Name: induction of maternal immune activation in mice at mid-gestation stage with viral mimic poly(i:c)
Uploaded: 2016-03-25T13:00:00.000+00:00
Duration: 7 min 13 s
Description: Watch this Scientific Journal Video about Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C at JoVE.com

Vorremmo onorare il compianto Dr. Paul H. Patterson per il suo contributo al progresso del modello di MIA, l&#39;autismo, la schizofrenia e la ricerca. Riconosciamo Sarkis K. Mazmanian per il suo grande sostegno su questo protocollo; Ruben M. Bayon, Yvette Garcia-Flores, Karen C. Lencioni, e Leslie A. Neumann per l&#39;assistenza amministrativa; Ali Khoshnan e Jan C. Ko per l&#39;assistenza su riprese; Elaine Y. Hsiao e Natalia Malkova per i loro consigli su MIA induzione; Jeffrey S. Cochrane, Joaquin Gutierrez, Kwan F. Lee, Jaime Rodriguez, Lorena C. Sandoval, e Natalie A. Verduzco per la loro zootecnia esperto. Questo lavoro è stato supportato dal NIH Conte Centro Award (NIH 5P50MH086383-04, di Paul H. Patterson); Autism Speaks (# 7670, di Paul H. Patterson); Simons Foundation (# 322839, a Sarkis K. Mazmanian); NIH Formazione Grant (NIH / RIV T32GM07616 a K.-HC); Caltech Estate Undergraduate Research Fellowship (SURF) (a ZY); Amgen Scholars Program al Caltech (a ZY); e Postdoctoral Fellowship from Consiglio Nazionale delle Scienze, Taiwan (NSC 101-2917-I-564-039, per W.-LW).

Tutti i protocolli sono stati eseguiti sotto l&#39;approvazione del California Institute of Technology Comitato cura e l&#39;uso di animali Istituzionale (IACUC). 1. Preparazione per le coppie Timed-accoppiamento <ol><li> Utilizzare almeno 6 dighe per esperimenti comportamentali e 3 per l&#39;analisi di espressione genica. NOTA: utilizzare il doppio del numero di topi femmina stimato, in quanto solo il 50% dei topi verrà inserito dal temporizzata-accoppiamento. </li><li> Utilizzare topi di sesso femminile senza gravidanza prima e in 8-16 settimane di età. NOTA: i topi femmine che hanno esposizione sessuale prima di topi maschi, ma non sono stati in stato di gravidanza sono accettabili. </li><li> Casa topi femmine insieme e posizionare le gabbie accanto all&#39;altro in modo da sincronizzare il loro ciclo estrale. Utilizzare una gabbia topi di serie con un&#39;altezza di oltre 5,5 pollici e un pavimento interno 75 pollice quadrato per consentire abitazioni di 5 topi adulti. Etichettare ogni topo femmina con un pugno all&#39;orecchio. </li></ol> 2. Impostazione Timed-matING coppia <ol><li> Impostare il time-accoppiamento 0-2 ore prima dell&#39;inizio del ciclo di buio. Inserire due topi di sesso femminile in ogni gabbia. Estrarre i topi e pesare individualmente. Registrare il peso corporeo di ogni topo femmina. </li><li> Posizionare un topo maschio in ogni gabbia con i due topi di sesso femminile. Utilizzare trio di accoppiamento per minimizzare l&#39;effetto paterna confondimento. </li></ol> 3. Inserire vaginale Check (E0.5) <ol><li> Controllare la femmina topi 0-4 ore dopo il ciclo di buio finisce. Sollevare delicatamente il mouse femminile dalla base della coda e lasciare il mouse per afferrare la griglia metallica, cima della gabbia, o il bordo della gabbia. </li><li> Ricerca di segni di spina vaginale: spina vaginale è una massa biancastra visibile in apertura vaginale. Se la spina non è evidente, inserire delicatamente un 200 microlitri punta della pipetta nell&#39;apertura vaginale. La resistenza da coagulazione conferma la formazione spina vaginale. Controllare la spina vaginale una volta al giorno. NOTA: spina vaginale è solo una indicazione che si è verificato l&#39;accoppiamento, e, pertanto, non gla gravidanza uarantee. spina vaginale può essere difficile da identificare in alcuni ceppi di topi. Cercare l&#39;aiuto di un assistente topi con esperienza per aumentare la precisione dell&#39;identificazione spina. </li><li> Spostare i topi collegati ad una nuova gabbia e raggrupparli House. Definire il giorno della vagina aspetto spina come embrionali 0,5 giorni (E0.5). </li></ol> 4. E10.5-11.5, Controllare per la gravidanza <ol><li> Sollevare delicatamente la base della coda e lasciare il mouse per afferrare la griglia metallica, cima della gabbia, o il bordo della gabbia. Osservare zona addominale per verificare eventuali segni di gravidanza, ad esempio, un rigonfiamento nell&#39;addome (Figura 1). NOTA: Il segno di gravidanza è sempre più evidente a E11.5 rispetto a E10.5. </li><li> Pesare i topi per verificare la gravidanza. Il mouse incinta generalmente pesa circa il 20% più pesante E10.5 e il 30% più pesante E12.5 rispetto ad un mouse non incinta (Figura 2). Casa singola topi incinta in gabbie pulite. </li></ol> 5. 20 mg /kg Poly (I: C) Preparazione <ol><li> Utilizzare almeno 6 dighe per gruppo per esperimenti comportamentali e 3 per gruppo per l&#39;analisi di espressione genica. Preparare la quantità corrispondente di poli (I: C) soluzione. </li><li> Pesare il poli (I: C) in polvere in una provetta conica da 50 ml per effettuare una concentrazione finale di 40 mg / ml. Al fine di minimizzare e standardizzare lo stress causato dal volume di iniezione, iniettare 5 ml di poli (I: C) soluzione per ogni grammo di peso corporeo mouse (5 ml / g, o 5 ml / kg). Per indurre MIA, iniettiamo 20 mg di poli (I: C) per kg. NOTA: La concentrazione di poli (I: C) soluzione necessaria è (20 mg / kg) / (5 ml / kg) = 4 mg / ml. Poiché il poli (I: C) polvere contiene 10% di poli (I: C), è necessario effettuare una concentrazione finale di (I: C) (4 mg / ml) /0.1= 40 mg / ml di poli soluzione. ATTENZIONE: Il poli (I: C) polvere è molto leggero. Fare attenzione a non perdere la polvere durante il trasferimento dalla spatola al tubo conico. </li><li> Aggiungere il volume corrispondente allo 0,9% di cloruro di sodio (soluzione fisiologica) nella vasca conicaE e chiudere il tappo. Sciogliere la polvere da dolci goccia salina sopra il poli (I: C) in polvere fino a quando la soluzione è chiara. Centrifugare la provetta conica a 800 xg per 1 min (Figura 3C). Filtrare la poly: soluzione di 0,22 micron filtro (I C). Trasferire la poli: soluzione (I C) per un tubo di 1,5 o 2 ml microcentrifuga. Opzionale: utilizzare uno spettrofotometro per misurare il rapporto 260/280 del poli (I: C) soluzione. Assicurarsi che il rapporto è tra 1,54-1,82 (Figura 3), come descritto dal produttore. NOTA: La salina utilizzata per dissolvere poli (I: C) in polvere e servire come iniezione di controllo dovrebbe essere di grado sterile e farmaceutica. </li></ol> 6. Saline o poli (I: C) Iniezione su E12.5 <ol><li> Pesare il mouse sulla scala e rimetterlo nella gabbia. Utilizzare la seguente formula per calcolare il volume di iniezione sia salina e poli (I: C) topi: peso (g) moltiplicato per 5 = volume di iniezione desiderato (microlitri). Utilizzare un isol 0,3 mlin siringa di redigere il volume calcolato di soluzione salina o 20 mg / kg poli (I: C) soluzione, ad esempio, 150 ml per un 30 g del mouse. </li><li> Delicatamente sollevare il mouse la base della coda e posizionarlo sulla parte superiore della gabbia. Maneggiare il mouse delicatamente per evitare effetti confondenti indotti da stress. Inserire l&#39;ago, smussata, a circa 20 ° rispetto al mouse al centro dei due nippli superiori (Figura 4), ​​e iniettare il (I: C) salina o poli soluzione. NOTA: L&#39;ago deve essere sostituito per ogni mouse dopo l&#39;iniezione. </li><li> Posizionare il mouse alla sua gabbia casa. Mettere le gabbie in una tranquilla stanza basso traffico per evitare gli altri effetti confondenti. </li></ol> 7. The Day After poli (I: C) Injection (E13.5) <ol><li> Controllare i topi iniettati la mattina seguente. Utilizzare una scala per misurare il peso corporeo. NOTA: Poli (I: C) iniettato topi in gravidanza di solito guadagnano meno peso rispetto salina iniettato topi il giorno dopo injessione. </li><li> Non disturbare i topi fino a quando la prole sono nati. </li></ol> 8. Calibro di MIA Offspring <ol><li> Per ridurre al minimo gli effetti confondenti di MIA induzione, seguire le linee guida elencate di seguito per misurare l&#39;utilizzo di MIA prole. <ol><li> Escludere le cucciolate da pretermine o la nascita in ritardo, o se la dimensione della cucciolata è più piccolo di 5. </li><li> Euthanize i cuccioli eccessive di CO 2 se la dimensione cucciolata è più grande di 10. </li></ol></li></ol> 9. Opzionale: Esaminate la risposta infiammatoria acuta Dopo MIA <ol><li> Sacrificare la gravidanza topi 3 ore dopo la salina o poli (I: C) di iniezione con il metodo descritto nel protocollo commissione ha approvato la cura degli animali dell&#39;istituzione. NOTA: dislocazione cervicale è spesso preferito in quanto riduce al minimo l&#39;effetto di CO 2 farmaci / eutanasia sulla diga e dell&#39;embrione. </li><li> Raccogliere la milza dai topi in gravidanza adulti e isolare la placenta e il reggiseno del fetoin con lo stereomicroscopio. <ol><li> Per la raccolta della milza, giaceva il mouse sulla piastra di dissezione e spruzzare il 70% di etanolo sulla zona addominale dei topi in gravidanza. Utilizzare un paio di forbici sterili per fare un taglio verticale al centro della zona addominale sotto la gabbia toracica attraverso la pelle. </li><li> La milza si trova nella sinistra quadrante addominale superiore. Utilizzare pinze per isolare la milza. Stendere la milza su delicati tergicristalli compito di rimuovere il tessuto adiposo intorno l&#39;organo. Raccogliere circa 50 mg di tessuto della milza e metterli singolarmente in tubi Eppendorf con 500 ml di RNA stabilizzare tampone o TRIzol per evitare la degradazione degli acidi nucleici. Conservare i campioni a -80 ° C freezer fino al momento dell&#39;uso. </li><li> Per la raccolta di placenta, estrarre delicatamente le corna uterine dal corpo. Collocare le corna uterine in una capsula di Petri riempita con fosfato salino autoclavato tamponata (PBS) e lasciare il piatto su ghiaccio. Utilizzare due pinze per strappare aperto la parete uterina ed esporre il sacco amniotico. <ol><li>usare con cautela le pinze per aprire il sacco amniotico senza danneggiare la placenta e il feto. Separare la placenta dal feto e tagliare il cordone ombelicale più vicino alla placenta possibile. Rimuovere i detriti sacco amniotico dalla placenta. Posizionare la placenta individualmente in tubi Eppendorf con 500 ml di RNA stabilizzano tampone o TRIzol. Conservare il campione a -80 ° C freezer fino al momento dell&#39;uso. </li></ol></li><li> Per la raccolta cervello fetale, memorizzare il feto nel RNA stabilizzare tampone dal punto 9.2.2 fino dissezione. Tease fuori la membrana pial dal ventricolo del cervello utilizzando delicati # 5 pinze con lo stereomicroscopio. Rimuovere con attenzione tutto la membrana del cervello fetale. Posizionare il cervello del feto individualmente in tubi Eppendorf con 500 ml di RNA stabilizzano tampone o TRIzol. Conservare il campione a -80 ° C freezer fino al momento dell&#39;uso. </li></ol></li><li> Estrarre il RNA dal tessuto e convertire l&#39;RNA a cDNA. Analizzare l&#39;espressione del gene IL6 nel cDNA mediante real-time PCR. </li><li> Estrarre il RNA dai tessuti seguendo il protocollo di fabbricazione di TRIzol. Se i tessuti sono memorizzati nel buffer di stabilizzazione dell&#39;RNA. Scongelare il campione e spin down del buffer. Trasferire il tessuto da punta ad un altro eppendorf con 500 microlitri TRIzol e continuare l&#39;estrazione di RNA. </li><li> Utilizzare uno spettrofotometro per misurare la concentrazione, 260/280 e 260/230 rapporto tra RNA. Assicurarsi che i rapporti sono al di sopra 2.0. </li><li> Eliminare la contaminazione del DNA dai trattati RNA con DNasi I. Mix RNA 1 mg, 1 microlitri 10X tampone di reazione, 1 ml RNase-Free DNase e acqua priva di nucleasi ad un volume finale di 10 pl. Incubare la master mix a 37 ° C per 30 min. Aggiungere 1 soluzione di stop DNasi microlitri di interrompere la reazione. Incubare la miscela a 65 ° C per 10 min. </li><li> Reverse trascrivere l&#39;RNA a cDNA. Utilizzare 20 reazioni microlitri, e fino a 1 mg di RNA totale per reazione. Mix 4 microlitri 5x invertire buffer di trascrizione (RT) mix di reazione, 1 ml revERSE trascrittasi, 11 stampo di RNA ml dopo il trattamento con DNasi I e 4 microlitri priva di nucleasi H 2 O per ottenere una soluzione finale di 20 ml. Programmare le condizioni termociclatore per RT. Una condizione generica includono: Fase 1: 25 ° C per 5 minuti; Fase 2: 42 ° C per 30 min; Fase 3: 85 ° C per 5 min; Fase 4: tenere a 4 ° C. Diluire il cDNA 5 volte. Per la conservazione a breve termine, conservare il cDNA a 4 ° C. Per la conservazione a lungo termine, congelare il cDNA a -20 ° C. </li><li> Analizzare l&#39;espressione del gene IL6 nel cDNA mediante real-time PCR e normalizzare l&#39;espressione del gene IL6 di beta-actina espressione genica. <ol><li> Fare un master mix per IL-6 e la rilevazione β-actina. Utilizzare 25 microlitri reazione, il master mix per ogni gene include 12,5 ml SYBR Green Mix, 0,5 l di 10 micron di primer in avanti, 0,5 l di 10 micron di primer inverso e 6,5 microlitri priva di nucleasi H 2 O. </li><li> Mettere 5 ml 5x diluito cDNA unt il fondo del pozzetto della piastra ottica reazione 96 pozzetti. Pipettare 20 ml di master mix per ogni bene per fare un finale di 25 microlitri PCR mix. Triplicato ogni gene per ogni campione. Sigillare la piastra utilizzando pellicola adesiva ottica. Centrifugare la piastra a 800 xg per 3 min a 4 ° C Utilizzare il programma standard di PCR in tempo reale: Fase 1: 50 ° C per 2 min; Fase 2: 95 ° C per 10 min; Fase 3: 40 cicli di 95 ° C per 15 sec e 60 ° C per 1 min. Aggiungere un ulteriore passo per la curva di dissociazione. </li></ol></li></ol> 10. esaminare le anomalie comportamentali nella prole MIA adulti (opzionale): <ol><li> Eseguire i test di comportamento all&#39;età di 8-12 settimane. Cambiare la biancheria da letto gabbia almeno 3 giorni prima della prova. Acclimatare i topi alla sala test comportamentali almeno 30 minuti prima del test. </li><li> Per prepulse inibizione (PPI), trattiene i topi nel cilindro plexiglass con un sensore piezoelettrico sotto. Acclimatare i topi al PPIcamera per 5 min. Esporre i topi con 6 prove di 120 dB rumore bianco (startle). </li><li> Poi esporre i topi con miscele randomizzati di 14 studi di rumore di fondo, 14 studi di Startle, 14 studi su prepulse 5 dB (5 dB più alto del rumore di fondo) + Startle (PPI5), e 14 studi di prepulse 15 dB (15 dB più alti di rumore di fondo + Startle (PPI15). </li><li> In media il risposte di allarme per i primi 6 prove di 120 prove dB. In media il risposte di allarme per gli altri stimoli individuali. Covert il valore di inibizione prepulse da (Startle - PPI5 o PPI15) / Startle. </li><li> Per la prova campo aperto, posizionare il mouse in un angolo di un&#39;arena piazza aperta (50 x 50 x 50 cm 3). Registrare la traiettoria del mouse per 10 min attraverso una telecamera soffitto. Definire il centro dell&#39;arena come la zona centrale (17 x 17 cm 2). Quantificare la distanza percorsa, le voci zona centrale, e il tempo trascorso in zona centrale manualmente o basato su immagini software di analisi automatica. </li&gt; <li> Per la prova sepoltura di marmo, acclimatare il mouse in una gabbia di prova con profondo, pulito, Aspen biancheria da letto di pino 5 centimetri compressa per 10 min. Ritorna il mouse per la sua homecage. Posizionare delicatamente 20 blu navy 1,5 cm biglie di vetro del diametro nel campo della moda di 4 x 5 Trattamento. Posizionare il mouse avanti alla gabbia test con marmi per 10 min. Contare i marmi che è stato sepolto nella durata test 10 min. </li></ol> 11. Esaminare il cerebellare Neuropatologia nella prole MIA adulti (opzionale): <ol><li> Euthanize la soluzione salina e MIA figli adulti da anestetico. Profumato il mouse con 50 ml di PBS e successivamente 50 ml 4% paraformaldeide attraverso il sistema cardiovascolare. </li><li> Rimuovere accuratamente il cervello e postfix il cervello in 4% paraformaldeide notte a 4 ° C. Risciacquare il cervello con PBS per tre volte e memorizzare il cervello in PBS con sodio azide 0,02% in C frigorifero 4 ° fino al momento dell&#39;uso. NOTA: Il cervello postfissati può essere memorizzato in PBS con 0,02% SODazide IUM in frigorifero per un massimo di un mese. </li><li> Sezione del cervello in 50 micron fette sottili sagittalmente utilizzando vibratome. Raccogliere le sezioni di cervello utilizzando una penna spazzola morbida. Terminare l&#39;attività perossidasica endogena incubando le sezioni di perossido di idrogeno 0,6% per 30 minuti a temperatura ambiente. </li><li> Incubare le sezioni in anticorpo anti-calbindin diluito nel tampone di bloccaggio (siero di capra 10% con 0,1% Triton X-100 e 0,02% di sodio azide in PBS) per una notte a temperatura ambiente. Incubare le sezioni in corrispondenti biotinilato anticorpo secondario diluito in tampone di bloccaggio per 2 ore a temperatura ambiente. </li><li> Incubare le sezioni in DH avidina - biotinilato perossidasi tampone complesso H per rilevare la biotina per 1 ora a temperatura ambiente. Sviluppare sezioni utilizzando substrato della perossidasi per visualizzare l&#39;antigene. Lavare le sezioni con PBS per tre volte tra i gradini. Montare le sezioni su vetrini da microscopio. Immagine sezioni sotto il microscopio. </li></ol&gt;

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+maternal+immune+system+induces+endocrine+changes+in+the+placenta+via+IL-6.">Activation of the maternal immune system induces endocrine changes in the placenta via IL-6.</a> Brain Behav Immun. 25 (4), 604-615 (2011).</li><li>Wu, W. L., 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=The+interaction+between+maternal+immune+activation+and+alpha+7+nicotinic+acetylcholine+receptor+in+regulating+behaviors+in+the+offspring.">The interaction between maternal immune activation and alpha 7 nicotinic acetylcholine receptor in regulating behaviors in the offspring.</a> Brain Behav Immun. , (2015).</li><li>Garay, P. A., Hsiao, E. Y., Patterson, P. H., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+immune+activation+causes+age-+and+region-specific+changes+in+brain+cytokines+in+offspring+throughout+development.">Maternal immune activation causes age- and region-specific changes in brain cytokines in offspring throughout development.</a> Brain Behav Immun. 31, 54-68 (2013).</li><li>Garbett, K. A., Hsiao, E. Y., Kalman, S., Patterson, P. H., Mirnics, K. <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+maternal+immune+activation+on+gene+expression+patterns+in+the+fetal+brain.">Effects of maternal immune activation on gene expression patterns in the fetal brain.</a> Transl Psychiatry. 2, e98 (2012).</li><li>Hsiao, E. Y., 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=Microbiota+modulate+behavioral+and+physiological+abnormalities+associated+with+neurodevelopmental+disorders.">Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders.</a> Cell. 155 (7), 1451-1463 (2013).</li><li>Naviaux, R. 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=Antipurinergic+therapy+corrects+the+autism-like+features+in+the+poly(IC)+mouse+model.">Antipurinergic therapy corrects the autism-like features in the poly(IC) mouse model.</a> PLoS One. 8 (3), e57380 (2013).</li><li>Pratt, L., Ni, L., Ponzio, N. M., Jonakait, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+inflammation+promotes+fetal+microglial+activation+and+increased+cholinergic+expression+in+the+fetal+basal+forebrain:+role+of+interleukin-6.">Maternal inflammation promotes fetal microglial activation and increased cholinergic expression in the fetal basal forebrain: role of interleukin-6.</a> Pediatr Res. 74 (4), 393-401 (2013).</li><li>Schwartzer, J. 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=Maternal+immune+activation+and+strain+specific+interactions+in+the+development+of+autism-like+behaviors+in+mice.">Maternal immune activation and strain specific interactions in the development of autism-like behaviors in mice.</a> Transl Psychiatry. 3, e240 (2013).</li><li>Khan, 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=Long-term+effects+of+maternal+immune+activation+on+depression-like+behavior+in+the+mouse.">Long-term effects of maternal immune activation on depression-like behavior in the mouse.</a> Transl Psychiatry. 4, e363 (2014).</li><li>Workman, A. D., Charvet, C. J., Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+transformations+of+neurodevelopmental+sequences+across+mammalian+species.">Modeling transformations of neurodevelopmental sequences across mammalian species.</a> J Neurosci. 33 (17), 7368-7383 (2013).</li><li>Abazyan, B., 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=Prenatal+interaction+of+mutant+DISC1+and+immune+activation+produces+adult+psychopathology.">Prenatal interaction of mutant DISC1 and immune activation produces adult psychopathology.</a> Biol Psychiatry. 68 (12), 1172-1181 (2010).</li><li>Meyer, U., 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=Adult+behavioral+and+pharmacological+dysfunctions+following+disruption+of+the+fetal+brain+balance+between+pro-inflammatory+and+IL-10-mediated+anti-inflammatory+signaling.">Adult behavioral and pharmacological dysfunctions following disruption of the fetal brain balance between pro-inflammatory and IL-10-mediated anti-inflammatory signaling.</a> Mol Psychiatry. 13 (2), 208-221 (2008).</li><li>Vuillermot, S., 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=Prenatal+immune+activation+interacts+with+genetic+Nurr1+deficiency+in+the+development+of+attentional+impairments.">Prenatal immune activation interacts with genetic Nurr1 deficiency in the development of attentional impairments.</a> J Neurosci. 32 (2), 436-451 (2012).</li><li>Bechard, A., Nicholson, A., Mason, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Litter+size+predicts+adult+stereotypic+behavior+in+female+laboratory+mice.">Litter size predicts adult stereotypic behavior in female laboratory mice.</a> J Am Assoc Lab Anim Sci. 51 (4), 407-411 (2012).</li><li>Harvey, L., 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=A+stereological+comparison+of+GAD67+and+reelin+expression+in+the+hippocampal+stratum+oriens+of+offspring+from+two+mouse+models+of+maternal+inflammation+during+pregnancy.">A stereological comparison of GAD67 and reelin expression in the hippocampal stratum oriens of offspring from two mouse models of maternal inflammation during pregnancy.</a> Neuropharmacology. 62 (4), 1767-1776 (2012).</li></ol>

The authors have nothing to disclose.

MIA induzione a differenti finestre temporali perturba diversi eventi di sviluppo del cervello nei roditori, e di conseguenza porta a diverse anomalie comportamentali e neuropatologie nella prole. Qui, abbiamo descritto un protocollo per indurre MIA nei topi con poli (I: C) iniezione E12.5. Questo metodo di MIA induzione porta a comportamentale, neurologiche, immunologiche, e le anomalie gastrointestinali associati con l&#39;autismo e la schizofrenia nella prole 8,10,11,16. È importante notare che, a causa M...

Il concetto di attivazione immunitaria materna (MIA) proviene dagli studi epidemiologici sulla associazione tra infezione materna con autismo e la schizofrenia 1. A causa dell&#39;assenza di patogeni virali rilevabili replicanti nella placenta o cervello fetale dopo l&#39;infezione virale materna 2,3, l&#39;effetto dell&#39;infezione sulla prole è ipotizzato essere causato dalla attivazione del sistema immunitario materno piuttosto che i patogeni stessi . Per chiarire il rapporto causa-effetto tra MIA e disturbi psichiatrici, l&#39;iniezione di sintesi chimica, virale mimica doppio polyinosinic RNA incagliato: acido policitidilico (poli (I: C)) in roditori in gravidanza è stato ampiamente utilizzato come modello animale per MIA 4,5. Poli (I: C) è riconosciuto da toll-like receptor 3 (TLR3), e la somministrazione sistemica di poli (I: C) induce risposta infiammatoria acuta virale simile. Uno dei meccanismi attraverso i quali poli (I: C) produces anomalie comportamentali e neuropatologie nella prole è causando uno squilibrio di citochine pro-infiammatorie e anti-nell&#39;asse materno-placentare-fetale 6. Diversi gruppi hanno adottato il modello MIA per comprendere l&#39;eziologia dei disturbi psichiatrici 7, ed a causa dei diversi interessi tra gruppi di ricerca, vari momenti di attivazione immunitaria sono stati utilizzati per ottenere diverse perturbazioni sullo sviluppo del cervello e comportamenti 7. Il laboratorio di Paul H. Patterson presso il California Institute of Technology adotta la strategia di iniezione di poli (I: C) in topi incinta a embrionali 12,5 giorni (E12.5), che ha dimostrato con successo che MIA è in grado di indurre comportamentale, neurologica, e cambiamenti immunologici nella prole che sono associati con l&#39;autismo e la schizofrenia 8-11. I nostri lavori precedenti mostrano che MIA prole visualizzare anomalie comportamentali (ad esempio, impairmen socialit, deficit di comunicazione, comportamento ripetitivo, comportamenti ansia-like, e latente deficit di inibizione 8,10,12), immunitario disregolazione e citochine squilibrio 8,13,14, alterazione del feto espressione genica del cervello 15, la perdita di cellule di Purkinje nel lobo VII di cervelletto 11, alterazione delle proprietà sinaptiche nell&#39;ippocampo 9, l&#39;interazione gene x ambiente 13, alterazione della permeabilità intestinale, intestino e la composizione del microbiota 16. Inoltre, strategie terapeutiche e profilattiche sono anche sviluppati da questa 13,16,17 sistema modello. Inducendo MIA a E12.5, altri hanno dimostrato che MIA produce attivazione della microglia fetale e colinergica alterazione dello sviluppo nel prosencefalo basale 18, ceppo specifico interazione 19, cervello sinaptosomiali cerebrale alterazioni ultrastrutturali, cerebrali respiratoria mitocondriale abnormalties catena iperfunzione, sottoregolazione di molecole sinaptosomiali cerebrali 17 ,comportamenti depressivi-like, compromissione della cognizione e ippocampo potenziamento a lungo termine (LTP) e deficit di adulti neurogenesi ippocampale 20. Qui, forniamo un metodo dettagliato di come indurre MIA a E12.5 da poli (I: C), così come paradigmi di come applicare questo modello per studiare l&#39;eziologia di autismo e la schizofrenia. È importante notare che MIA è un fattore di rischio per una varietà di disturbi 4, ed i suoi risultati sono estremamente sensibili al tempo e metodo di induzione e l&#39;allevamento delle dighe in gravidanza. Come tale, anche incongruenze minori tra i laboratori spesso sfociano in bassa riproducibilità e / o differenti fenotipi nella prole. Il nostro metodo è specificamente progettato per chi è interessato a studiare MIA come un fattore di rischio ambientale per l&#39;autismo e la schizofrenia, e la descrizione dettagliata fornita aiuterà i ricercatori a migliorare la riproducibilità dei dati.

<table><tbody><tr><td>Polyinosinic&amp;ndash;polycytidylic acid potassium salt</td><td>SIGMA</td><td>P9582</td><td></td></tr><tr><td>0.9% sodium chloride INJ. USP</td><td>HOSPIRA</td><td>NDC 0409-4888-10</td><td></td></tr><tr><td>MONOJECT insuline syrinage 3/10 ml 29 G x 1/2&quot;</td><td>COVIDIEN</td><td>8881600145</td><td></td></tr><tr><td>50 ml conical screw cap tubes</td><td>USA SCIENTIFIC</td><td>1500-1211</td><td></td></tr><tr><td>Nanodrop 1000 spectrophotometer</td><td>THERMO SCIENTIFIC</td><td>1000</td><td>Optional</td></tr><tr><td>Stereomicroscope</td><td>Wild Heerbrugg</td><td>M5A</td><td>Optional</td></tr><tr><td>Dumont #5 Forceps Inox Tip Size .10 X .06 mm</td><td>Roboz</td><td>RS-5045</td><td>Optional</td></tr><tr><td>RNAlater RNA stabilization reagent</td><td>Qiagen</td><td>76104</td><td>Optional</td></tr><tr><td>TRIzol reagent</td><td>Life Technologies</td><td>15596-026&amp;nbsp;</td><td>Optional</td></tr><tr><td>RQ1 Rnase-free DNase</td><td>Promega</td><td>M610A</td><td>Optional</td></tr><tr><td>iScript cDNA synthesis kit</td><td>Bio-Rad</td><td>170-8891</td><td>Optional</td></tr><tr><td>FastStart universal SYBR green master mix with ROX&amp;nbsp;</td><td>Roche</td><td>4913922001</td><td>Optional</td></tr><tr><td>Real-time PCR</td><td>ABI</td><td>7300</td><td>Optional</td></tr><tr><td>Primer: Il6 forward</td><td>Life Technologies</td><td>TAGTCCTTCCTACCCCAATTTCC</td><td>Optional</td></tr><tr><td>Primer: Il6 Reverse</td><td>Life Technologies</td><td>TTGGTCCTTAGCCACTCCTTC</td><td>Optional</td></tr><tr><td>Primer: beta-actin forward</td><td>Life Technologies</td><td>AGAGGGAAATCGTGCGTGAC</td><td>Optional</td></tr><tr><td>Primer: beta-actin Reverse</td><td>Life Technologies</td><td>CAATAGTGATGACCTGGCCGT</td><td>Optional</td></tr><tr><td>MicroAmp optical 96-well reaction plate</td><td>Life Technologies</td><td>4306737</td><td>Optionl</td></tr><tr><td>MicroAmp optical adhesive film&amp;nbsp;</td><td>Life Technologies</td><td>4311971</td><td>Optionl</td></tr><tr><td>EthoVision</td><td>Noldus</td><td>EthoVision</td><td>Optionl</td></tr><tr><td>SR-LAB apparatus (PPI)</td><td>San Diego Instruments&amp;nbsp;</td><td>SR-LAB</td><td>Optionl</td></tr><tr><td>Marbles</td><td>PENN-PLAX</td><td>Blue gem stones marbles</td><td>Optionl</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>Life Technologies</td><td>21600-069</td><td>Optionl</td></tr><tr><td>Paraformaldehyde</td><td>MACRON</td><td>2621-59</td><td>Optionl</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000 S</td><td>Optionl</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>Optionl</td></tr><tr><td>Triton x-100</td><td>Sigma</td><td>X100</td><td>Optionl</td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma</td><td>18312</td><td>Optionl</td></tr><tr><td>Goat serum</td><td>Vector Laboratories</td><td>S-1000</td><td>Optionl</td></tr><tr><td>Rabbit anti-calbindin antibody</td><td>Abcam</td><td>ab11426</td><td>Optionl</td></tr><tr><td>Biotinlyated goat anti-rabbit IgGantibody</td><td>Vector Laboratories</td><td>BA-1000</td><td>Optionl</td></tr><tr><td>VECTASTAIN ABC Kit</td><td>Vector Laboratories</td><td>PK-4000</td><td>Optionl</td></tr></tbody></table>

induction of maternal immune activation in mice at mid-gestation stage with viral mimic poly(i:c)

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.

<html> L&#39;iniezione di 20 mg / kg poli (I: C) a E12.5 potrebbe evocare una risposta infiammatoria acuta nell&#39;asse materno-placentare-fetale e precipitare un effetto cronica allo sviluppo del cervello e fenotipi comportamentali 12,13. Elevati livelli di citochine proinfiammatorie una, interleuchina (IL) -6, è un indicatore affidabile della risposta infiammatoria acuta dopo MIA. Il momento di picco per IL6 espressione genica nella placenta e nel cervello del feto...

Watch this Scientific Journal Video about Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C at JoVE.com

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

Induzione di materna immunitario attivazione nei topi a Mid-gestazione stage con Viral Mimic poli (I: C)

Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

induction-maternal-immune-activation-mice-at-mid-gestation-stage-with

Research

JoVE Journal

Immunology and Infection

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

18.2K Views.  California Institute of Technology. Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

Video: Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Un modello murino di Nell&#39;utero Trapianto

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Ricerca

Medicina

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

Valutazione della materna vascolare rimodellamento durante la gravidanza in utero mouse

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Biologia dello sviluppo

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Uso di<em&gt; In vivo</em&gt; Imaging per monitorare la progressione della Sperimentale mouse infezione da citomegalovirus nei neonati

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Immunologia e infezioni

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Dissezione Innate Immune Signaling in Evasion virale di produzione di citochine

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

Manipolazione CRISPR mirata alla placenta del mouse in vivo

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Genetica

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

L&#39;isolamento dei leucociti di tessuti murini all&#39;interfaccia materno-fetale

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

Intracerebroventricular e intravascolare Iniezione di Viral particelle e fluorescenti microperle nel cervello neonatale

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Neuroscienze

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, termed chorioamnionitis. Chorioamnionitis exposes the developing fetus to a high risk of organ injuries, perinatal morbidity, and mortality, as well as life-long neurobehavioral impairments and other non-neurological developmental issues. The two most frequent subtypes of GBS isolates from maternal and fetal tissues are serotypes Ia (13%-23%) and III (25%-53%). Our lab has developed and characterized a rat model of GBS-induced chorioamnionitis to study subsequent impacts on the central nervous system of the developing fetus and to understand underlying mechanistic aspects. This article presents the design as well as uses of the preclinical rat model, which closely reproduces the hallmark of GBS-induced chorioamnionitis in humans. This article aims to help scientists reproduce the experimental design as well as to provide support through examples of troubleshooting. The present model may also contribute to potential discoveries through uncovering causes, mechanisms, and novel therapeutic avenues, which remain unsettled in many developmental impairments arising from chorioamnionitis. Furthermore, the use of this model may be extended to the studies of perinatal non-neurological common and severe morbidities affecting, for instance, the retina, bowel, lung, and kidney. The main interest of this research is in the field of GBS-induced fetal neurodevelopmental impairments such as cerebral palsy (CP), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD). The rationale supporting this model is presented in this article, followed by procedures and results.

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus GBS

The goal of this protocol is to describe a preclinical animal model of Group B Streptococcus (GBS)-induced chorioamnionitis. The study is designed to investigate mechanistic processes, potential causal links with developmental impairments, and finally to develop translational anti-inflammatory placento- and neuro-protective treatments.

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Un modello murino di esposizione fetale all'infiammazione materna per studiare gli effetti della corioamionite acuta sullo sviluppo intestinale appena nato

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in offspring. Animal models of MIA are used to test causality, investigate mechanisms, and develop diagnostics and treatments for these disorders. Despite their widespread use, many MIA models suffer from a lack of reproducibility and almost all ignore two important aspects of this risk factor: (i) many offspring are resilient to MIA, and (ii) susceptible offspring can exhibit distinct combinations of phenotypes. To increase reproducibility and model both susceptibility and resilience to MIA, the baseline immunoreactivity (BIR) of female mice before pregnancy is used to predict which pregnancies will result in either resilient offspring or offspring with defined behavioral and molecular abnormalities after exposure to MIA. Here, a detailed method of inducing MIA via intraperitoneal (i.p.) injection of the double stranded RNA (dsRNA) viral mimic poly(I:C) at 12.5 days of gestation is provided. This method induces an acute inflammatory response in the dam, which results in perturbations in brain development in mice that map onto similarly impacted domains in human psychiatric and neurodevelopmental disorders (NDDs).

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring

Maternal infection is a risk factor for neurodevelopmental disorders. Mouse models of maternal immune activation (MIA) may elucidate infection's impact on brain development and function. Here, general guidelines and a procedure are provided to produce reliably resilient and susceptible offspring exposed to MIA.

Generazione di un modello riproducibile di attivazione immunitaria materna a metà gestazionale utilizzando poli (I: C) per studiare la suscettibilità e la resilienza nella prole

Induzione di materna immunitario attivazione nei topi a Mid-gestazione stage con Viral Mimic poli (I: C)

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