Gli autori desiderano ringraziare il personale dagli impianti di risonanza magnetica presso l&#39;Semmes-Murphey Clinic, Memphis, Tennessee così come il reparto di Neurochirurgia presso l&#39;Università del Tennessee Health Science Center a Memphis, Tennessee.

1. Preparazione di gel Phantoms e Dye <ol><li> Preparare 0,2% gel di agarosio sciogliendo 2 g di 0,1% agarosio in polvere in 1000 ml di acqua deionizzata. Agitare la soluzione per circa 1 min per assicurare la corretta miscelazione; e forno a microonde immediatamente la soluzione in 3 min intervalli di 9 minuti o fino a quando chiara, mescolando tra gli intervalli. </li><li> Mentre il gel di agarosio è liquido, versare la soluzione in 5 cm x 5 cm x 5 centimetri contenitori. Lasciare spazio nella parte superiore del contenitore di aggiungere acqua e lasciare il gel di agarosio raffreddare e decantare. </li><li> Una volta che il gel agarosio si è solidificato (circa 1-2 ore), aggiungere 1 cm di acqua alla superficie del gel e conservare in frigorifero. E &#39;meglio usare il gel entro 24-48 h di miscelazione, ma può essere conservato per un massimo di una settimana refrigerato 10. </li><li> Preparare un colorante di contrasto radio in una siringa da 60 ml con 50 ml di 0,017% blu di bromofenolo colorante (BPB), e 2 mM di Gadoteridolo mezzi di contrasto radio. <ol><li> Combina 8.5 mg di BPB tintura a 50 ml acqua deionizzata per creare una soluzione di BPB 0,017%. </li><li> Aggiungere 0,2 ml di 0,5 M magazzino Gadoteridolo alla soluzione BPB 50 ml 0,017% per creare una soluzione di Gadoteridolo 2 mM. </li></ol></li></ol> 2. Preparazione del Sistema di Infusione <ol><li> Sistema di infusione pompa a siringa (metodo preferito): Per la preparazione pompa a siringa, collegare il catetere di infusione direttamente alla siringa attraverso il sensore di pressione, riducendo il volume morto della linea di infusione. La funzione di spurgo della pompa siringa può essere utilizzata per pulire la linea di aria utilizzando un bolo superiore al volume di priming del catetere ad una velocità di 10 ml / min. </li><li> Sistema di infusione pompa Tube (metodo alternativo): Collegare la siringa contenente il colorante radio contrasto con la pompa per infusione. Fissare il sensore di pressione alla mandata della pompa con il trasduttore collegato al monitor IV. Attaccare un catetere di infusione 16 G all&#39;estremità aperta del sensore di pressione. Nota: La punta del catetere G 16 di infusione ha un diametro internometro di 0,2 mm e un diametro esterno di 0,35 mm. La punta è fatta di silice fusa e la lunghezza della punta è di 3 mm. Aumenta di a circa 0,75 mm, continua per 15 mm, il catetere poi passi in modo conico a 1,6 mm o 16 G. </li><li> Preparare per infusione da spurgo del sistema per circa 15 min a 16.667 ml / min per rimuovere eventuali bolle d&#39;aria. Non superare la portata di 16.667 ml / min, in quanto la macchina cesserà di infusione a causa della pressione alta linea. Seguendo il fissaggio del catetere di infusione alla linea che esce dalla pompa di infusione, linee di spurgo d&#39;aria usando la funzione &quot;bolo&quot; della pompa di infusione. </li><li> Fissare il supporto del catetere di infusione e telaio traiettoria al contenitore gel phantom (5 centimetri x 5 cm x 5 cm) e posto in MRI. </li></ol> 3. CED Gel Infusion e MR scansione <ol><li> Azzerare il valore della pressione (mmHg) registrati dal monitor IV prima di iniziare l&#39;infusione. </li><li> Inserire il catetere di infusione nel gel di agarosio with la pompa di infusione esecuzione alla portata più bassa, in questo caso 1.667 ml / min. </li><li> Iniziare la scansione MR, utilizzando i parametri elencati nella Tabella 1, e continuare l&#39;infusione ad una velocità di 1.667 ml / min. Infondere il gel ad una velocità costante fino al volume totale infuso raggiunge i 60 microlitri (circa 38 minuti). </li><li> Eseguire la scansione del gel continuamente in 3 minuti e 50 secondi intervalli. Registrare le letture della pressione ogni 60 sec. Una volta che il volume infuso raggiunge i 60 microlitri, spegnere la pompa di infusione; e la scansione MR completo, pur continuando a registrare valori di pressione. </li></ol> 4. MR Analisi dei dati <ol><li> Per analizzare le immagini RM, utilizzare un visualizzatore DICOM appropriata con funzionalità di segmentazione ROI. </li><li> Selezionare il frame corretto in ogni scansione segnato dalla sezione trasversale del catetere come si vede nella Figura 1. </li><li> Utilizzando il &quot;ROI - bottone&quot; Strumento, selezionare la parte più grande del gel che non include alcunparte del sito di infusione. La volontà di uscita software una densità di pixel media con deviazione standard. Trovare il valore pari a tre deviazioni standard dalla media. Questo valore viene utilizzato come soglia per determinare quando il contrasto è presente con una confidenza del 99,7%. </li><li> Utilizzando il &quot;ROI - circolo&quot; strumento, circondare il sito di infusione con una abbastanza grande cerchio e dare a questo un nome univoco. </li><li> Selezionare il cerchio e con il &quot;ROI - impostare i valori dei pixel a&quot; utensile, valori di soglia d&#39;ingresso si trovano in fase 4.3 in &quot;se il valore corrente è maggiore:&quot; la casella e segno di spunta solo questa linea. Poi, in &quot;a questo nuovo valore:&quot; la casella, immettere un valore grande (25.000). Aggiorna la densità di pixel per selezionare l&#39;area compresa all&#39;interno soglia precedentemente definito. </li><li> Successivamente, utilizzando il &quot;ROI - crescere regione (2D/3D segmentazione)&quot; strumento, selezionare la regione 2D in crescita, algoritmo di fiducia con il parametro raggio iniziale = 2, e pennello ROI. Fare clic all&#39;interno del sito di infusione per il software per calcolare la superficie totale of questa regione. </li><li> Assumendo una nuvola infusione sferica, calcolare il volume di diffusione dalla zona tramite la seguente equazione: V = 4/3π (√ (Area / π)) 3 </li></ol>

<ol>
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R., 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=Successful+and+safe+perfusion+of+the+primate+brainstem:+in+vivo+magnetic+resonance+imaging+of+macromolecular+distribution+during+infusion.">Successful and safe perfusion of the primate brainstem: in vivo magnetic resonance imaging of macromolecular distribution during infusion.</a> Journal of neurosurgery. 97, 905-913 (2002).</li><li>Raghavan, R., 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=Convection-enhanced+delivery+of+therapeutics+for+brain+disease,+and+its+optimization.">Convection-enhanced delivery of therapeutics for brain disease, and its optimization.</a> Neurosurg Focus. 20, (2006).</li><li>Sillay, 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=Benchmarking+the+ERG+valve+tip+and+MRI+Interventions+Smart+Flow+neurocatheter+convection-enhanced+delivery+system's+performance+in+a+gel+model+of+the+brain:+employing+infusion+protocols+proposed+for+gene+therapy+for+Parkinson's+disease.">Benchmarking the ERG valve tip and MRI Interventions Smart Flow neurocatheter convection-enhanced delivery system's performance in a gel model of the brain: employing infusion protocols proposed for gene therapy for Parkinson's disease.</a> Journal of neural engineering. 9, (2012).</li><li>Schomberg, D., Wang, A., Marshall, H., Sillay, K., Miranpuri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ramped-Rate+vs.+continuous+rate+infusions:+An+in+vitro+comparison+of+Convection+Enhanced+Delivery+protocols.">Ramped-Rate vs. continuous rate infusions: An in vitro comparison of Convection Enhanced Delivery protocols.</a> Annals of Neurosciences. 20, (2013).</li><li>Sillay, K. 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Gli autori dichiarano di non avere interessi finanziari in competizione.

<html> I passaggi critici per assicurare il successo dell&#39;infusione sono: spurgo della linea di infusione di aria, mescolando il gel di agarosio, analizzando i dati di RM, con piccoli diametri catetere interno, utilizzando intensificato disegni catetere per minimizzare riflusso, e riducendo al minimo la pressione percepita dal gel o tessuto in cui viene infusa farmaco. Come affermato in precedenza, il danno principale al successo della infusione è aria linea di infusione. Correttamente e completamente lo spurgo dell...

Convezione-enhanced consegna (CED) è stata proposta come opzione di trattamento per un ampio spettro di disturbi neurologici, tra cui tumori maligni cerebrali, epilessia, disordini metabolici, malattie neurodegenerative (come il morbo di Parkinson) 1, ictus e trauma 2. CED impiega bulk flow pressione positiva per la distribuzione di un farmaco o altro infusate. CED fornisce consegna sicura, affidabile e omogenea di composti con peso molecolare, che vanno dal basso verso l&#39;alto, a volumi clinicamente rilevanti 3. Somministrazione di farmaci tradizionali al tessuto cerebrale è fortemente limitata dalla barriera emato-encefalica 4. Formata dalle giunzioni strette tra le cellule endoteliali che costituiscono i capillari nel cervello, i blocchi barriera ematoencefalica polare e molecole ad alto peso molecolare di entrare nel parenchima del cervello. Diretto infusione cerebrale intraparenchimale via CED in grado di superare i limiti delle precedenti modalità di consegna della droga terapeutichee permette l&#39;uso di agenti terapeutici che non attraversa la barriera emato-encefalica, e quindi stato precedentemente non disponibili come opzioni di trattamento vitale 5. I ricercatori del National Institutes of Health degli Stati Uniti (NIH) hanno descritto CED nei primi anni 1990 come un mezzo per raggiungere maggiori concentrazioni terapeutiche di farmaco che da sola diffusione 6-8. I primi metodi di CED coinvolti impiantare uno o più cateteri nel cervello, collegando una pompa per infusione al catetere, e pompando gli agenti terapeutici direttamente nella regione di destinazione. La frazione di distribuzione maggiore e concentrazione relativamente stabile è riportata, come la pressione positiva creata dalla pompa di infusione provoca i tessuti si dilatano e permettono di permeazione del farmaco 9. La tecnica fondamentale per CED rimane in gran parte lo stesso che è stato descritto. I progressi nella progettazione catetere 10, tecnica di infusione <sup&gt; 11, il monitoraggio della pressione linea 2, e il monitoraggio MRI in tempo reale per correggere turno cervello 12, 13, ottimizzare le infusioni multiple collineari 14, e monitor per la perdita infusate 15 hanno aumentato la sicurezza e l&#39;efficacia del trattamento 10. Ulteriori importanza è stata posta nella progettazione del catetere e la strategia di infusione compreso la portata. Successo CED, con limitata reflusso catetere e danni ai tessuti, è stata correlata con il disegno del catetere e la velocità di infusione. L&#39;uso di un catetere con un diametro ridotto e una bassa velocità di infusione di limitare riflusso lungo l&#39;interfaccia cervello-catetere nonché limitare i danni alla punta del catetere 16. RM fornisce una conferma visiva della posizione corretta per il posizionamento del catetere di infusione, e quindi la consegna della droga, consentendo anche per la correzione di infusione di reflusso o di consegna aberrante 17. MR immagini possono anche essere utilizzato per stimare e monitorare i volumi di distribuzione (Vd) Del farmaco infuso. Il Vd è calcolato utilizzando un valore di intensità di segnale RM superiore a tre deviazioni standard sopra la media dal gel non infusa circostante come soglia per la segmentazione 18. Il Vd è una misura utile per CED perché rappresenta il volume del farmaco distribuiti nel cervello. Insieme al volume infuso (Vi), un rapporto può essere generato (Vd / Vi) quantificare il volume coperto dal farmaco infuso. Fantasmi gel di agarosio imitano diversi cruciali proprietà meccaniche del cervello umano importanti per la comprensione CED quali: Vd, interazioni gel-catetere, proprietà poroelastic e infusione nube morfologia 10. Miscugli di 0,2% gel di agarosio hanno dimostrato di mimare cambiamenti in vivo in frazione poro locale causate da gel dilatazione a causa CED. Una frazione poro simile a cervello umano promuove interazioni simili e misure accurate di Vd 19. Inoltre, simili concentrazioni di ungel garose come 0,6% e 0,8% hanno mostrato profili di pressione di infusione simili al cervello 20. Inoltre, i gel di agarosio traslucide forniscono il vantaggio di visualizzazione in tempo reale di posizionamento del catetere e l&#39;infusione reflusso. Fantasmi gel di agarosio sono relativamente poco costosi da produrre. Il costo dei fantasmi gel di agarosio può essere la chiave per il futuro della formazione diffusa in tutto neurochirurgia. Grazie a queste proprietà, gel di agarosio forniscono un utile surrogato, replicando molti degli attributi chiave di infusioni cervello umano senza l&#39;uso del tessuto cerebrale. Come detto sopra, guidata dalle immagini CED in modelli di gel di agarosio fornisce un vantaggioso metodo in vitro per la prova, la ricerca, e la formazione. Lo scopo di questo articolo è quello di descrivere come ricreare fantasmi gel agarosio, a delineare opportuni protocolli di test e analisi CED, e per affrontare gli errori più comuni affrontate durante le infusioni CED per il trattamento della malattia neurologica.

<table border="0" cellpadding="0" cellspacing="0" width="590">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Prohance</td>
			<td style="width:71px;">Bracco</td>
			<td style="width:119px;"></td>
			<td style="width:195px;">Gadoteridol radio contrast media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Bromophenol Blue Dye</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-0404</td>
			<td>Dye for infusate visualization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Agarose Gel Powder</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-3101EDU</td>
			<td>Agarose powder for creating gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Veris MR Vital Signs Monitor</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pressure monitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">16 Gauge SmartFlow Catheter</td>
			<td style="width:71px;">SurgiVision</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Continuum MR Infusion System</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">SMART Frame MRI Guided Trajectory Frame</td>
			<td style="width:71px;">ClearPoint</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter frame</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:205px;">Osirix Imaging Software and DICOM Viewer</td>
			<td style="width:71px;">Osirix Imaging Software</td>
			<td style="width:119px;"></td>
			<td>OsiriX 32-bit DICOM Viewer</td>
		</tr>
	</tbody>
</table>

image-guided convection-enhanced delivery into agarose gel models of the brain

Convezione avanzata consegna (CED) è stata proposta come opzione di trattamento per una vasta gamma di malattie neurologiche. Neuroinfusion catetere CED permette di flusso di massa pressione positiva per fornire maggiori quantità di terapie per un obiettivo intracranica rispetto ai metodi di somministrazione dei farmaci tradizionali. L&#39;utilità clinica del reale tempo MRI guidate CED (rCED) si trova nella capacità di colpire con precisione, monitorare la terapia e identificare le complicanze. Con la formazione, rCED è efficiente e le complicanze possono essere minimizzati. Il modello gel di agarosio del cervello fornisce uno strumento accessibile per i test CED, la ricerca e la formazione. Cervello simulato rCED permette la pratica della chirurgia finto fornendo anche un feedback visivo della infusione. Analisi di infusione consente di calcolare la frazione di distribuzione (Vd / Vi) permettendo l&#39;allievo per verificare la somiglianza del modello rispetto al tessuto cerebrale umano. In questo articolo si descrive il nostro gel di agarosio phantom cervello e delinea importante metrics durante una CED protocolli di infusione e di analisi, mentre affrontando le insidie ​​più comuni affrontate durante CED di infusione per il trattamento della malattia neurologica.

Interpretazione e analisi di infusi CED coinvolgono diversi fattori importanti quali la frazione di distribuzione e reflusso infusate. Il calcolo frazione distribuzione dipende pesantemente sul calcolo del Vd. Pertanto, l&#39;interpretazione accurata delle immagini RM è fondamentale. Proponiamo un metodo semi-automatico per riprodurre in modo affidabile queste misure, come riportato sopra. Questi metodi oggettivamente determinare l&#39;area della sezione trasversale della nube infusate e un raggio approssimativo. Mentr...

Watch this Scientific Journal Video about Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain at JoVE.com

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convezione avanzata consegna (CED) è stata proposta come opzione di trattamento per una vasta gamma di malattie neurologiche. Al fine di preparare professionisti sanitari per l&#39;adozione del CED, sono necessari modelli di formazione accessibili. Descriviamo l&#39;uso di gel di agarosio come un tale modello di cervello umano per il test, la ricerca, e la formazione.

Immagine-guida di consegna convezione-enhanced in gel di agarosio Modelli del cervello

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

image-guided-convection-enhanced-delivery-into-agarose-gel-models

Research

JoVE Journal

Medicine

11.3K Views.  University of Tennessee Health Science Center.  Convezione avanzata consegna (CED) è stata proposta come opzione di trattamento per una vasta gamma di malattie neurologiche. Al fine di preparare professionisti sanitari per l'adozione del CED, sono necessari modelli di formazione accessibili. Descriviamo l'uso di gel di agarosio come un tale modello di cervello umano per il test, la ricerca, e la formazione. 

Video: Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Ricerca

Medicina

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Eterotopico mucosa engrafting Procedura di Drug Delivery diretto al cervello nei topi

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Mouse trapianto renale: Modelli di Allograft Rifiuto

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

Transposon Mediated Integrazione di DNA plasmide nella zona subventricolare di topi neonati a generare modelli Novel di Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Metodo ottimizzato di LC-MS/MS per l'analisi di High-throughput di campioni clinici di Ivacaftor, suoi principali metaboliti e Lumacaftor nei fluidi biologici di pazienti affetti da fibrosi cistica

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Migliorato il metodo per la creazione di un In Vitro barriera emato - encefalica modello basato sulle cellule endoteliali Porcine cervello

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Immagine-Guida di resezione del Glioblastoma e l'impianto intracranico di terapeutica delle cellule staminali-seminato ponteggi

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

Predire la risposta di trattamento alle terapie immagine-guida utilizzando Machine Learning: un esempio per Trans-arteriosa trattamento dell'epatocarcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Un'immagine guidata Transtral Valve Opuscolo Puntura Modello di Puntura del Sovraccarico di Volume Controllato da Rigurgitazione Mitrale nel Ratto

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

Somministrazione intratracheale della formulazione di polvere secca nei topi

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...