compare the results with an aged matched dataset from the Nationwide Inpatient Sample (NIS). From August 2008 to December 2012, 22 elective aneurysm clippings on patients &#8804;55 years of age were performed by the same dual fellowship-trained cerebrovascular/endovascular neurosurgeon. One patient (4.5%) experienced transient post-operative complications. 18 of 22 patients returned for follow-up imaging and there were no recurrences through an average duration of 22 months. A search in the NIS database from 2008 to 2010, also for patients aged &#8804;55 years of age, yielded 1,341 hospitalizations for surgical clip ligation of unruptured cerebral aneurysms. Inpatient length of stay and hospital charges at our institution using the minimally invasive thumb-sized pterional technique were nearly half that of NIS (length of stay: 3.2 vs 5.7 days; hospital charges: $52,779 vs. $101,882). The minimally invasive thumb-sized pterional craniotomy allows good exposure of unruptured small and medium-sized supraclinoid anterior circulation aneurysms. Cerebrospinal fluid drainage from key subarachnoid cisterns and constant bimanual microsurgical techniques avoid the need for retractors which can cause contusions, localized venous infarctions, and post-operative cerebral edema at the retractor sites. Utilizing this set of techniques has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

Minimally invasive thumb-sized pterional craniotomy for aneurysm clipping has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

Minimamente invasiva Thumb dimensioni Pterional Craniotomy per chirurgica legatura della clip di non rotto Anteriori aneurismi circolazione

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

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 ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Deficit motore riproducibile a seguito dell&#39;occlusione aortica in un modello di ratto dell&#39;ischemia del midollo spinale

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Un modello come minimo dilagante per analizzare Endochondral frattura la guarigione nei topi in condizioni standardizzate di biomeccaniche

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

Localizzazione di noduli transtoracici di navigazione elettromagnetica per chirurgia toracica minimamente invasiva

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Un modello di ratto preclinico per lo studio delle lesioni ischemia-reperfusione in microchirurgia ricostruttiva

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment of end-tidal CO2 (ETCO2), its accuracy is commonly validated using commercial kits composed of a capnography monitor and its matching disposable nasal cannula sampling lines. The purpose of this study was to assess the compatibility and accuracy of cross-paired capnography sampling lines with a single portable bedside capnography monitor. A series of 4 bench tests were performed to evaluate the tensile strength, rise time, ETCO2 accuracy as a function of respiratory rate, and ETCO2 accuracy in the presence of supplemental O2. Each bench test was performed using specialized, validated equipment to allow for a full evaluation of sampling line performance. The 4 bench tests successfully differentiated between sampling lines from different commercial sources and suggested that due to increased rise time and decreased ETCO2 accuracy, not all nasal cannula sampling lines provide reliable clinical data when cross-paired with a commercial capnography monitor. Care should be taken to ensure that any cross-pairing of capnography monitors and disposable sampling lines is fully validated for use across respiratory rates and supplemental O2 flow rates commonly encountered in clinical settings.

The goal of this study was to evaluate the accuracy of capnography sampling lines used in conjunction with a portable bedside capnography monitor. Sampling lines from 7 manufacturers were evaluated for tensile strength, rise time, and ETCO2 accuracy as a function of respiratory rate or supplemental oxygen flow rate.

Valutazione della compatibilità e dell'accuratezza della linea di campionamento capnografica se utilizzata con un monitor capnografico portatile

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

Un metodo non invasivo per generare il modello di lesione cartilaginea intra-articolare indotta dal carico ciclico del ginocchio di ratto

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...