Dr. Carvalho Arbeit wurde durch ein Forschungsstipendium aus dem Office of Research am Women `s Health und National Institute of Child Health and Human Development der National Institutes of Health (5K12 HD043452) unterstützt. Dr Angst erhalten Lieferungen (On-Q PainBuster Post-Op Pain Relief System) und Fonds (für die biochemischen Assays) von I-Flow (Lake Forest, Kalifornien).

Ein. Study Protocol <ul><li> Erhalten Institutional Review Board Genehmigung und schriftlicher Einwilligung. </li><li> Wählen Sie eine geeignete chirurgische Bevölkerung Studiendurchführung. Diese Methode wurde nur Kaiserschnitt Wunden angewendet worden, aber die meisten chirurgischen Wunden angemessen wäre. </li><li> Betrachten Einschluss-und Ausschlusskriterien für Ihre Teilnahme an der Studie. Patienten und Verfahren mit hohen chirurgischen Wundinfektionen möglicherweise nicht geeignet. </li><li> Standardisieren Anästhesie und postoperative Behandlung für alle Patienten, um mögliche Störfaktoren zu vermeiden. </li></ul> 2. Nozizeptiven und Inflammatory Biochemical Mediator Sammlung <ul><li> Legen Sie die ON-Q PainBuster mit ON-Q SilverSoaker (I-Flow, Lake Forest, CA) in die subkutane Schicht kurz vor der Schließung (andere Gewebeschichten können berücksichtigt werden) gewickelt. Die ON-Q PainBuster Pain Relief-System liefert kontinuierlich physiologische Kochsalzlösung mit einer Rate von 2 ml / h. Der Träger kann KammsINED mit oder im Vergleich zu Drogen von Interesse, wie ein Lokalanästhetikum und nicht-steroidale Entzündungshemmer. </li><li> Integrieren Sie eine Standard-Drei-Wege-Hahn in dieses System, um eine Aspiration von Wundexsudat zu festgelegten Zeitpunkten zu ermöglichen. Die kontinuierliche Infusion verhindert den Katheter von Gerinnungszeit und verbessert die Zuverlässigkeit des Systems, um Exsudat Proben zu erzeugen. Falls Aspiration von Exsudat ist schwer (ca. 5% der Fälle), sollten Sie sanft über der Wunde, das Thema zu wechseln die Position (z. B. sitzt der Patient oder liegend sie flach) mit einem 0,5-1 ml normaler Kochsalzlösung oder Aberkennung der Katheters 1-2 cm. </li><li> Zeitpunkten durch die Protokoll (z. B. 1, 4, 6, 24 und 48 h nach Kaiserschnitt) angegeben, ansaugen und abheben 1 ml Wundexsudat. Übertragen Sie das Exsudat in einer Polyethylen-Becher mit 30 ul Proteinase-Inhibitor. Wir haben komplette Proteinase-Inhibitor (Roche Bioscience, Palo Alto, CA) für unsere Zwecke verwendet. </li><li&gt; Auf den gleichen Zeitintervallen vom Studienprotokoll festgelegt, sammeln 10 ml Blut. Wir haben ein Röhrchen mit Lithium-Heparin verwendet, an unserer Hochschule ist eine grüne-Spitze Glasrohr. Fügen Sie eine weitere 300 ul Proteinase-Inhibitor in die Blutproben. </li><li> Innerhalb von 1 h-Sammlung, legte Proben auf Eis und Zentrifuge bei 3.000 rpm für 10 min. </li><li> Entfernen Serum und Wunde Überstand und in einem Standard-Mikrozentrifugenröhrchen und bei -20 ° C. </li></ul> 3. Assay-Analyse <ul><li> Sobald die gesamte Studie Exsudat und Serumproben gesammelt und gespeichert werden, auftauen und Analyse der Proben zur gleichen Zeit. </li><li> Wir haben die Cytokine und Chemokine unter Verwendung eines Multiplex-Immunoassay gemessen wird. Das Multiplex-Immuno-Assay-Technik ermöglicht es uns, Assay zahlreichen Analyten aus kleinen (50 ul) Probenvolumina. Die Multiplex produzieren Ergebnisse vergleichbar zu denen mit ELISA erzielt. Wir haben die 17-plex Bio-PlexTM System (BioRad, Herc verwendetModule, CA) für unsere Analyse. Dieser Assay ist für die Messung von Tumornekrosefaktor α (TNF), Interferon α (INFα), Interleukin 1β (IL-1β), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin-5 (IL-5 ), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 17 (IL-17), Granulozyten-Kolonie stimulierender Faktor (G-CSF), Granulozyten-Makrophagen-Kolonie-stimulierenden Faktor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1) und Makrophagen inflammatorisches Protein 1 (MIP-1β). Nerve growth factor wurde mit NGF DY256 Antikörper, indem Sie es an die 17-plex Platte mit dem Bio-Plex Amin Kupplung Kit gemessen. </li><li> Um eine gute Qualität Analyse zu gewährleisten, sollte jede Messung doppelt gemacht werden. Standardkurven für jeden Analyten unter Verwendung Bezugnahme Analyten durch den Hersteller (bei Konzentrationen von 0,20, 0,78, 3,13, 12,5, 50, 200, 800, 3200 pg / ml plus erzeugteneine Null-Standard für die normale Kochsalzlösung). Standardkurven werden in jedem Durchlauf und Probenkonzentrationen sind mit Bio-Plex Manager-Software berechnet. </li><li> Substanz P und Prostaglandin E2 wurden mit hoch Sensibilität ELISA Kits. Wir haben Assay Designs, Inc. (Ann Arbor, Michigan) für diese Analyse verwendet. Führen Sie die Analyse in zweifacher Ausfertigung nach den Angaben des Herstellers. </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>

Keine Interessenskonflikte erklärt.

Diese Methodik skizzierten hat sich als sensitiv genug, um entzündliche und biochemische Mediatoren in nociceptiven Wundexsudat detektieren. Probenahme an verschiedenen Zeitpunkten hat uns auch die Möglichkeit, Änderungen dieser Mediatoren im Laufe der Zeit. 1,2 Wir sind keine Kenntnis von einem anderen menschlichen Operationswunde Assay, seriell messen kann das Freisetzungsprofil von entzündlichen und nozizeptiven Mediatoren in der chirurgischen Wundexsudat beobachten. Ergebnisse unserer Methode von Wundexsudat Sammlung von dem chirurgischen Inzisionsstelle unterscheidet sich von Untersuchungen unter Verwendung fließt aus dem Hüftgelenk, wonach 7,8 subkutane Wunde Mediatorfreisetzung kann sich von intraartikulären oder andere Körperhöhlen biochemischen Mediatorfreisetzung schlägt. Darüber hinaus können die Ergebnisse in die Kanalisation erhalten durch den Blutverlust in die Wunde verwechselt werden, und deshalb können sie nicht genau widerspiegeln lokalen biochemischen Mediatorfreisetzung. Die schwache Korrelation zwischenWund-und Serumspiegel zeigt die Bedeutung der Bestimmung ortsspezifische Freisetzung beim Studium Operationswunden. Lokale Änderungen können nicht systemisch, wegen der vielen Faktoren, einschließlich Differential systemische und lokale Produktion als Reaktion auf chirurgische Trauma und Stress reflektiert werden. Obwohl wir haben sich für eine bestimmte Auswahl von Pro-und Anti-inflammatorische Zytokine und Chemokine sowie NGF, SP und PG-E2 aus Kaiserschnitt Wundexsudat, 1,2 Diese Methodik kann auf andere chirurgische Modelle angewendet werden und viele andere messen biochemischen Mediatoren. Operationswunde Lokalanästhetikum Instillation ist eine häufig verwendete Technik zur Verbesserung der postoperativen Schmerzen. 9 Anwendung der Methodik konnten wir, dass Lokalanästhetika Instillation in Operationswunden zu demonstrieren hat Wunde Biochemie beeinflussen. 2 Eine Abnahme der IL-10 und der Erhöhung der Substanz P in Wundflüssigkeit mit Bupivacain Instillation schlägt eine Störung of entzündungshemmende Modulation. Peripher verabreicht Lokalanästhetika kann dadurch Einfluss auf die entzündlichen Prozesse, 10,11, die teilweise für die lokale Anästhesie-induzierten Knorpelschwund in den Gelenken verantwortlich nach der Operation kann. 12 ON-Q PainBuster Pain Relief System ist eine US-amerikanische Food and Drug Administration (FDA) zugelassen Gerät seit 1998, die für die Lieferung von Lokalanästhetika auf die chirurgische Wunde verwendet wird, um Schmerzen nach der Operation zu reduzieren. Kontinuierliche Wunde Instillation nutzen diese und andere Katheter wurde nicht mit einem erhöhten Wundinfektionen in Verbindung gebracht. 9,13 ON-Q PainBuster mit ON-Q SilverSoaker wurde entwickelt, um Medikamente einzuflößen und nicht wiederholt probieren Wundexsudat. Wir hatten zwar keine Wundinfektionen mit dieser Technik haben, betonen wir eine aseptische beim Abtasten Wundexsudat aus diesem System. Unsere vorgeschlagene Änderung der Probenahme zu erleichtern, sollte für wissenschaftliche Zwecke reserviert und nicht für die routinemäßige klinische use. Wir empfehlen auch, dass die Dauer der Katheter bleibt in-situ zu 48-72 Stunden begrenzt werden. Dieses chirurgische Website Bioassay-Technik bietet ein gutes Modell, um die Beziehung zwischen entzündlichen und nozizeptiven biochemischen Mediatoren in der postoperativen Schmerztherapie zu verstehen. Wir werden die Methodik in zukünftigen Studien verwenden, um Effekte von lokal verabreicht nicht-steroidalen Antirheumatika und Opioide untersuchen. Die Technik kann auch auf Untersuchungen eignet sich die Prüfung Wundheilung, übermäßiger Narbenbildung und chirurgischen Wundinfektionen.

Nach einem chirurgischen Eingriff, wohnhaft und Migration entzündlichen Zellen freizusetzen pro-und anti-inflammatorischen Zytokinen, Prostanoide, Neuropeptide und Chemokine, die wichtig für die Einleitung und Aufrechterhaltung Schmerzen. 3,4 Die Möglichkeit, Site-spezifische biochemische Marker zu sammeln, ist entscheidend für die Bestimmung Beziehung zwischen den Ebenen im Serum und Operationswunde, verstehen die komplexen Beziehungen zwischen Mediatorfreisetzung und chirurgische Schmerzen, und in der Lage sein, um die Wirkung von sowohl systemischer und peripheren Arzneimittelverabreichung auf Operationswunde Biochemie auszuwerten. Studien haben sich auf Messungen von systemischen biochemische Mediatoren konzentriert und nicht als ortsspezifische Freisetzung. 5,6 Serum Messungen von Cytokinen und Chemokinen eventuell nicht reflektierenden lokaler Operationswunde biochemischen Ereignisse und zeigen nur eine globale systemischen Reaktion auf chirurgische Verletzung. Dieser Artikel bietet eine detaillierte und einzigartige visuelle Beschreibung der Methodik, die Tannen warst 2008 im Journal of Pain verwiesen. 1 Das Ziel dieser JoVE Artikels ist es, eine einzigartige und optisch verbesserte Beschreibung der Methoden liefern.

collecting and measuring wound exudate biochemical mediators in surgical wounds

Wir beschreiben eine Methode, mit der wir in der Lage zu erfassen und zu messen biochemischen entzündlichen und nozizeptiven Mediatoren an der chirurgischen Wunde. Sammeln site-spezifische biochemische Marker ist wichtig, die Beziehung zwischen den Ebenen in Serum und Operationswunde zu verstehen, ermitteln die Assoziationen zwischen Mediatorfreisetzung, Schmerz, Analgesie und andere Ergebnisse von Interesse, und bewerten die Wirkung der systemischen und periphere Verabreichung des Arzneimittels auf Operationswunde Biochemie. Diese Methode wurde bei gesunden Frauen nach elektiven Kaiserschnitt mit Spinalanästhesie angewendet worden. Wir haben Wundexsudat und Serum Mediatoren in den gleichen Zeitintervallen wie Patienten Schmerz-Scores und Analgetika Verbrauch für bis zu 48 Stunden nach der Kaiserschnitt gemessen. Mit dieser Methode ist es uns gelungen, verschiedene biochemische Mediatoren einschließlich Nervenwachstumsfaktor (NGF), Prostaglandin E2 (PG-E2) Substanz P, IL-1β, IL-2, IL-4, IL-6, IL-7 zu detektieren, IL-8,IL-10, IL-12, IL-13, IL-17, TNF, INFγ, G-CSF, GM-CSF, MCP-1 und MIP-1β. Studien, die diese humanen Operationswunde Bioassay haben keine Korrelationen zwischen Wunde und Serum Cytokinkonzentrationen oder deren zeitliche Freisetzungsprofil gefunden (J Pain 2008;. 9 (7) :650-7) 1 Wir haben auch die Nützlichkeit der Technik dokumentiert, um zu identifizieren. Drogen-vermittelte Veränderungen in der Wunde Zytokin-Gehalt (Anesth Analg 2010; 111:1452-9). 2

Mit dieser Methode ist es uns gelungen, alle biochemischen Mediatoren oben mit Ausnahme von IL-5 in Wundexsudat und Plasma umrissen detektieren. 1,2 Einige zusätzliche Cytokine wurden im Plasma nicht erkannt (IL-4, IL-17, TNF, INFγ , GM-CSF). 1. Der zeitliche Verlauf der gemessenen Zytokine und Chemokine in berichteten Studien waren konsistent und erreichte ein Plateau innerhalb der ersten Stunden, die für das 24-Stunden Beobachtungszeitraum aufrechterhalten wurde .. 1,2 Jedoch erhöhte NGF stetig über die 24-Stunden Erfassungszeitraum, während PGE2 innerhalb von 1 Stunde erreichte und dann stetig gesunken. 1 Besonders bemerkenswerte Ergebnisse sind deutlich Spiegel von IL-10 verringert und ein höheres Maß an Substanz P in Wunden mit dem Lokalanästhetikum Bupivacain, kompatibel mit eingeflößt eine umfassende Pro- inflammatorische Wirkung des Medikaments (Abbildung 1). 2 In einer anderen Studie haben wir gezeigt, dass die analgetische Verbrauch nach Kaiserschnitt deLackierung Operation wurde negativ mit Wundexsudat IL-6, IL-1 β und G-CSF-Konzentrationen (Abbildung 2) korreliert. 1 <img alt="Abbildung 1" fo:content-width="3in" fo:src="/files/ftp_upload/50133/50133fig1highres.jpg" src="/files/ftp_upload/50133/50133fig1.jpg" /> Abbildung 1. Interleukin-10 (IL-10) und Substanz P gewickelt Exsudatmengen Ebenen (pg / ml) in 1 gemessen, 3, 5, 7 und 24 Stunden nach Kaiserschnitt. Die physiologische Kochsalzlösung Gruppe ist grau Diamanten angegeben, und die Gruppe wird Bupivacain mit schwarzen Quadraten gezeigt. * = Signifikant verschieden zwischen den Gruppen (P &lt;0,005). <img alt="Abbildung 2" src="/files/ftp_upload/50133/50133fig2.jpg" /> Abbildung 2. Korrelation zwischen Interleukin-6 (pg / ml) und Morphin Verbrauch in den ersten 24 Stunden nach Kaiserschnitt.

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

Dieser Artikel enthält eine detaillierte und visuelle Beschreibung einer Methode zur Sammlung und Messung biochemischer entzündlichen und nozizeptiven Mediatoren an der chirurgischen Wunde nach Kaiserschnitt. Diese menschliche Bioassay wurde verwendet, um Korrelationen zwischen Wunde und Serum Cytokinkonzentrationen und Drug-vermittelten Änderungen Wunde Zytokinen, Chemokinen und neuropetides bestimmen.

Sammeln und Messen Wundexsudat Biochemische Mediatoren In chirurgische Wunden

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 Aufrufe.  Stanford University School of Medicine. Dieser Artikel enthält eine detaillierte und visuelle Beschreibung einer Methode zur Sammlung und Messung biochemischer entzündlichen und nozizeptiven Mediatoren an der chirurgischen Wunde nach Kaiserschnitt. Diese menschliche Bioassay wurde verwendet, um Korrelationen zwischen Wunde und Serum Cytokinkonzentrationen und Drug-vermittelten Änderungen Wunde Zytokinen, Chemokinen und neuropetides bestimmen.

Serie: 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).

Biochemische Measurement of Neonatal Hypoxie

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

Forschung

Medizin

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.

Maus-Modell der Intraluminal MCAO: Hirninfarkt Evaluation durch Cresylviolett 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. ...

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 Messung der Herzfrequenz in Mouse von Nichtinvasive M-Mode-Echokardiographie

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.

Intravitalen Mikroskopie von Tumor-assoziierten Gefäßsystem mit erweiterten dorsalen Hautfalte Fenster Kammern an transgenen Mäusen fluoreszierende

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.

Verwendung von In-vitro- Live Cell Imaging zur Erkundung Chemotherapeutika geliefert durch Lipid-basierte Nanopartikel

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.

Sammeln von Haarproben für Cortisol Haaranalyse bei Afroamerikanern

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.

Protokoll zur Herstellung chronischer Wunden bei diabetischen Mäusen

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.

Epitheliale Korneaabnutzung mit okulärer Burr als Modell für die Hornhaut Wundheilung

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.

Ausrichtung auf graue Rami Communicantes im selektiven chemischen lumbale Sympathektomie

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