Gli autori sono grati a Dan Firl, Chris Corbett e Jesse Denson, PhD. Il finanziamento è stato fornito dal NIH NINDS R01 NS060765 per SR, il P30 Cobre programma pilota di LJ e la firma del programma Child Health di LJ presso l&#39;Università del New Mexico.

Cura istituzionale e Usa Comitati all&#39;ospedale sia dei bambini di Boston e l&#39;Università del New Mexico Health Sciences Center hanno approvato tutte le procedure sperimentali. NOTA: Prima di iniziare la procedura, sigillo, sterilizzare in autoclave e tutti gli strumenti chirurgici e teli chirurgici. Inoltre, preparare i farmaci post-operatorie in fiale sterili, tra cui 0,125% bipivucaine e 0,1 mg / kg di buprenorfina. Preparare anche la soluzione lipopolisaccaride (LPS) sterilmente: 0,04 mg / ml (LPS 0111: B4) in soluzione fisiologica sterile contenente diluire colorante blu di Evan. 1. Anestesia <ol><li> Indurre anestesia in un giorno embrionale 18 (E18) Sprague Dawley incinta ratto con una miscela di 3% isoflurano bilanciato 70% di azoto e del 30% di ossigeno. </li><li> Rimuovere ratto dalla camera di induzione e posizionare il supina ratto su chirurgica coperta acqua circolante drappeggiata a 37 ° C. Trasferimento anestesia a cono e ridurre isofluraLivello ne al 2%. </li><li> Applicare delicatamente pomata oftalmica per ciascun occhio per evitare l&#39;essiccazione della cornea. Durante la procedura di continuo monitoraggio della temperatura, frequenza respiratoria e la frequenza cardiaca dell&#39;animale. Fisiologia materna dovrebbe rimanere stabile durante tutta la procedura. </li></ol> 2. chirurgico Prep e Scrub <ol><li> Utilizzando piccoli tagliatori animali rimuovere tutti i capelli nella regione addominale inferiore. Shave in un modello rettangolare con cura per evitare di intaccare i capezzoli o la generazione di eruzioni cutanee rasoio che può essere irritante per il futuro di cura di cuccioli nati vivi. </li><li> Preparare la pelle addominale alternando applicazione di povidone-iodio e il 70% di etanolo scrub con tamponi di cotone sterili. Ripetere la macchia in modo che iodopovidone e il 70% di etanolo sono ciascuno applicato 3x in modo alterno. Lasciate asciugare. </li><li> Confermare profondità dell&#39;anestesia via dell&#39;assenza di convergenza pizzico reflex. In assenza di riflessi e stimoli al dolore, ridurre il livello di isoflurano al 1%. </li><li> Usando i tovaglioli chirurgici sterili,drappeggio l&#39;animale. Fare attenzione a posizionare le tende in un angolo appropriato tale da massimizzare la quantità di liquido di irrigazione che assorbono pur non ostacolare il flusso di sangue per le corna uterine. </li></ol> 3. addominale laparotomia <ol><li> Usando un bisturi fare una incisione mediana 3 cm nella pelle addominale preparato. Senza mezzi termini sezionare lo strato di pelle dalla fascia addominale con le forbici. Utilizzando pinze e forbici chirurgiche, elevare lo strato fasciale addominale ed effettuare una incisione della linea alba avascolare per accedere alla cavità peritoneale. </li><li> Posizionare garza all&#39;esterno dell&#39;incisione e bagnare con soluzione fisiologica sterile. Utilizzando pinze smussato e pressione esterna sull&#39;addome, rimuovere delicatamente le corna uterine dalla cavità peritoneale e disporre sulla garza inumidita. </li><li> Evitare accuratamente entanglement e il contatto con gli intestini. Disporre feti con pinze contattando solo il tessuto muscolare in tra i singoli sacchi amniotici.Esporre e isolare i 4 arterie uterine utilizzando smussa. NOTA: Si deve prestare attenzione a sezionare le arterie uterine. Tessuto circostante e dei vasi stessi sono estremamente delicati. Danni ai vasi materni può causare sanguinamento, e nei casi più gravi, morte fetale e materna. </li></ol> 4. Posizionamento di clip per aneurisma <ol><li> Posizionare un G clip di topo 30 aneurisma dell&#39;arteria uterina ogni. Garantire la cessazione del flusso di sangue, tra cui prossimali e distali impulsi, e oscuramento dei vasi uterini tra cui singole placente. Coprire le corna a vista e tutto il campo chirurgico con garza ed irrigare con soluzione salina sterile. Fare attenzione a mantenere il campo umido con irrigazione circa ogni 10 min. </li><li> Dopo 60 minuti, rimuovere la garza e irrigare il campo. Assicurarsi che le corna uterine e le navi sono adeguatamente inumidite per la rimozione della clip di successo. Rimuovere delicatamente ogni clip aneurisma con pinze. Fare attenzione a non causare traumi alla nave, e maintl&#39;integrità dei tessuti ain durante la rimozione. </li><li> Lavare bene le corna uterine e campo, avendo cura di rimuovere eventuali discussioni randagi di garza dalle sacche amniotiche. </li></ol> 5. Iniezione di Lipopolisaccaride per Amniotic Sacs <ol><li> Alla base di ogni singolo sacco amniotico, solo anteriore alla piastra placentare, iniettare 100 ml di LPS (4 mg / sac) con colorante blu diluito di Evan per il liquido amniotico. Utilizzare pinze smussato per stabilizzare e ruotare ogni sacco amniotico per una posizione ottimale per l&#39;iniezione. Colorante blu di Evan è Diluire un mezzo di contrasto che è utile per confermare il corretto posizionamento siringa e l&#39;iniezione. NOTA: Usare solo un ultra-sottile siringa da 0,3 ml di insulina con annesso 8 millimetri 31 ago G per le iniezioni intra-amniotico. Usando aghi calibro maggiore comporterà la perdita del liquido amniotico cronica, morte fetale e il riassorbimento della gravidanza. Piccole quantità di perdite di liquido amniotico dopo l&#39;estrazione della siringa può essere mitigated dalla pressione diretta al sacco amniotico. Alcuni feti di ratto possono tollerare un certo grado di oligoidramnios. Tuttavia, la perdita di liquido amniotico acuta da puntura con i grandi aghi calibro, o puntura accidentale con conseguente perdita di fluido cronico, si traduce in perdita fetale e nei casi più gravi, la perdita di gravidanze vicini. </li><li> Irrigare il uterino corna 3x con soluzione salina sterile. </li></ol> 6. Chiusura della laparotomia <ol><li> Utilizzando pinze, tornare attentamente le corna uterine alla cavità peritoneale. Garantire un adeguato spazio tra le sacche amniotiche e l&#39;incisione mediana, nuovamente ravvicinare le bordi del livello muscolofasciale utilizzando una esecuzione di sutura 3-0 seta. Essere consapevoli del posizionamento dei sacchi amniotici durante la chiusura l&#39;incisione muscolo. Fare attenzione a non a suturare dentro o attraverso un sac. </li><li> Re-approssimare lo strato di pelle, usando una sutura 3-0 esecuzione seta, chiudendo lo strato di pelle. NOTA: La laparotomia deve essere chiuso in due strati di sutura continua per consentireper la pelle e l&#39;espansione muscolare con l&#39;aumentare della gestazione. Suture continue consentono di tensione ferita uniformemente distribuito. Punti staccati sono meno desiderati come più nodi sono irritanti e possono essere facilmente masticati dal topo in recupero da anestesia. Punti chirurgici non sono desiderati. Code dei nodi chirurgici devono essere tagliati molto corti (&lt;3 mm). </li><li> Iniettare 1 ml di 0,125% bupivacaina per via sottocutanea bordi intorno ferita con un ago 26 G. Somministrare una dose di 0,1 mg / kg per via sottocutanea alla buprenorfina nuca. </li><li> Spegnere isoflurano e asciugamano ratto asciutto, se necessario. Mettere in gabbia casa pulita e monitorare il recupero dall&#39;anestesia. Garantire ratto non diventi ipotermico. </li></ol> 7. postoperatoria recupero e cura <ol><li> Monitorare il ratto ogni 8-12 ore per 72 ore e poi tutti i giorni fino cuccioli sono nati (circa E22 o E23). Somministrare ulteriori dosi di buprenorfina q8-12 ore / 72 ore o prn come dettato dalla IACUC. </li><li> Monitorare ratti per segni di dolore, disagio, sanguinamento vaginale o sanguinamento dal sito chirurgico. Ispezionare le suture e incisione e fare attenzione a garantire topo non è masticare o rimuovendo i loro punti di sutura prematuramente. Anche se eccezionalmente raro, i ratti che compromettono i loro punti di sutura sono a rischio di deiscenza della ferita. NOTA: A volte i ratti possono ingerire quantità eccessive di biancheria da letto o di generi non alimentari, chiamato pica, come effetto collaterale di amministrazione buprenorfina. Anche se molto raro, topi devono essere monitorati per la pica e potenziale conseguente occlusione intestinale. </li></ol> 8. Tissue Processing e criosezionamento <ol><li> Per preparare Ematossilina e eosina (H &amp; E) la colorazione del cervello post-natale, rimuovere i cuccioli dalla loro gabbia casa il postnatale giorno 2 (P2). Utilizzando le forbici chirurgiche decapitano cuccioli di ratto e delicatamente rimuovere il cervello dal cranio. </li><li> Goccia correzione cervello in un tubo da 15 ml contenente 7 ml di 4% paraformaldeide in tampone fosfato(PBS). Posizionare il cervello a 4 ° C e fissare per 72 ore. </li><li> Dopo 72 ore, trasferire cervelli di una soluzione sterile di PBS contenente il 30% di saccarosio (w / v) e ritornare a 4 ° C. NOTA: Una volta cervelli goccia nella soluzione di saccarosio sono pronti per essere sezionato su un criostato. </li><li> Rapidamente congelare il cervello e il montaggio su pestello criostato per l&#39;acquisizione di sezioni coronali congelate. Tagliare 20 micron sezioni coronali congelate e montare su vetrini. Assicurarsi che le sezioni sono raccolti in serie. </li><li> Lasciare i vetrini si asciughino notte a temperatura ambiente. Conservare scivola a -20 ° C. </li></ol> 9. ematossilina &amp; eosina <ol><li> Prendere scivolo montato sezioni congelate e caldo a temperatura ambiente. NOTA: Preparare tutte le soluzioni fresco. </li><li> Collocare i vetrini in una diapositiva set più calda a 50 ° C per 2 ore. </li><li> Trasferire i vetrini in un rack per colorazione. Dip scivola 10x in acqua bidistillata deionizzata (DDH 2 O). </li><li> Incubare i vetrini in 100% ematossilina per 5 min. Time in ematossilina può essere ottimizzata a seconda del grado di colorazione viola. </li><li> Immergere i vetrini 4x in rubinetto H 2 O, e lasciare riposare in un ambiente pulito rubinetto H 2 O per 1 min. </li><li> Dip scivola 15x in alcool acido (250 ml 70% di etanolo + 1 ml di acido cloridrico). </li><li> Immergere i vetrini 4x in rubinetto H 2 O, e lasciare riposare in un ambiente pulito rubinetto H 2 O per 1 min. </li><li> Incubare in 1% di carbonato di litio per 2 min. </li><li> Immergere i vetrini 4x in rubinetto H 2 O, e lasciare riposare in un ambiente pulito rubinetto H 2 O per 1 min. </li><li> Incubare in 95% di etanolo per 1 min. </li><li> Immergere i vetrini 7x in 100% eosina. Numero di cali di Eosina può essere modificato a seconda del grado di colorazione rosa. </li><li> Immergere i vetrini in 5x 95% di etanolo. </li><li> 5x Dip diapositiva in una nuova variazione del 95% di etanolo. </li><li> Incubare i vetrini in etanolo al 100% per 1 min. </li><li> Incubare i vetrini in una nuova variazione di etanolo 100% per 1 min. </li><li> Incubare i vetrini in 100% xilene per 15 min. NOTA: passaggi xilene emontavetrini dovrebbe avvenire in una cappa aspirante. </li><li> Incubare i vetrini a nuove modifiche di xilene per 15 min. </li><li> Coverslip con Permount e lasciare asciugare in cappa. </li><li> Immagine vetrini con un microscopio ottico. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+loss+of+gamma+aminobutyric+acid+pathway+expression+after+human+perinatal+brain+injury.">Neonatal loss of gamma aminobutyric acid pathway expression after human perinatal brain injury.</a> J Neurosurg. 104, 396-408 (2006).</li><li>Boksa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prenatal+infection+on+brain+development+and+behavior+a+review+of+findings+from+animal+models.">Effects of prenatal infection on brain development and behavior a review of findings from animal models.</a> Brain Behav Immun. 24, 881-897 (2010).</li><li>Burd, I., Brown, A., Gonzalez, J. M., Chai, J., Elovitz, M. A. <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+term+chorioamnionitis+unraveling+causes+of+adverse+neurological+outcomes.">A mouse model of term chorioamnionitis unraveling causes of adverse neurological outcomes.</a> Repro Sci. 18, 900-907 (2011).</li><li>Bergeron, J. D., 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=White+matter+injury+and+autistic+like+behavior+predominantly+affecting+male+rat+offspring+exposed+to+group+B+streptococcal+maternal+inflammation.">White matter injury and autistic like behavior predominantly affecting male rat offspring exposed to group B streptococcal maternal inflammation.</a> Devel Neurosci. 35, 504-515 (2013).</li><li>Uchida, 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=Effects+of+Ureaplasma+parvum+lipoprotein+multiple+banded+antigen+on+pregnancy+outcome+in+mice.">Effects of Ureaplasma parvum lipoprotein multiple banded antigen on pregnancy outcome in mice.</a> J Reprod Immunol. 100, 118-127 .</li></ol>

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Encefalopatia di prematurità è difficile da modellare negli animali a causa della complessa interazione di eziologie, decorso dello sviluppo neurologico, complessità di formazione delle reti cerebrale umana, meccanismi di danno che si sovrappongono, e le diverse fenotipi di CNS insulti manifestano nei neonati prematuri umani. EoP è associato con specifiche vulnerabilità delle cellule di tipo (cioè oligodendrociti immaturi) 21, così come diversi percorsi evolutivamente regolamentati (cioè<...

Con oltre il 12% dei bambini nati negli Stati Uniti prima di 37 settimane di età gestazionale stimata 1, danno cerebrale perinatale (PBI) da prematurità è una causa importante di invalidità permanente. PBI da prematurità, encefalopatia anche chiamato di prematurità (EOP), colpisce tutto il sistema nervoso centrale (SNC). Lesioni del sistema nervoso centrale spesso inizia in utero, ed è aggravata da processi prenatali tra cui corionamnionite e le complicanze post-natali, quali ipossia e sepsi. PBI da insulti sistemici altera neurosviluppo e porta alla paralisi cerebrale, epilessia, ritardo cognitivo e numerosi disturbi neuropsichiatrici che interessano la regolazione emotiva, la memoria e la funzione esecutiva 1,2. Anche se sono stati fatti molti progressi, una comprensione limitata rimane di come le conseguenze cellulari e molecolari delle lesioni del sistema nervoso centrale da parto pretermine traducono alla moltitudine di sequele neurologiche nei bambini che sono nati pretermine. Questa mancanza di conoscenze posterioriERS diagnosi in tempo reale del sistema nervoso centrale la gravità delle lesioni e il dosaggio informato degli interventi emergenti. Inoltre, adeguate all&#39;età strategie terapeutiche per questa popolazione di pazienti vulnerabili rimangono elusivi. Infiammazione intrauterina è molto comune in estrema prematurità e coinvolge una cascata infiammatoria materno-fetale-placentare complesso 3. Infezione intrauterina è spesso subclinica. Risultati placentari specifiche coerenti con infiammazione acuta, o corionamnionite istologico, sono i principali determinanti della risposta infiammatoria fetale e sono coincidenti con lesioni cerebrali associati alla nascita pretermine 3-5. In effetti, la risposta infiammatoria fetale ha implicazioni cliniche distinte per i risultati a lungo termine da parto pretermine. I bambini che sono piccoli per l&#39;età gestazionale (SGA) o che sperimentano infezione sono particolarmente vulnerabili alle deficit neurologico 3,4. Corionamnionite è una diagnosi patologica tipica dopo la nascita pretermine <sup&gt; 6,7, e l&#39;esame istologico rivela segni di infiammazione nel 70% dei bambini nati da placente molto pretermine 4. Inoltre, corionamnionite è associato a deterioramento cognitivo a due anni 8. La prova di ipoperfusione vascolare materno nella placenta dei bambini nati estremamente pretermine è anche associata a paralisi cerebrale durante l&#39;infanzia 9. L&#39;impatto sinergica di corionamnionite e difetti di perfusione placentare è ben illustrato dal notevolmente alto rischio di esiti neurologici anormali in questa popolazione di pazienti in due anni di 10,11 anni. Per simulare difetti umani placentare perfusione sistemici e corionamnionite associati con l&#39;infiammazione patogeno-indotta, abbiamo sviluppato un modello di prenatale transitorio sistemica ipossia-ischemia (TSHI) in combinazione con lipopolisaccaride intra-amniotico (LPS) nei ratti. Il nostro obiettivo era quello di adattare il nostro modello di TSHI solo nei ratti 12-16 per includere infiammazione intrauterina,per facilitare la modellazione preclinico di lesioni del sistema nervoso centrale associato a nascita pretermine. TSHI solo ha rivelato la perdita persistente di cellule lignaggio oligodendrogliali, neuroni corticali, aumento della morte cellulare, e elevati livelli di citochine pro-infiammatorie, con intervalli ischemici progressivi che portano a un modello graduato di lesioni compatibili con lesioni cerebrali prenatale 16. Le modifiche ai componenti ischemiche di questo modello hanno anche dimostrato deficit nella codifica della memoria, a breve e memoria a lungo termine e le alterazioni muscolo-scheletriche lievi nei ratti come 17-19 invecchiano. In effetti, abbiamo precedentemente dimostrato che la combinazione di TSHI + LPS ricapitola le caratteristiche fisiopatologiche della EoP, tra cui oligodendrociti e perdita neuronale, danno assonale, infiammazione cellulare e anomalie funzionali 20.

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modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats

Encefalopatia di prematurità (EOP) è un termine che comprende il sistema nervoso centrale (SNC) anomalie associate alla nascita pretermine. Per migliori obiettivi anticipo traslazionali e scoprire nuove strategie terapeutiche per le lesioni del cervello associata alla nascita pretermine, modelli preclinici di EoP devono includere meccanismi simili di lesioni globale prenatale osservato negli esseri umani e coinvolgere più componenti del sistema materno-placentare-fetale. Idealmente, i modelli dovrebbero produrre un simile spettro di deficit funzionali nell&#39;animale maturo e ricapitolare molteplici aspetti della fisiopatologia. Per simulare difetti umani sistemici placentari perfusione placentare, ipoperfusione e / o corioamnionite associate a infiammazione patogeno-indotta nei primi mesi del parto pretermine, abbiamo sviluppato un modello di prenatale transitorio sistemica ipossia-ischemia (TSHI) in combinazione con lipopolisaccaride intra-amniotico (LPS). In gravidanza ratti Sprague Dawley, TSHI via uterina occlusione dell&#39;arteria on giorno embrionale 18 (E18) induce un difetto ipoperfusione placentare graduata associata con l&#39;aumento danno del sistema nervoso centrale del feto. Se combinato con iniezioni intra-LPS amniotico, infiammazione placentare è aumentata e il danno del sistema nervoso centrale è aggravato con associati materia, andatura e di imaging anomalie bianchi. Prenatale TSHI e TSHI + LPS insulti prenatali incontrano molti dei criteri di un modello EoP compreso ricapitolando l&#39;insulto intrauterina, causando la perdita di neuroni, oligodendrociti e degli assoni, la perdita di piastra e deficit funzionali in animali adulti che imitano quelli osservati nei bambini nati estremamente pretermine. Inoltre, questo modello consente la dissezione di infiammazione indotta da tipi di lesioni divergenti.

A seguito di TSHI + LPS a E18, ematossilina e eosina rivela significative alterazioni istopatologiche sia la placenta (Figura 1) e nel cervello (Figura 2). Placente esaminate su E19 e E21 sono grossolanamente edematosi con micro-emorragie e necrosi in tutto il decidua e labirinto. Si osserva anche infiltrato infiammatorio e significativo aumento della vascolarizzazione. Brains esaminati sulla P2 rivelano ventricolomegalia, così come la perdita di materia bianca e piastra neuroni rispet...

Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modellazione Encefalopatia di prematurità Utilizzando prenatale ipossia-ischemia con Lipopolisaccaride intra-amniotico in ratti

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

modeling-encephalopathy-prematurity-using-prenatal-hypoxia-ischemia

Research

JoVE Journal

Medicine

11.2K Views.  University of New Mexico. Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Video: Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Corsa di modellazione in Mouse - Medio occlusione dell&#39;arteria cerebrale con il Modello Filament

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Ricerca

Medicina

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Imaging non invasiva e analisi di ischemia cerebrale in ratti Living Utilizzando Positron Emission Tomography con<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Un modello di Piglet neonatale Hypoxic-ischemica Encefalopatia

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Valutare l&#39;encefalopatia spongiforme trasmissibile Specie Barriere con un<em&gt; In Vitro</em&gt; Prion Protein Assay Conversion

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Simultanea Imaging PET / MRI Durante mouse cerebrale ipossia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

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 ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Circoscritto capsulare Infarct Modeling usando una tecnica Photothrombotic

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Preparazione di impalcature renali decellularizzate nei ratti

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Analisi di campioni di rizoma di Cyperi grezzi e lavorati mediante cromatografia liquida-spettrometria di massa tandem in ratti con dismenorrea primaria

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

Manipolazione standardizzata di Tuina per l'artrosi del ginocchio nei ratti

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Autore in primo piano: Comprendere il meccanismo della manipolazione del tuina nei ratti 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Manipolazione del tuina per ridurre l'infiammazione e la perdita di cartilagine nei ratti affetti da osteoartrite del ginocchio

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...