Il lavoro del Dr. Carvalho è stato sostenuto da una borsa di studio presso l&#39;Ufficio di Ricerca sulla salute della Donna e il National Institute of Child Health e lo Sviluppo Umano del National Institutes of Health (5K12 HD043452). Angst Dr ricevuto forniture (On-Q PainBuster Post-Op sistema sollievo dal dolore) e fondi (per i saggi biochimici) da I-Flow (Lake Forest, California).

1. Protocollo di studio <ul><li> Ottenere l&#39;approvazione istituzionale Comitato Etico e il consenso informato scritto. </li><li> Selezionare una popolazione chirurgica appropriata per il comportamento di studio. Questa metodologia è stata applicata solo alle ferite parto cesareo, ma la maggior parte delle ferite chirurgiche sarebbe opportuno. </li><li> Considerare i criteri di inclusione ed esclusione per la partecipazione allo studio. I pazienti e le procedure con alti tassi di infezione della ferita chirurgica non può essere appropriato. </li><li> Standardizzare l&#39;anestesia e la gestione post-operatoria per tutti i pazienti per evitare potenziali fattori confondenti. </li></ul> 2. Nocicettivo e infiammatorie Collezione mediatore biochimico <ul><li> Inserire l&#39;interruttore ON-Q PainBuster con ON-Q SilverSoaker (I-Flow, Lake Forest, CA) nello strato sottocutaneo appena prima di ferire la chiusura (strati di tessuto di altri può essere considerato). L&#39;ON-Q Sistema Pain Relief PainBuster eroga in continuo soluzione fisiologica ad una velocità di 2 ml / h. Il vettore può essere pettineinato con o rispetto ai farmaci di interesse come un anestetico locale e non-steroidei anti-infiammatori. </li><li> Incorporano uno standard valvola a tre vie in questo sistema per permettere l&#39;aspirazione di essudato in punti temporali specifici. L&#39;infusione continua impedisce al catetere di coagulazione e migliora l&#39;affidabilità del sistema di produrre campioni di essudato. Se l&#39;aspirazione di essudato è difficile (circa il 5% dei casi), si consideri spingendo delicatamente sopra la ferita, cambiando la posizione del soggetto (ad esempio, il paziente seduto o sdraiato su di loro piatto), usando un normale filo 0,5-1 ml di soluzione fisiologica o di ritiro del catetere 1-2 cm. </li><li> A tempi previsti dal protocollo (ad esempio, 1, 4, 6, 24, e 48 ore dopo il parto cesareo), aspirare e prelevare 1 ml di essudato. Trasferire l&#39;essudato in una tazza di polietilene contenente 30 ml di inibitore di proteinasi. Abbiamo usato Inhibitor Complete proteinasi (Bioscience Roche, Palo Alto, CA) per i nostri scopi. </li><li&gt; Al stessi intervalli di tempo specificati dal protocollo di studio, raccogliere 10 ml di sangue. Abbiamo usato un tubo di raccolta del sangue contenente eparina al litio; nel nostro istituto si tratta di un verde ripiano in tubo di vetro. Aggiungere un ulteriore 300 ml di inibitore di proteinasi nei campioni di sangue. </li><li> Entro 1 ora di collezione, mettere i campioni in ghiaccio e centrifugare a 3000 rpm per 10 min. </li><li> Rimuovere sopranatante siero e ferita e posto in una provetta da microcentrifuga standard e conservare a -20 ° C. </li></ul> 3. Saggio di analisi <ul><li> Una volta che tutto l&#39;essudato studio e campioni di siero sono raccolti e memorizzati, scongelare e analizzare i campioni nello stesso momento. </li><li> Abbiamo misurato le citochine e chemochine utilizzando un immunodosaggio multiplex. L&#39;immuno-analisi tecnica di multiplex ci permette di dosaggio numerosi analiti da piccoli (50 pl) campioni di volume. Il multiplex producono risultati comparabili con quelli ottenuti con ELISA. Abbiamo usato il 17-plex Bio-PlexTM sistema (BioRad, Hercduli, CA) per la nostra analisi. Questo test è in grado di misurare il fattore di necrosi tumorale α (TNFa), interferone α (INFα), interleuchina 1β (IL-1β), interleuchina 2 (IL-2), interleuchina 4 (IL-4), interleuchina 5 (IL-5 ), interleuchina 6 (IL-6), interleuchina 7 (IL-7), interleuchina 8 (IL-8), interleuchina (IL-10), interleuchina 12 (IL-12), interleuchina 13 (IL-13), interleuchina 17 (IL-17), fattore stimolante colonie di granulociti (G-CSF), granulociti-macrofagi fattore stimolante le colonie (GM-CSF), monocyte chemoattractant proteina 1 (MCP-1) e proteina macrofagica infiammatoria 1 (MIP-1β). Fattore di crescita dei nervi è stata misurata con NGF DY256 anticorpo aggiungendolo al 17-plex piastra con il Bio-Plex kit ammina accoppiamento. </li><li> Per garantire una buona analisi di qualità, ogni misurazione deve essere effettuata in duplicato. Curve standard per ciascun analita sono generati utilizzando analiti di riferimento forniti dal produttore (a concentrazioni di 0,20, 0,78, 3,13, 12,5, 50, 200, 800, 3200 pg / ml, piùuno standard zero per la soluzione fisiologica). Le curve standard sono inclusi in ogni seduta e concentrazione del campione sono calcolati con Bio-Plex software di gestione. </li><li> La sostanza P e prostaglandina E2 sono stati misurati utilizzando altamente sensibilità kit ELISA. Abbiamo usato Assay Designs, Inc. (Ann Arbor, Michigan) per questa analisi. Effettuare l&#39;analisi in duplicato in base alle specifiche del costruttore. </li></ul>

<ol>
	<li>Carvalho, B., Clark, D. J., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+Systemic+Release+of+Cytokines,+Nerve+Growth+Factor,+Prostaglandin+E2,+and+Substance+P+in+Incisional+Wounds+and+Serum+Following+Cesarean+Delivery.">Local and Systemic Release of Cytokines, Nerve Growth Factor, Prostaglandin E2, and Substance P in Incisional Wounds and Serum Following Cesarean Delivery.</a> J. Pain. 9, 650-657 (2008).</li><li>Carvalho, B., Clark, D. J., Yeomans, D. C., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+subcutaneous+instillation+of+bupivacaine+compared+to+saline+reduces+interleukin+10+and+increases+substance+P+in+surgical+wounds+after+cesarean+delivery.">Continuous subcutaneous instillation of bupivacaine compared to saline reduces interleukin 10 and increases substance P in surgical wounds after cesarean delivery.</a> Anesth. Analg. 111, 1452-1459 (2010).</li><li>Verri, W. A., Cunha, T. M., Parada, C. A., Poole, S., Cunha, F. Q., Ferreira, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypernociceptive+role+of+cytokines+and+chemokines:+targets+for+analgesic+drug+development.">Hypernociceptive role of cytokines and chemokines: targets for analgesic drug development.</a> Pharmacol. Ther. 112, 116-138 (2006).</li><li>Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+of+central+nervous+system+functions:+from+sickness+responses+to+pathological+pain.">Immune regulation of central nervous system functions: from sickness responses to pathological pain.</a> J. Intern. Med. 257, 139-155 (2005).</li><li>Beilin, B., Bessler, H., Mayburd, E., Smirnov, G., Dekel, A., Yardeni, I., Shavit, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+preemptive+analgesia+on+pain+and+cytokine+production+in+the+postoperative+period.">Effects of preemptive analgesia on pain and cytokine production in the postoperative period.</a> Anesthesiology. 98, 151-155 (2003).</li><li>Wu, C. T., Jao, S. W., Borel, C. O., Yeh, C. C., Li, C. Y., Lu, C. H., Wong, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+epidural+clonidine+on+perioperative+cytokine+response,+postoperative+pain,+and+bowel+function+in+patients+undergoing+colorectal+surgery.">The effect of epidural clonidine on perioperative cytokine response, postoperative pain, and bowel function in patients undergoing colorectal surgery.</a> Anesth. Analg. 99, 502-509 (2004).</li><li>Buvanendran, A., Kroin, J. S., Berger, R. A., Hallab, N. J., Saha, C., Negrescu, C., Moric, M., Caicedo, M. S., Tuman, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+prostaglandin+E2+and+interleukins+in+the+central+nervous+system+and+peripheral+tissue+during+and+after+surgery+in+humans.">Upregulation of prostaglandin E2 and interleukins in the central nervous system and peripheral tissue during and after surgery in humans.</a> Anesthesiology. 104, 403-410 (2006).</li><li>Kristiansson, M., Soop, M., Sundqvist, K. G., Soop, A., Suontaka, A. M., Blomback, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+vs.+systemic+immune+and+haemostatic+response+to+hip+arthroplasty.">Local vs. systemic immune and haemostatic response to hip arthroplasty.</a> Eur. J. Anaesthesiol. 15, 260-270 (1998).</li><li>Liu, S. S., Richman, J. M., Thirlby, R. C., Wu, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+continuous+wound+catheters+delivering+local+anesthetic+for+postoperative+analgesia:+a+quantitative+and+qualitative+systematic+review+of+randomized+controlled+trials.">Efficacy of continuous wound catheters delivering local anesthetic for postoperative analgesia: a quantitative and qualitative systematic review of randomized controlled trials.</a> J. Am. Coll. Surg. 203, 914-932 (2006).</li><li>Eriksson, A. S., Sinclair, R., Cassuto, J., Thomsen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+lidocaine+on+leukocyte+function+in+the+surgical+wound.">Influence of lidocaine on leukocyte function in the surgical wound.</a> Anesthesiology. 77, 74-78 (1992).</li><li>Clark, J. D., Shi, X., Li, X., Qiao, Y., Liang, D., Angst, M. S., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+reduces+local+cytokine+expression+and+neutrophil+infiltration+after+incision.">Morphine reduces local cytokine expression and neutrophil infiltration after incision.</a> Mol. Pain. 3, 28 (2007).</li><li>Webb, S. T., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-articular+bupivacaine:+potentially+chondrotoxic.">Intra-articular bupivacaine: potentially chondrotoxic.</a> Br. J. Anaesth. 102, 439-441 (2009).</li><li>Scott, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+infiltration+for+surgery.">Wound infiltration for surgery.</a> Anaesthesia. 65, 67 (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

Questo metodo di cui sopra è stato trovato per essere abbastanza sensibile da rilevare biochimici mediatori infiammatori e nocicettivo in essudato. Campionamento a diversi punti temporali ci ha anche permesso di osservare i cambiamenti di questi mediatori nel corso del tempo. 1,2 Non sono a conoscenza di qualsiasi altro test umana ferita chirurgica in grado di misurare il profilo seriale rilascio di mediatori infiammatori e nocicettivi in essudato della ferita chirurgica. Risultati del nostro metodo di raccolta essudato della ferita dal sito di incisione chirurgica è diverso da studi che utilizzano scarichi dall&#39;articolazione dell&#39;anca, 7,8 che suggerisce che il rilascio ferita sottocutanea mediatore può essere diverso da cavità intrarticolari o altro rilascio mediatore biochimico. Inoltre, i risultati ottenuti nelle acque di scarico possono essere confusi da perdita di sangue nella ferita, e pertanto non possono essere diverse da locale rilascio biochimico mediatore. La scarsa correlazione traferita e livelli sierici dimostra l&#39;importanza di determinare site-specific di rilascio quando si studia ferite chirurgiche. Modifiche locali non può essere riflessa sistemica a causa di molti fattori, tra cui la produzione differenziale sistemica e locale in risposta al trauma chirurgico e lo stress. Anche se abbiamo scelto di misurare una particolare selezione di citochine pro e anti-infiammatorie e chemochine come NGF, SP e PG-E2 essudato dalla ferita cesareo, 1,2 questa metodologia può essere applicata ad altri modelli chirurgici e molti altri mediatori biochimici. Instillazione ferita chirurgica in anestesia locale è una tecnica comunemente utilizzata per migliorare il dolore post-operatorio. 9 Applicando la metodologia, siamo stati in grado di dimostrare che l&#39;instillazione di anestetico locale in ferite chirurgiche influisce biochimica ferita. 2 Una diminuzione della IL-10 e l&#39;aumento della sostanza P negli essudati della ferita con bupivacaina instillazione suggerisce una interruzione of antinfiammatorio modulazione. Perifericamente somministrati anestetici locali possono quindi influenzare i processi infiammatori, 10,11 che può parzialmente rappresentano anestesia locale indotta condrolisi nei giunti dopo l&#39;intervento 12. ON-Q Sistema PainBuster Pain Relief è un US Food and Drug Administration (FDA) ha approvato dispositivo dal 1998 che viene utilizzato per la somministrazione di anestetici locali per il sito della ferita chirurgica per ridurre il dolore dopo l&#39;intervento. Instillazione ferita continua utilizzando cateteri questo ed altri non è stata associata con infezione maggiore. 9,13 ON-Q PainBuster con ON-Q SilverSoaker è progettato per infondere farmaci e non per campionare ripetutamente essudato. Anche se non abbiamo avuto infezioni della ferita con questa tecnica, sottolineiamo una tecnica asettica, mentre il campionamento essudato da questo sistema. La modifica proposta da facilitare la campionatura dovrebbe essere riservato a scopo di ricerca e non per la routine clinica uSE. Si consiglia inoltre la durata del catetere rimane in situ essere limitata a 48-72 ore. Questa tecnica chirurgica bioassay sito offre un buon modello per comprendere la relazione tra mediatori biochimici infiammatori e nocicettivo del dolore post-operatorio. Useremo la metodologia in studi futuri per indagare gli effetti di amministrati localmente non-steroidei anti-infiammatori e oppioidi. La tecnica può anche prestarsi alle indagini esaminare la guarigione delle ferite, infezioni chirurgiche eccessive cicatrici e ferite.

Dopo una incisione chirurgica, residenti e la migrazione di cellule infiammatorie rilasciano citochine pro-e anti-infiammatori, prostanoidi, neuropeptidi e chemochine che sono importanti per l&#39;avvio e il mantenimento del dolore. 3,4 La capacità di raccogliere marcatori biochimici specifici del sito è fondamentale per determinare il rapporto tra i livelli di siero e della ferita chirurgica, comprendere il complesso rapporto tra rilascio di mediatori e dolore chirurgico, e di essere in grado di valutare l&#39;effetto di entrambi sistemico e somministrazione del farmaco periferica sulla biochimica ferita chirurgica. Gli studi si sono concentrati su misure sistemiche di mediatori biochimici e non sono considerate specifiche del sito di rilascio. 5,6 misurazioni sierici di citochine e chemochine potrebbero non essere riflettente di eventi locali della ferita chirurgica biochimici e solo indicare una risposta globale sistemica al danno chirurgico. Questo articolo fornisce una descrizione dettagliata e visivo unico di questa metodologia che è stata abetest fa riferimento nel 2008 nel Journal of Pain. 1 L&#39;obiettivo di questo articolo JoVE è di fornire una descrizione univoca e visivamente migliore dei metodi.

collecting and measuring wound exudate biochemical mediators in surgical wounds

Descriviamo un metodo mediante il quale siamo in grado di raccogliere e misurare biochimici mediatori infiammatori e nocicettivi nel sito della ferita chirurgica. Raccolta di marcatori biochimici specifici del sito è importante per capire la relazione tra i livelli di siero e ferita chirurgica, determinare eventuali associazioni tra rilascio di mediatori, il dolore, l&#39;uso di analgesici e altri esiti di interesse, e di valutare l&#39;effetto della somministrazione per via sistemica e periferica sulla ferita chirurgica biochimica. Questa metodologia è stata applicata a donne sane sottoposte a parto cesareo elettivo con anestesia spinale. Abbiamo misurato l&#39;essudato e mediatori di siero a intervalli di tempo stessi punteggi del dolore del paziente e il consumo di analgesici per fino a 48 ore dopo parto cesareo. Usando questa metodologia siamo stati in grado di rilevare vari mediatori biochimici compreso il fattore di crescita nervoso (NGF), prostaglandina E2 (PG-E2) sostanza P, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8,IL-10, IL-12, IL-13, IL-17, TNFa, INFγ, G-CSF, GM-CSF, MCP-1 e MIP-1β. Gli studi che applicano il presente saggio biologico umano ferita chirurgica non hanno trovato correlazioni tra ferita e le concentrazioni sieriche di citochine o il loro tempo a rilascio profilo (J Pain 2008;. 9 (7): 650-7) 1 Abbiamo anche documentato l&#39;utilità della tecnica da identificare. farmaco-mediata cambiamenti nel contenuto della ferita (citochine Anesth Analg 2010; 111:1452-9). 2

Usando questa metodologia siamo stati in grado di rilevare tutti i mediatori biochimici sopra delineati con l&#39;eccezione di IL-5 in essudato e plasma. 1,2 Alcune citochine addizionali non sono stati rilevati nel plasma (IL-4, IL-17, TNFa, INFγ , GM-CSF). 1 L&#39;andamento nel tempo di citochine e chemochine misurati in studi riportati sono stati coerenti, raggiungendo un plateau entro le prime ore che è stato sostenuto per il periodo di osservazione 24 ore .. 1,2 Tuttavia, NGF aumento costante delle 24 ore periodo di raccolta, mentre PGE2 raggiungeva il picco entro 1 ora e quindi in costante calo. 1 risultati particolarmente degni di nota sono notevolmente diminuiti i livelli di IL-10 e di un aumento dei livelli di sostanza P nelle ferite instillato con l&#39;anestetico locale bupivacaina, compatibile con un complessivo pro- infiammatorio del farmaco (Figura 1). 2 In un altro studio abbiamo dimostrato che il consumo di analgesici dopo cesareo dechirurgia livrea era correlata negativamente con l&#39;essudato della ferita IL-6, IL-1 β, e G-CSF concentrazioni (Figura 2) 1. <img alt="Figura 1" fo:content-width="3in" fo:src="/files/ftp_upload/50133/50133fig1highres.jpg" src="/files/ftp_upload/50133/50133fig1.jpg" /> P Figura 1. L&#39;interleuchina-10 (IL-10) e la sostanza avvolto livelli di essudato livelli (pg / ml) misurati a 1, 3, 5, 7, e 24 ore dopo il parto cesareo. Il gruppo salina normale è indicata in diamanti grigi, e il gruppo bupivacaina è mostrato con quadrati neri. * = Significativamente diversi tra i due gruppi (P &lt;0,005). <img alt="Figura 2" src="/files/ftp_upload/50133/50133fig2.jpg" /> Figura 2. Correlazione tra l&#39;interleuchina-6 (pg / ml) e il consumo di morfina nelle prime 24 ore dopo il parto cesareo.

Watch this Scientific Journal Video about Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds at JoVE.com

Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

Questo articolo fornisce una descrizione dettagliata e visiva di una metodologia per la raccolta e la misurazione biochimici mediatori infiammatori e nocicettivi nel sito della ferita chirurgica dopo il parto cesareo. Questo saggio biologico umano è stato utilizzato per determinare le correlazioni tra ferita e concentrazioni sieriche di citochine e farmaco-mediata cambiamenti ferita citochine, chemochine e neuropetides.

La raccolta e la misurazione mediatori biochimici essudato nelle ferite chirurgiche

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. ...

collecting-measuring-wound-exudate-biochemical-mediators-surgical

Nanyang Technological University

Research

JoVE Journal

Medicine

11.9K Views.  Stanford University School of Medicine. Questo articolo fornisce una descrizione dettagliata e visiva di una metodologia per la raccolta e la misurazione biochimici mediatori infiammatori e nocicettivi nel sito della ferita chirurgica dopo il parto cesareo. Questo saggio biologico umano è stato utilizzato per determinare le correlazioni tra ferita e concentrazioni sieriche di citochine e farmaco-mediata cambiamenti ferita citochine, chemochine e neuropetides.

Video: Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Misure biochimiche di ipossia neonatale

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Ricerca

Medicina

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Modello murino di MCAO intraluminale: valutazione infarto cerebrale da colorazione viola cresile

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

In utero Misurazione della frequenza cardiaca in mouse da non invasiva ecocardiografia M-mode

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Videomicroscopia microscopia del sistema vascolare del tumore-associati utilizzando avanzate dorsale dello Skinfold finestra Chambers su topi transgenici fluorescenti

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Utilizzo In Vitro cellule vive di Imaging per esplorare chemioterapici consegnato da nanoparticelle di basata sui lipidi

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

Raccolta dei campioni di capelli per capelli cortisolo analisi negli afro-americani

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Protocollo per creare ferite croniche nei topi diabetici

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

Guarigione delle ferite di abrasione epiteliale corneale con oculare Burr come un modello per Cornea

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Targeting grigio Rami Communicantes nello Sympathectomy lombare chimico selettivo

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...