Grazie a Jessica Ottmar per i suggerimenti protocollo e contributi per i dati rappresentativi. Un ringraziamento speciale a Sabrina Blackledge, Lauren Kohoutek e Kerisa Shelton per dimostrare questo protocollo.

1. Impostare <ol><li> Tutte le ricerche devono essere approvate dal Consiglio appropriato Institutional Review o revisione umana soggetti comitato prima della raccolta dei dati. </li><li> Idealmente, due camere dovrebbero essere disponibili per condurre il TSST. Una confortevole sala d&#39;attesa dovrebbero essere a disposizione dei partecipanti prima di iniziare la TSST e durante i periodi di recupero. Una sala colloquio separato deve essere usato durante la preparazione del discorso, la performance vocale e porzioni di matematica del TSST di introdurre novità e uncontrolability. </li><li> Disporre sala delle udienze in modo che il partecipante si troverà ad affrontare intervistatori confederate e video posizione o apparecchiature di registrazione audio nel campo visivo del partecipante. Camici dovrebbero essere disponibili per gli intervistatori da indossare durante la TSST per aumentare lo stress durante la TSST. Istruire gli intervistatori a mantenere il contatto visivo con i partecipanti e di astenersi dal fare emotive espressioni facciali durante la TSST. </li><li> Considerare attentamente °e domanda di ricerca (s) quando si seleziona misure dipendente. Linee guida per l&#39;analisi e la pubblicazione di varie metrature di risposta allo stress sono forniti da diverse fonti (linee guida per la pubblicazione e l&#39;analisi delle misure psicofisiologiche sono forniti dalla Società per la Ricerca psicofisiologica: <a href="http://www.sprweb.org/journal/index.cfm#guidelines" target="_blank">http://www.sprweb.org/journal/index. cfm # guidelines</a> ). 5 Consultazione di queste risorse durante la progettazione di esperimenti è raccomandato. </li><li> Impostare tutte le attrezzature necessarie per la registrazione fisiologica, psicologica o risposte endocrine al TSST. Questa apparecchiatura può includere (ma non solo) elettrodi e apparecchiature elettroniche e / o software per registrare la frequenza cardiaca, la reattività cardiovascolare, conduttanza cutanea o una risposta, batter d&#39;occhio, la contrazione muscolare / tono, frequenza respiratoria, carta e matita o versioni elettroniche di strumenti psicologici come l&#39;ansia di tratto Stato Inventory; 6 tubi e materiali per la raccolta di altri campioni di urina, sangue o saliva;. o apparecchiature di registrazione video Per questa dimostrazione verrà utilizzato tre derivazioni dell&#39;elettrocardiogramma (ECG) elettrodi per misurare la frequenza cardiaca durante il TSST, e la somministrazione ripetuta del Stato Trait Anxiety Inventory per misurare la risposta psicologica prima e dopo l&#39;evento stressante. Ci si avvale di un sistema di base di registrazione progettato per misurare le risposte fisiologiche (Biopac MP36 di sistema) per registrare la frequenza cardiaca. </li><li> Se i campioni di sangue (piuttosto che saliva) saranno raccolti durante l&#39;esperimento, gli sforzi per minimizzare il dolore e il disagio durante la raccolta deve essere fatta. Procedure di raccolta del sangue può introdurre ulteriori stress fisico o psicologico, la risposta alla confusione TSST. </li><li> Quando il partecipante arriva, loro il benvenuto al laboratorio. Ottenere il consenso informato ed il sedile del partecipante comodamente. </li><li> Raccogliere dati demografici, le informazioni di screening o supplemisure arrivo nale che sono richiesti dal vostro esperimento. Verificare che il partecipante non ha consumato cibi o bevande (a parte l&#39;acqua), nell&#39;ultima ora se la raccolta misurazioni del cortisolo. Un breve periodo di digiuno è consigliato se vi sarà la raccolta di campioni di saliva per l&#39;analisi. Le variazioni di pH associati a cibi acidi o bevande o di contaminazione visibile può interferire con la misurazione accurata dei marcatori salivari. Lo screening tossicologico delle urine può essere necessaria se le misure di interesse possono essere contaminati da farmaci o altri metaboliti. Informazioni sui fattori che possono influire sulla risposta allo stress (ad esempio farmaci, malattie, sonno, eventi stressanti recenti, fobia sociale) dovrebbe essere considerato quando i partecipanti screening. </li></ol> 2. Misure pre-stress <ol><li> Istruire il partecipante ad aspettare comodamente per 45 minuti nella sala d&#39;attesa. Si può scegliere di fornire al partecipante con materiale di lettura, come riviste contenenti emonalmente contenuto indipendente, o un&#39;altra attività emotivamente neutro durante questo periodo. </li><li> Dopo il periodo di 45 minuti di attesa, amministrare il Trait Anxiety Inventory Stato per valutare lo stress pre-ansia. 6 In alternativa, altri strumenti che sono progettati per valutare le variazioni dello stato di stress, ansia o rilassamento possono essere somministrati (ad esempio scale visive analogiche). </li><li> Raccogliere un campione &quot;pre-stress&quot; saliva. Istruire i partecipanti a sciacquare la bocca con acqua. Dare al partecipante una sezione 5-10 cm di cannuccia e un ambiente pulito, fiala monouso in polipropilene 2,0 ml. Istruire il partecipante di inclinare la testa in avanti per consentire la saliva di piscina sul pavimento della bocca poi passare la saliva attraverso la cannuccia nel flaconcino. Raccogliere circa 1,5-2,0 ml di saliva dal partecipante. In alternativa, la saliva possono essere raccolti usando assorbenti stoppino dentale (o materiale simile) e istruire i partecipanti a masticare o saturare il materiale con la saliva. Se possibile, lasciate la stanza di fornire al partecipante con la privacy. Etichetta e conservare tutti i campioni di saliva a -20 ° C nel più breve tempo possibile. </li><li> Registrare 5 minuti di misurazioni fisiologiche pre-stress. </li></ol> 3. Il TSST <ol><li> Se si prevede di utilizzare una stanza separata intervista (per aumentare gli effetti dello stress del TSST), portare il partecipante dalla sala d&#39;attesa nella sala colloqui. </li><li> Per iniziare TSST il periodo di preparazione vocale, leggere il seguente script al partecipante: &quot;Questa è la parte di preparazione discorso del compito, si sta per preparare mentalmente un discorso di cinque minuti che descrivono perché vi sarebbe un buon candidato per il tuo lavoro ideale. Il suo discorso sarà videoregistrata e rivista da un panel di giudici addestrati nel parlare in pubblico. Hai dieci minuti per preparare il vostro tempo e comincia adesso &quot;. </li><li> Registrare le misurazioni fisiologiche durante il periodo di 10 minuti discorso di preparazione. Impostare un timer digitale per 10 minuti e lasciare la stanza. </li&gt; <li> Dopo 10 minuti, tornate alla stanza indossa un camice da laboratorio. Leggi il seguente script al partecipante: &quot;Questa è la parte vocale del compito Siete a un discorso che descrive il motivo per cui sarebbe un buon candidato per il tuo lavoro ideale si deve parlare per l&#39;intero il periodo di cinque minuti di tempo... il tempo comincia adesso &quot;. La videocamera prop dovrebbe essere ora attivato, per aumentare valutativa / prestazioni stress. </li><li> Registrare le misurazioni fisiologiche durante i 5 minuti di discorso prestazioni. Se il partecipante smette di parlare durante il discorso, le consenta di rimanere in silenzio per 20 secondi. Se lui o lei non riprende parlando, richiesta ai partecipanti di continuare a parlare, insegnando loro: &quot;Hai ancora tempo rimanente.&quot; </li><li> Al termine dei 5 minuti di discorso di prestazioni, leggere il seguente script al partecipante: &quot;Durante gli ultimi cinque minuti parte la matematica di questo compito vi sarà chiesto di sottrarre in sequenza il numero 13 da 1.022 Potrai verbale.Ly segnalare le vostre risposte a voce alta, e sarà chiesto di ricominciare da 1.022 se viene commesso un errore. . Vostro tempo comincia adesso &quot;Se il partecipante fa un errore, li prompt con:&quot; Questo non è corretto, si prega di ricominciare da 1.022 &quot;Impostare un timer digitale per 5 minuti.. </li><li> Registrare le misurazioni fisiologiche durante i 5 minuti di matematica prestazioni. </li><li> Al termine del periodo di matematica prestazioni del TSST, raccogliere un campione &quot;post-stress&quot; saliva. </li><li> Amministrare lo stress post-Trait Anxiety Inventory Stato. </li></ol> 4. Dopo lo stress di recupero Misure <ol><li> Ritorna partecipanti alla stanza originale attesa. Durante il periodo di recupero prima, istruire il partecipante ad aspettare comodamente per 20 minuti. Impostare un timer digitale per 20 minuti e lasciare la stanza. </li><li> Ritorna alla sala d&#39;attesa dopo 20 minuti e raccogliere un &quot;recupero 1&quot; campione di saliva. </li><li> Durante il periodo di recupero secondo, istruire il partecipante di aspettare ancora comodamente per 20 minuti. Impostare un timer digitale per 20 minuti e lasciare la stanza. </li><li> Ritorna alla sala d&#39;attesa dopo 20 minuti e raccogliere un &quot;recupero 2&quot; campione di saliva. </li><li> Istruire i partecipanti di completare il tratto &quot;recupero&quot; State Anxiety Inventory. </li><li> Debriefing del partecipante per quanto riguarda la vera natura dell&#39;esperimento. Informa il partecipante che la loro performance non è stata registrata e che nessuna analisi del loro linguaggio o le prestazioni di matematica sarà condotta. Spiegate ai partecipanti che i compiti con la quale sono stati presentati sono stati straordinariamente difficile, e non riflettere sulle attitudini del partecipante o abilità. </li></ol> 5. Analisi dei dati <ol><li> Per analizzare le misure psicologiche, il punteggio di ogni Trait Anxiety Inventory Stato dal pre-stress, post-stress e il tempo di recupero punti 6. </li><li> Per analizzare le misure fisiologiche, calcolare un punteggio medio della frequenza cardiaca per ciascuno dei 5 minuti di &quot;stress pre&quot;, &quot;preparazione del discorso&quot;, &quot;performance vocale&quot; e &quot;math &quot;periodi del TSST. In alternativa, calcolare i punteggi medi per ogni minuto della TSST o della frequenza cardiaca misura in punti tempo discreto durante il TSST. </li><li> Per analizzare le misure neuroendocrini, i campioni di test salivare per la concentrazione dell&#39;ormone usando i kit EIA disponibile in commercio. </li></ol> 6 Opzionale:. Aggiuntive cortisolo salivare Information Protocol Assay (riprodotto da Salimetrics salivari inserire kit dosaggio del cortisolo) <ol><li> Portare tutti i reagenti del kit test salivari a temperatura ambiente. </li><li> Preparare il tampone di lavaggio in base alle istruzioni del kit. </li><li> Determinare layout del pannello e pipettare 25 ml di standard e controlli nei pozzetti in duplicato. </li><li> Campioni di saliva disgelo, vortex e centrifugare a 1.500 XG (@ 3.000 rpm) per 15 minuti. </li><li> Pipettare 25 ml di liquido chiaro da campioni centrifugati in pozzetti in lamiera (in duplice copia). </li><li> Preparare enzima coniugato e pipettare 200 ul in tutti i pozzetti. </li&gt; <li> Mescolare piastra per 5 minuti a 500 rpm e incubare per altri 55 minuti. </li><li> Lavare e asciugare la piastra di 4 volte con tampone di lavaggio. </li><li> Aggiungere 200 ul tetrametilbenzidina (TMB) in ogni pozzetto utilizzando una pipetta multicanale. </li><li> Mescolare la piastra per 5 minuti a 500 rpm e incubare al buio per altri 25 minuti. </li><li> Aggiungere 50 ul di soluzione di stop a ogni bene e mescolare per 3 minuti a 500 rpm per fermare le reazioni. </li><li> Pulire il fondo piatto pulito e leggere la piastra in un lettore di piastre a 450 nm entro 10 minuti dall&#39;aggiunta della soluzione di stop. </li><li> Per calcolare la concentrazione di cortisolo, calcolare la densità media ottica per tutti i pozzetti duplicati. Sottrarre la densità ottica media dei pozzi legame non specifico dalla densità ottica media dello zero, standard, controllo e pozzetti dei campioni. </li><li> Calcolare il tasso di legame (B / Bo) per ogni controllo standard e campione dividendo la densità media ottico di questi pozzi (B) dal Averetà densità ottica dei pozzi zero (Bo). Determinare la concentrazione dei controlli e dei campioni per interpolazione utilizzando un software in grado di logistica. </li></ol> 7. Rappresentante dei risultati: I dati qui presentati rappresentante è stato compilato da una revisione della letteratura e due studi che sono stati condotti nel nostro laboratorio durante il pomeriggio con i partecipanti adulti maschi sani. Questi dati sono destinati a rappresentare circa i risultati che potrebbero essere trovati utilizzando il protocollo presentato in questo articolo. Questo protocollo TSST (Figura 1) induce aumenti di auto-riferito ansia (come misurata dal State Trait Anxiety Inventory; Figura 2), la frequenza cardiaca (Figura 3), e le concentrazioni di cortisolo salivare (Figura 4). Il protocollo sperimentale illustrato nella figura 1 è pensato per fornire un esempio di come varie misure potrebbe essere fatto durante una sessione di raccolta dati con il TSST. I tempi di MISUREts e l&#39;inclusione di diverse misure saranno dettate dalla domanda di ricerca specifici. Se si utilizza un intervento che ha lo scopo di ridurre l&#39;ansia e / o la risposta allo stress, aumenta smussati o attenuati in queste misure sono previsti. <img alt="Figura 1" src="/files/ftp_upload/3238/3238fig1.jpg" /> Figura 1. Protocollo sperimentale Nota: Stress Test Trier sociale (TSST); State Trait Anxiety Inventory (STAI), 5 minuti di stress Pre (PS), 10 minuti di stress anticipatoria (preparazione vocale) (AS), 5 minuti di discorso. (S), 5 minuti di matematica (M). <img alt="Figura 2" src="/files/ftp_upload/3238/3238fig2.jpg" /> Figura 2. Rappresentante punteggi ansia di stato. Nota: Ansia punteggio calcolato Stato dal 20-Iten misura ansia di stato di Trait Anxiety Inventory Stato (STAI) punteggi Rappresentante riflettere i dati raccolti nel nostro laboratorio e pubblicati in letteratura.. Figura 3. Rappresentante dati di frequenza cardiaca Nota:.. La frequenza cardiaca in battiti per minuto (BPM) valori rappresentativi riflettere i dati raccolti nel nostro laboratorio e pubblicati in letteratura. <img alt="Figura 4" src="/files/ftp_upload/3238/3238fig4.jpg" /> Figura 4. Rappresentante dei dati cortisolo salivare Nota:.. Concentrazione di cortisolo in UL / DL valori rappresentativi riflettere i dati raccolti nel nostro laboratorio e pubblicato in letteratura.

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+salivary+hormones.">Assessment of salivary hormones.</a> Methods in Social Neuroscience. , 17-44 (2009).</li><li>Kudielka, B. M., Hellhammer, D. H., W&#252;st, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+we+respond+so+differently?+Reviewing+determinants+of+human+salivary+cortisol+responses+to+challenge.">Why do we respond so differently? Reviewing determinants of human salivary cortisol responses to challenge.</a> Psychoneuroendocrinology. 34, 2-18 (2009).</li><li>Buske-Kirschbaum, A., Jobst, S., Wustmans, A., Kirschbaum, C., Rauh, W., Hellhammer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+free+cortisol+response+to+psychosocial+stress+in+children+with+atopic+dermatitis.">Attenuated free cortisol response to psychosocial stress in children with atopic dermatitis.</a> Psychosom. Med. 59, 419-426 (1997).</li></ol>

Nessun conflitto di interessi dichiarati.

In questo articolo abbiamo dimostrato come condurre la Social Stress Test Trier in un volontario sano. Il TSST è un fattore di stress laboratorio standardizzato sociale che induce un aumento robusto e affidabile in psicologici, misure fisiologiche e neuroendocrini. Il TSST è una valida alternativa a fattori di stress fisici, durante i test pressa a freddo a piedi o tapis roulant, e riproduce lo stress psicologico più naturalistica delle prestazioni in presenza di un pubblico valutativa. M...

<table border="1"><tbody><tr><th> Il nome del dispositivo / alimentazione </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> BSL Psicofisiologia del sistema, Mac OS Incluso MP36 Data Acquisition Unit e tre elettrodi dell&#39;elettrocardiogramma di piombo </td><td> BIOPAC Systems Inc. </td><td> BSLPSY-M </td></tr><tr><td> Stato Trait Anxiety Inventory </td><td> Mente Giardino, Inc. </td><td> Staid-B </td></tr><tr><td> Il cortisolo salivare Enzyme Immuno Assay Kit </td><td> Salimetrics </td><td> 1-3002 </td></tr><tr><td> 2,0 ml in polipropilene fiale </td><td> Fisher Scientific </td><td> 05 40B 146 </td></tr><tr><td> BioRad Microplate Reader, Modello 680, con software di gestione per micropiastre </td><td> BioRad </td><td> Piastra Lettore: 168 1000 Software: 1706800 </td></tr></tbody></table>

the trier social stress test protocol for inducing psychological stress

Questo articolo viene illustrato un protocollo di stress psicologico per l&#39;utilizzo in un ambiente di laboratorio. Protocolli che permettono ai ricercatori di studiare i percorsi biologici della risposta allo stress in salute e malattia sono fondamentali per il progresso della ricerca in stress e ansia. 1 Sebbene esistano numerosi protocolli per indurre risposta allo stress in laboratorio, molti trascurano di fornire un contesto naturalistico o di integrare gli aspetti di stress sociale e psicologico. Dei protocolli di stress psicologico, meta-analisi suggerisce che la Social Stress Test Trier (TSST) è il protocollo più utile ed opportuno standardizzata per lo studio della reattività allo stress ormonale. 2 Nella descrizione originale del TSST, i ricercatori hanno cercato di progettare e valutare una procedura in grado di indurre una risposta affidabile di stress nella maggior parte dei volontari sani. 3 Questi ricercatori hanno trovato aumenti della frequenza cardiaca, pressione sanguigna e lo stress endocrino diversi markers in risposta al TSST (un fattore di stress psicologico) rispetto ad una iniezione di soluzione salina (un fattore di stress fisico) 3. Sebbene il TSST è stato modificato per soddisfare le esigenze dei vari gruppi di ricerca, si compone in genere di un periodo di attesa dopo l&#39;arrivo, parola di anticipazione preparazione, la performance vocale e verbale aritmetica periodi di prestazioni, seguito da uno o più periodi di recupero. Il TSST richiede ai partecipanti di preparare e consegnare un discorso, e verbalmente rispondere a un problema di aritmetica impegnativo in presenza di un pubblico socialmente valutativa 3. Valutazione sociale e incontrollabilità sono stati identificati come componenti chiave di induzione dello stress dal TSST. 4 In uso per oltre un decennio, l&#39;obiettivo del TSST è di indurre una risposta allo stress sistematicamente al fine di misurare differenze di reattività, l&#39;ansia e l&#39;attivazione dell&#39;asse ipotalamo-ipofisi-surrene (HPA) o simpatico-surrenale-midollare (SAM) asse durante il compito 1. Researcsuo genere valutare i cambiamenti in auto-riferito ansia, misure fisiologiche (ad esempio frequenza cardiaca), e / o indici neuroendocrini (per esempio il cortisolo, l&#39;ormone dello stress) in risposta al TSST. Molti ricercatori hanno adottato per il campionamento salivare marcatori di stress come il cortisolo e alfa-amilasi (un marker di attivazione del sistema nervoso autonomo) come alternativa al prelievo di sangue per ridurre lo stress confondenti di sangue tecniche di raccolta. In aggiunta ai cambiamenti sperimentato da un individuo completando il TSST, i ricercatori possono confrontare le modifiche tra diversi gruppi di trattamento (ad esempio clinico campioni di controllo rispetto ai sani) o l&#39;efficacia di interventi di riduzione dello stress 1.

Watch this Scientific Journal Video about The Trier Social Stress Test Protocol for Inducing Psychological Stress at JoVE.com

The Trier Social Stress Test Protocol for Inducing Psychological Stress

Questo articolo descrive un protocollo per indurre stress psicologico nei partecipanti, che consente ai ricercatori di misurare psicologico, le risposte fisiologiche e neuroendocrina allo stress all&#39;interno di singoli partecipanti o tra i gruppi.

The Social Trier Protocollo di prova dello stress per indurre stress psicologico

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

the-trier-social-stress-test-protocol-for-inducing-psychological

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JoVE Journal

Medicine

55.6K Views.  Northern Arizona University. Questo articolo descrive un protocollo per indurre stress psicologico nei partecipanti, che consente ai ricercatori di misurare psicologico, le risposte fisiologiche e neuroendocrina allo stress all'interno di singoli partecipanti o tra i gruppi.

Video: The Trier Social Stress Test Protocol for Inducing Psychological Stress

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

Utilizzo dei metodi visivi e narrativi a Raggiungere processo equo in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

Ricerca

Medicina

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

Sviluppo di un unilateralmente lesionata 6-OHDA modello murino della malattia di Parkinson

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Impianto di un bracciale carotidea per innescare Shear-stress aterosclerosi indotta in topi

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

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 ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Stress Test cardiaca indotta da dobutamina e monitorato da cateterizzazione cardiaca nei topi

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Riduzione del ventricolo sinistro stress di parete e di miglioramento della funzione in mancanza di Cuori con Algisyl-LVR

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Un tumore modello murino di stress chirurgico per esplorare i meccanismi di immunosoppressione post-operatorio e valutare Novel perioperatoria Immunoterapie

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

A Modified<em&gt; In vitro</em&gt; Invasion test per determinare il ruolo potenziale di ormoni, citochine e / o fattori di crescita nella mediazione Cancer Invasion cellulare

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Glutammato e ipossia come modello di stress per l&#39;isolato perfuso vertebrati Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Induzione e analisi dello stress ossidativo nelle cellule epiteliali del pigmento retinico umano trasforato dalla bella addormentata nel bosco

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...