Gli autori desiderano ringraziare il Dr. Hui Zheng (Baylor College of Medicine) per la APP cDNA. Questo lavoro è stato finanziato da sovvenzioni NIH R21AG031948 (DB, JMB), F30AG030878 (ESS), R01DK073394 (AFO), il John Sealy Memorial Fondo di dotazione per la ricerca biomedica (AFO), e il C. Jean e William D. Willis Neuroscience Research Endowment (ESS). JMB è uno studioso nel Programma Translational Research Scholar e membro della University of Texas Medical Branch Claude E. Independence Pepper Centro anziani americani (supportato da sovvenzioni NIH UL1RR029876 e P30-AG-024832, rispettivamente).

<html> 1. L&#39;espressione di APP ricombinante dominio intracellulare (AICD) <ol><li> Transform E. coli ceppo BL21 con AICD umana (residui 649-695 di APP, isoforma neuronale numerazione) clonato nel vettore pGEX-4T-1 (GE Healthcare). Questo vettore esprimerà AICD la porzione C-terminale di una proteina di fusione di glutatione-S-transferasi (GST). Questo vettore codifica anche una sequenza trombina clivaggio per facilitare la rimozione della porzione GST. Dettagli della clonazione AICD in pGEX-4T-1 può essere trovata nella nostra precedente pubblicazione 4. </li><li> Da una singola colonia, inoculare 10 ml di brodo LB con ampicillina (100 ug / ml), e incubare una notte a 37 ° C con agitazione vigorosa. </li><li> Il mattino seguente, inoculare 400 ml LB / ampicillina in una beuta da un litro con 5 ml di coltura durante la notte. </li><li> Incubare a 37 ° C con agitazione vigorosa fino a quando la densità ottica a 600 nm ha raggiunto 0,4-0,6. Questo normalmente 2-2,5 ore. </li></ol>contenuto &quot;&gt; A questo punto può essere desiderabile per prendere un piccolo campione (~ 10 pl) di seguire la purificazione mediante SDS-PAGE (Figura 1A, B). Ogni fase successiva purificazione deve anche avere una piccola aliquota rimossi per l&#39;analisi mediante SDS-PAGE. <ol start="5"><li> Indurre l&#39;espressione di GST-AICD aggiungendo 0,4 mM di isopropil-beta-D-tiogalattopiranoside (IPTG). Incubare a 37 ° C con agitazione vigorosa per 5 ore. </li><li> Centrifugare le cellule a 6.000 xg per 15 min a 4 ° C. Il pellet cellulare può essere conservato a -80 ° C per diversi mesi, a questo punto del procedimento. </li></ol> 2. Purificazione di GST-AICD <ol><li> Preparare 50 ml di tampone di lisi come segue: 1 mM ditiotreitolo 1 mM acido etilendiamminotetraacetico (EDTA), 0,1% Triton-X100, 1 mM fenilmetilsulfonil fluoruro (PMSF), Complete cocktail inibitore di proteasi (Roche Applied Science), e tampone fosfato. Freddo sul ghiaccio. </li><li> Scongelare pellet batterico (se precedentemente congelato) sullaghiaccio. </li></ol> A questo punto, è fondamentale che tutti i passi vengono eseguiti a 4 ° C per limitare proteolisi. Tutti i tubi, tamponi, e altri reagenti devono essere pre-refrigerati. Se si utilizza una pressa francese o emulsionante per lisi, questi dovrebbero essere pre-refrigerati pure. <ol start="3"><li> Accuratamente risospendere il pellet batterico in 20 ml di tampone di lisi. Una combinazione di vortex e triturazione con pipetta 10 ml di ghiaccio assicurerà completa risospensione del pellet. </li><li> Far passare le cellule risospese batteriche attraverso un emulsionante (ad esempio, Avestin Emulsiflex-C3) o cellule pressione francese, pre-refrigerati a 4 ° C a 30.000 psi. Passare il lisato attraverso l&#39;emulsionante una seconda volta. Raccogliere il lisato in un pre-refrigerati tubo da 50 ml. </li></ol> Il vantaggio principale di questi due strumenti è che minimizzare il riscaldamento del campione. La tecnica comunemente usata di sonicazione genera calore sulla punta della sonda sonicazione epuò causare aggregazione di proteine ​​ricombinanti. <ol start="5"><li> Centrifugare il lisato a 15.000 xg per 30 min. Separare e mantenere il supernatante in un pre-raffreddata tubo da 50 ml. </li><li> Aggiungere 500 pl di un impasto al 50% di glutatione-agarosio (Sigma-Aldrich). Ruotare per 30 min a 4 ° C. Mentre il campione sta ruotando, fanno tampone fresco eluizione: 50 mM Tris 7,8, 0,1% Triton-X100, 0,5 mM di ditiotreitolo, 10 mM glutatione ridotto. </li></ol> L&#39;unico passo del protocollo che viene eseguita a temperatura ambiente è la fase di eluizione. Così, il tampone di eluizione viene mantenuta a temperatura ambiente. Buffer di eluizione deve essere preparato fresco. <ol start="7"><li> Versare il lisato e dei liquami in un usa e getta 5 &quot;colonna polistirolo cromatografia con grossolani (90-130 micron) filtri (Evergreen scientifico). Lavare la colonna 5 volte con 3 ml di tampone fosfato. </li><li> Tappare il fondo della colonna e aggiungere 500 microlitri di tampone di eluizione temperatura ambiente. Cap all&#39;inizio dellacolonna e ruotare per 5 min a temperatura ambiente. Raccogliere l&#39;eluato in una provetta da microcentrifuga refrigerata 1,5 ml. Ripetere l&#39;eluizione altre due volte (1.5 ml eluato totale). </li><li> Dializzare l&#39;eluato in un molecolare 10000 membrana di dialisi taglio di peso (ad esempio, Thermo Scientific Slide-A-lizzatore dialisi cassette) in 4 litri di soluzione salina tamponata per una notte a 4 ° C. Dializza contro un ulteriore 4 litri di tampone fosfato a 4 ° C per 1 ora il giorno seguente. </li><li> Rimuovere la proteina dalla cassetta dialisi ed eseguire un test per determinare la quantificazione delle proteine ​​resa di GST-AICD. Una purificazione tipico da 400 ml di coltura comporta rendimenti di&gt; 20 mg di proteina purificata a concentrazioni di 10-20 mg / ml. Aliquotare la proteina in 200 pl aliquote e congelati a -80 ° C. </li></ol> 3. Cleavage trombina di GST-AICD e Purificazione di AICD <ol><li> Scongelare 200 ml di acqua di GST-AICD e aggiungere 20 ml di 1 U / mL thrombtrovi Incubare per una notte a 37 ° C. </li></ol> La qualità e la purezza della trombina varia considerevolmente tra i costruttori, e può essere una fonte di contaminazione. Usiamo trombina da GE Healthcare (vedi tabella dei reagenti). Dopo incubazione per una notte,&gt; 95% della proteina di fusione deve essere scisso (Figura 1B). <ol start="2"><li> Dopo clivaggio notte con trombina, è probabile che una frazione del AICD liberato subito aggregazione e la soluzione può apparire torbida. Cancellare il materiale aggregato mediante centrifugazione a 20.000 xg per 30 min a 4 ° C. Rimuovere il surnatante in un pre-raffreddata provetta per microcentrifuga. </li><li> Aggiungere 50 ml di 50% di glutatione-agarosio slurry e 50 pl 50% p-aminobenzamidine-agarosio slurry per rimuovere GST e trombina, rispettivamente. Incubare con rotazione per 5 min a temperatura ambiente. </li><li> Centrifugare brevemente per sedimentare le perline di agarosio, e rimuovere il surnatante in una nuova provetta. Estratto tGST ha quattro volte più con glutatione-agarosio. Una singola estrazione con p-aminobenzamidine-agarosio è sufficiente a rimuovere il 20 U di trombina. </li><li> Eseguire un test di quantificazione delle proteine, il 5 microlitri della AICD purificato. Rese tipiche di una cultura 400 ml sono 50-100 mcg ad una concentrazione di 0,2 a 0,5 mg / ml. Rese più elevate sono possibili, con l&#39;adesione più grandi volumi di GST-AICD. </li></ol> Purificato AICD deve essere preparato fresco per la caratterizzazione biochimica / biofisica. Materiale non utilizzato deve essere eliminato. È anche possibile concentrare il AICD dalla liofilizzazione del campione e la risospensione in un volume più piccolo di tampone 6. Se la rilevazione AICD richiede assorbente (ad esempio dopo una trappola filtro o saggio dot blot), il recupero antigene sulla membrana deve essere eseguita come descritto da Pimplikar e Suryanarayana 12. 4. Aggregazione AICD per microscopia a forza atomica (AFM) <ol><li> Diluire appena preparata AICD in phosphate salina tamponata ad una concentrazione di 0,1 mg / ml in un volume finale di 15 microlitri. A questo punto, vari composti e / o le proteine ​​possono essere aggiunti per determinare se essi inibiscono l&#39;aggregazione AICD. Per esempio, abbiamo dimostrato che le concentrazioni equimolari di ubiquilin-1 prevenire l&#39;aggregazione di AICD in questo test 4. </li></ol> È anche possibile aggregare grandi volumi di reazione per esperimenti biochimici come saggi trap filtrazione e diffusione della luce 4. Abbiamo avuto successo eseguendo saggi trappola filtro su miscele di reazione più grande a 400 ml con AICD diluito a concentrazioni a partire da 1 pM. <ol start="2"><li> Inducono aggregazione termica incubando i campioni a 43 ° C con agitazione a 800 rpm in un Thermomixer Eppendorf per 48 ore. Questa temperatura è stato scelto sulla base di test di chaperone che esaminano l&#39;aggregazione termica del modello citrato sintasi accompagnatore substrato 4,13. </li></ol><p class="jove_content"Aggregazione&gt; termico è un metodo comunemente usato per indurre la transizione strutturale di una proteina da un nativo a una struttura non nativo (che di solito è ricca foglietto beta) che si traduce nella formazione di aggregati intermolecolari. Aggregazione termica è anche utilizzato per esaminare la funzione di chaperone molecolare, che proteggono i segmenti idrofobi da formare appropriato interazioni intermolecolari (e quindi ritardare o prevenire l&#39;aggregazione termica). <ol start="3"><li> Diluire il campione di 20 volte in acqua deionizzata e posto su 2 microlitri mica appena spaccati. Essiccare il campione in essiccatore notte. Sarà necessario determinare empiricamente la diluizione ottimale per immagini, e quindi diluizioni diverse da 10 - a 100 volte sarebbe prudente. </li><li> Visualizza la AICD aggregato AFM utilizza il sistema operativo in tapping mode 14. I cantilever utilizzati sono leve micro oro rivestite con nitruro di taglienti (MSNL, Bruker) con un raggio di punta nominale di 2 nm. Tipica toccando ampiezze duranteing imaging sono 10-20 nm alla frequenza di risonanza del cantilever (30-50 kHz). </li></ol> Abbiamo trovato che la qualità dell&#39;immagine e il contrasto di aggregati proteici differenti dipendeva dalla forza applicata e la durata dell&#39;esperimento. Per minimizzare perturbazione campione dalla punta, abbiamo mantenuto la forza applicata relativamente basso mantenendo un set-point rapporto superiore al 95% e limitando la scansione di ogni campione per 5-10 min. L&#39;elaborazione delle immagini è stata effettuata con il software WSxM (Nanotec). Elaborazione immagine standard consisteva piano sottrazione e appiattimento. I nostri laboratori utilizzano una &quot;casa-costruita&quot; AFM 15 interfacciato ad un sistema commerciale di sonda di controllo microscopio a scansione (Nanotec). 5. Risultati rappresentativi L&#39;espressione e arricchimento relativo di GST-AICD è mostrato nella Figura 1A. Dopo lisi, la maggior parte della proteina di fusione è presente nella frazione solubile e quindi estrazione da Inclusiil corpo non è necessaria. Il materiale eluito dalla colonna di glutatione agarosio è&gt; 95% puro. Nella preparazione mostrato nella figura 1, la concentrazione di proteina eluita dalla colonna era 14,5 mg / ml, e la resa era 22 mg (da una coltura iniziale di 400 ml). Clivaggio di 200 pl di questa preparazione overnight con 20 U di trombina comportato quasi il 100% clivaggio della proteina di fusione (Figura 1B). La quantità di trombina utilizzata è sufficientemente basso da non essere rilevabile mediante colorazione con Coomassie blue (vedi corsia &quot;Cleaved&quot;, Figura 1B). Rimozione della trombina e GST provocato una perdita di materiale, tuttavia era&gt; 90% puro con una resa in questa preparazione di 70 pg di AICD ad una concentrazione di 0,35 mg / ml. Figura 1C mostra il diagramma di flusso per l&#39;aggregazione di AICD e successiva analisi. Aggregati AICD ripreso da AFM sono tipicamente sferoide / amorfo, e la gamma nel formato da 50 a 100 nM (Figura 1D, F). Aggregatomateriale non è rilevabile quando una quantità equimolare di chaperone ubiquilin-1 viene aggiunto alla reazione di aggregazione (Figura 1E) 4. <img alt="Figura 1" src="/files/ftp_upload/4204/4204fig1.jpg" /> Figura 1. Purificazione e aggregazione di AICD. A) Coomassie colorazione blu di campioni raccolti in ogni fase del processo di purificazione. Per i campioni uninduced e indotta, 20 pl di batteri totali sono stati eseguiti sul gel. Per la frazione supernatante, 5 microlitri di lisato (da un volume totale di 20 ml) sono stati analizzati su gel. Per la frazione di pellet, il pellet è stato risospeso in 20 ml di tampone di lisi, e 5 microlitri di questo materiale sono stati sonicati in un sonicatore bagno di acqua per 30 minuti a 4 ° C prima di caricare il gel. Per l&#39;eluato, 5 microlitri sono stati eseguiti sul gel. Le quantità di eluato caricati sul gel sono indicati. B) Coomassie blu gel colorato di uncleaved e spaccati GST-AICD (2 ml) e 1 e 5 &amp; mu, l purificato AICD. C) Diagramma di flusso di purificazione AICD, aggregazione e analisi. D, E) Rappresentante AFM immagine termicamente aggregate AICD in assenza (D) e presenza (E) del chaperone molecolare ubiquilin-1. F) Istogramma della distribuzione delle dimensioni degli aggregati AICD.

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Nessun conflitto di interessi dichiarati.

In questo protocollo abbiamo delineato una procedura per ottenere AICD elevata purezza per analisi strutturali, biofisica e biochimica. Questa procedura non richiede attrezzature sofisticate cromatografia ed è quindi accessibile alla maggior parte dei laboratori. Altri gruppi hanno purificato AICD 5-7,16, compreso GST-AICD 17-19, per le analisi biochimiche / strutturale. Svantaggi ai protocolli precedenti includono scarsa solubilità di AICD 16, a meno di ideale purezza 17, e...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> pGEX-4T-1 </td><td> GE Healthcare </td><td> 28-9545-49 </td><td></td></tr><tr><td> Trombina </td><td> GE Healthcare </td><td> 27-0846-01 </td><td></td></tr><tr><td> Ampicillin </td><td> Fisher Scientific </td><td> BP1760 </td><td></td></tr><tr><td> Bradford proteine ​​reagenti del dosaggio </td><td> Bio-Rad </td><td> 500-0002 </td><td></td></tr><tr><td> Coomassie blu </td><td> Bio-Rad </td><td> 161-0786 </td><td></td></tr><tr><td> IPTG (isopropil-beta-D tiogalattopiranoside) </td><td> Sigma-Aldrich </td><td> I6758 </td><td></td></tr><tr><td> Glutatione-agarosio </td><td> Sigma-Aldrich </td><td> G4510 </td><td></td></tr><tr&gt; <td> p-aminobenzamidine-agarosio </td><td> Sigma-Aldrich </td><td> A7155 </td><td></td></tr><tr><td> Completa cocktail di inibitori delle proteasi </td><td> Roche </td><td> 11836170001 </td><td></td></tr><tr><td> Slide-A-lizzatore dialisi cassette </td><td> Thermo Scientific </td><td> 66380 </td><td></td></tr><tr><td> Cromatografia colonne </td><td> Evergreen scientifico </td><td> 208-3367-050 </td><td></td></tr><tr><td> Emulsionante </td><td> Avestin, Inc </td><td> Emulsiflex-C3 </td><td> Consigliatissimo </td></tr><tr><td> Eppendorf Thermomixer </td><td> Eppendorf </td><td> 022670107 </td><td></td></tr><tr><td> Mica dischi </td><td> Ted Pella </td><td> 50-12 </td><td></td></tr><tr><td> Cantilever AFM </td><td> Bruker </td><td> MSNL-10 </td><td></td></tr><tr><td> WSxM software </td><td> Nanotec </td><td> N / A </td><td> Pee scaricare </td></tr></tbody></table>

purification and aggregation of the amyloid precursor protein intracellular domain

Proteina precursore dell&#39;amiloide (APP) è un tipo di proteina transmembrana I associato alla patogenesi della malattia di Alzheimer (AD). APP è caratterizzata da un grande dominio extracellulare e un dominio breve citosolico chiamato il dominio intracellulare di APP (AICD). Durante la maturazione attraverso la via secretoria, APP può essere scissa dalla proteasi denominate α, β, e γ-secretasi 1. Sequenziale clivaggio proteolitico di APP con β e γ-secretasi porta alla produzione di un peptide piccolo proteolitico, Ap definito, che è amiloidogena e il componente principale delle placche senili. L&#39;AICD è liberata dalla membrana dopo trasformazione secretasi, e attraverso interazioni con Fe65 e Tip60, può traslocare al nucleo di partecipare nella regolazione della trascrizione di geni bersaglio più 2,3. Proteina-proteina interazioni che coinvolgono il AICD può influenzare il traffico, l&#39;elaborazione, e le funzioni cellulari di olo-APP e il suo C-terminaL frammenti. Abbiamo recentemente dimostrato che AICD può aggregare in vitro, e questo processo è inibito dalla AD-implicato chaperone molecolare ubiquilin-1 4. Coerentemente con questi risultati, il AICD ha esposto domini idrofobici ed è intrinsecamente disordinati in vitro 5,6, tuttavia ottiene stabile struttura secondaria quando si lega al Fe65 7. Abbiamo proposto che ubiquilin-1 impedisce appropriato interazioni inter e intramolecolari di AICD, impedendo l&#39;aggregazione in vitro e in cellule intatte 4. Mentre la maggior parte degli studi si concentrano sul ruolo di APP nella patogenesi di AD, il ruolo di AICD in questo processo non è chiara. Espressione di AICD ha mostrato di indurre apoptosi 8, per modulare i percorsi di segnalazione 9 e di regolare il calcio segnalazione 10. Over-espressione di AICD e Fe65 in un modello di topo transgenico induce Alzheimer come patologia 11, e recentemente AICD è stato rilevato in reggisenonei lisati mediante Western blotting utilizzando appropriate tecniche di recupero di antigene 12. Per facilitare studi strutturali, biochimici e biofisici del AICD, abbiamo sviluppato una procedura per produrre quantità elevate di ricombinante altamente puro proteine ​​AICD. Abbiamo inoltre descrivere un metodo per indurre l&#39;aggregazione in vitro termica AICD e analisi mediante microscopia a forza atomica. I metodi descritti sono utili per la caratterizzazione biochimica, biofisica e strutturale del AICD e gli effetti di chaperone molecolare sull&#39;aggregazione AICD.

Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Un metodo per la grande purificazione del dominio intracellulare APP (AICD) è descritto. Abbiamo anche descrivere la metodologia di indurre<em&gt; In vitro</em&gt; AICD aggregazione e visualizzazione mediante microscopia a forza atomica. I metodi descritti sono utili per la caratterizzazione biochimica / strutturale del AICD e gli effetti di chaperone molecolare sulla sua aggregazione.

Purificazione e aggregazione della proteina precursore dell&#39;amiloide dominio intracellulare

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

purification-aggregation-amyloid-precursor-protein-intracellular

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JoVE Journal

Medicine

11.7K Views.  University of Texas Medical Branch. Un metodo per la grande purificazione del dominio intracellulare APP (AICD) è descritto. Abbiamo anche descrivere la metodologia di indurre In vitro AICD aggregazione e visualizzazione mediante microscopia a forza atomica. I metodi descritti sono utili per la caratterizzazione biochimica / strutturale del AICD e gli effetti di chaperone molecolare sulla sua aggregazione.

Video: Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Esecuzione ed elaborazione di FNA anteriore Fat Pad per Amiloide

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

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Medicina

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Trapianto subretinale di cellule precursori del fotorecettore purificato MACS nella retina del topo adulto

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Utilizzo sinergico delle cellule neurali e precursori peptidi autoassemblanti in Sperimentale Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Valutazione di alternanza spontanea, riconoscimento novello di oggetti e attacco degli arti nei modelli di topo transgenico Modelli di neuropathologia di Amiloide-β e Tau

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetria su Human Lavato Piastrine: l&#39;effetto della Pannexin1-inibitore blu brillante FCF sul livello di aggregazione indotta dal collagene

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Valutazione del reticolo sarcoplasmico calcio intracellulare e riserva diastolica calcio rimozione nei cardiomiociti ventricolari isolati

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

In Vivo Imaging dei topi Reporter Cx3cr1gfp/gfp con tomografia a coerenza ottica Spectral-domain e scansione Laser oftalmoscopia

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Isolamento e purificazione dei Periciti Cardiaci Murine

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Isolamento basato su ultracentrifuga di vescicole extracellulari da ADSC umane

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Autore in primo piano: Standardizzare e migliorare l'estrazione e la purificazione delle vescicole extracellulari da ADSC umane

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Purificazione e caratterizzazione di vescicole extracellulari da cellule staminali mesenchimali di derivazione adiposa umana

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...