Les travaux de M. Carvalho a été soutenu par une subvention de recherche du Bureau de recherche sur la santé des femmes et de l&#39;Institut national de la santé infantile et le développement humain de la National Institutes of Health (5K12 HD043452). Angst Dr reçu des fournitures (On-Q PainBuster Post-Op Système de soulagement de la douleur) et des fonds (pour les dosages biochimiques) de I-Flow (Lake Forest, en Californie).

1. Protocole d&#39;étude <ul><li> Obtenir l&#39;approbation du Conseil institutionnel Donnez votre avis et consentement éclairé écrit. </li><li> Choisissez une population chirurgicale appropriée pour la conduite des études. Cette méthodologie a été appliquée qu&#39;à des plaies d&#39;accouchement par césarienne, mais la plupart des blessures chirurgicales serait approprié. </li><li> Tenir compte des critères d&#39;inclusion et d&#39;exclusion pour votre participation à l&#39;étude. Les patients et les procédures des taux élevés d&#39;infection de la plaie chirurgicale peut ne pas être approprié. </li><li> Normaliser l&#39;anesthésie et prise en charge postopératoire pour tous les patients à éviter les facteurs de confusion potentiels. </li></ul> 2. Nociceptive et inflammatoires Collection médiateur biochimique <ul><li> Insérez le PainBuster ON-Q avec ON-Q SilverSoaker (I-Flow, Lake Forest, CA) dans la couche sous-cutanée juste avant de refermer la plaie (d&#39;autres couches de tissu peut être considéré). Le Système ON-Q PainBuster Pain Relief délivre en continu une solution saline normale à un débit de 2 ml / h. Le support peut être peigneminé avec ou par rapport à des médicaments d&#39;intérêt comme un anesthésique local et un non-stéroïdiens anti-inflammatoires. </li><li> Intégrer une norme robinet à trois voies dans ce système pour permettre l&#39;aspiration des exsudats de la plaie à des moments spécifiés. La perfusion continue empêche la coagulation du cathéter et améliore la fiabilité du système pour produire des échantillons d&#39;exsudat. Si l&#39;aspiration de l&#39;exsudat est difficile (environ 5% des cas), pensez à pousser doucement au-dessus de la plaie, en changeant la position du sujet (par exemple, le patient assis ou allongé jusqu&#39;à leur plat), en utilisant un chasse 0,5-1 ml sérum physiologique ou le retrait de la cathéter 1-2 cm. </li><li> À des moments spécifiés par le protocole (par exemple, 1, 4, 6, 24, et 48 h après l&#39;accouchement par césarienne), aspirer et retirer 1 ml de exsudat de la plaie. Transfert de l&#39;exsudat dans une cupule en polyéthylène contenant 30 pl d&#39;inhibiteur de protéinase. Nous avons utilisé protéinase complet (Bioscience Roche, Palo Alto, CA) pour nos besoins. </li><li&gt; Aux mêmes intervalles de temps spécifiés par le protocole de l&#39;étude, de recueillir 10 ml de sang. Nous avons utilisé un tube de prélèvement sanguin contenant de l&#39;héparine de lithium; dans notre établissement il s&#39;agit d&#39;un tube de verre vert au sommet. Ajouter un supplément de 300 pl d&#39;inhibiteur de la protéinase dans les échantillons de sang. </li><li> Dans 1 heure de la collecte, mettre des échantillons sur la glace et centrifuger à 3000 tours par minute pendant 10 minutes. </li><li> Retirer le sérum et le surnageant de la plaie et dans un microtube standard et conserver à -20 ° C. </li></ul> 3. Analyse test <ul><li> Une fois que toutes les exsudats étude et des échantillons de sérum sont collectées et stockées, décongeler et analyser les échantillons en même temps. </li><li> Nous avons mesuré les cytokines et de chimiokines en utilisant un immunodosage multiplexe. Le multiplex immuno-essai technique nous permet de doser des analytes multiples à partir de petits échantillons de volume (50 pi). Le multiplex produire des résultats comparables à ceux obtenus avec la méthode ELISA. Nous avons utilisé le 17-plex bio-PlexTM système (BioRad, Herculesdules, CA) pour notre analyse. Ce dosage est capable de mesurer le facteur de nécrose tumorale α (TNF), l&#39;interféron α (INFα), l&#39;interleukine 1β (IL-1β), interleukine 2 (IL-2), l&#39;interleukine 4 (IL-4), l&#39;interleukine 5 (IL-5 ), l&#39;interleukine 6 (IL-6), l&#39;interleukine 7 (IL-7), l&#39;interleukine 8 (IL-8), l&#39;interleukine (IL-10), l&#39;interleukine 12 (IL-12), l&#39;interleukine 13 (IL-13), interleukine 17 (IL-17), le facteur stimulant les colonies de granulocytes (G-CSF), le facteur de colonies de granulocytes-macrophages (GM-CSF), protéine chimio-attractive des monocytes 1 (MCP-1) et protéine inflammatoire des macrophages 1 (MIP-1β). Facteur de croissance nerveuse a été mesurée avec le NGF DY256 anticorps en l&#39;ajoutant à la plaque 17-plex avec le kit Bio-Plex amine couplage. </li><li> Pour garantir une analyse de bonne qualité, chaque mesure doit être effectuée en double. Courbes d&#39;étalonnage pour chaque analyte sont générées en utilisant des analytes de référence fournis par le fabricant (à des concentrations de 0,20, 0,78, 3,13, 12,5, 50, 200, 800, 3.200 pg / ml plusune norme zéro pour la solution saline normale). Les courbes d&#39;étalonnage sont inclus dans chaque série et concentrations des échantillons sont calculées avec le logiciel de gestion de placements Bio-Plex. </li><li> La substance P et la prostaglandine E2 ont été mesurées à l&#39;aide de sensibilité hautement Kits ELISA. Nous avons utilisé Assay Designs, Inc (Ann Arbor, Michigan) pour cette analyse. Effectuer l&#39;analyse en double exemplaire selon les spécifications du fabricant. </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>

Aucun conflit d&#39;intérêt déclaré.

Cette méthodologie décrite ci-dessus a été jugée suffisamment sensibles pour détecter biochimiques médiateurs inflammatoires et nociceptifs dans exsudat de la plaie. L&#39;échantillonnage à divers points de temps nous a également permis d&#39;observer l&#39;évolution de ces médiateurs au fil du temps. 1,2 Nous ne sommes pas au courant d&#39;aucun autre essai blessure humaine chirurgicale qui peut en série mesurer le profil de libération de médiateurs inflammatoires et nociceptifs dans exsudat d...

Après une incision chirurgicale, résidents et migrateurs cellules inflammatoires libèrent des cytokines pro-et anti-inflammatoires, prostanoïdes, les neuropeptides et de chimiokines qui sont importants pour l&#39;initiation et le maintien de la douleur. 3,4 La capacité de recueillir spécifiques au site marqueurs biochimiques est essentiel de déterminer la relation entre le niveau de la plaie chirurgicale et le sérum, de comprendre la relation complexe entre la libération de médiateurs et de la douleur chirurgicale, et d&#39;être en mesure d&#39;évaluer l&#39;effet à la fois systémique et l&#39;administration du médicament périphérique sur la biochimie des plaies chirurgicales. Les études ont porté sur des mesures systémiques de médiateurs biochimiques et n&#39;ont pas tenu compte spécifique au site de libération. 5,6 mesures sériques de cytokines et de chimiokines peuvent ne pas refléter des événements locaux plaies chirurgicales biochimiques et indiquent seulement une réponse systémique globale d&#39;une blessure chirurgicale. Cet article fournit une description détaillée et visuelle unique de cette méthodologie qui a été sapinst référencé en 2008 dans le Journal of Pain. 1 Le but de cet article est JoVE de fournir une description unique et visuellement amélioré des méthodes.

collecting and measuring wound exudate biochemical mediators in surgical wounds

Nous décrivons une méthode par laquelle nous sommes en mesure de recueillir et de mesurer biochimiques médiateurs inflammatoires et nociceptifs au site de la plaie chirurgicale. Collecte spécifiques au site marqueurs biochimiques est important de comprendre la relation entre les niveaux de sérum de blessure et chirurgicaux, déterminer les associations entre la libération des médiateurs, la douleur, l&#39;utilisation d&#39;analgésiques et d&#39;autres résultats d&#39;intérêt, et d&#39;évaluer l&#39;effet de l&#39;administration systémique de médicaments et périphérique sur la plaie chirurgicale biochimie. Cette méthodologie a été appliquée à des femmes en bonne santé subissant une césarienne élective sous rachianesthésie. Nous avons mesuré l&#39;exsudat et les médiateurs de sérum dans les mêmes intervalles de temps que les scores de douleur du patient et de la consommation des analgésiques pour un maximum de 48 heures après l&#39;accouchement par césarienne. Grâce à cette méthodologie, nous avons été capables de détecter divers médiateurs biochimiques, y compris le facteur de croissance des nerfs (NGF), la prostaglandine E2 (PG-E2) de la substance P, l&#39;IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8,IL-10, IL-12, IL-13, IL-17, TNF, INFγ, G-CSF, GM-CSF, MCP-1 et MIP-1β. Les études appliquant le présent essai biologique plaie chirurgicale humaine n&#39;ont trouvé aucune corrélation entre la plaie et les concentrations de cytokines sériques ou de leur temps de diffusion du profil (J Pain 2008;. 9 (7) :650-7) 1 Nous avons également documenté l&#39;utilité de la technique à identifier. médicament à médiation changements dans le contenu des cytokines plaie (Anesth Analg 2010; 111:1452-9). 2

Grâce à cette méthodologie, nous avons été en mesure de détecter tous les médiateurs biochimiques décrites ci-dessus, à l&#39;exception de l&#39;IL-5 dans exsudat de la plaie et le plasma. 1,2 Certaines cytokines supplémentaires n&#39;ont pas été détectés dans le plasma (IL-4, IL-17, TNF, INFγ , GM-CSF). 1 L&#39;évolution dans le temps de cytokines et de chimiokines mesurées dans les études signalés correspondaient, pour atteindre un plateau dans les premières heures qui ont ét...

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

Cet article fournit une description détaillée et visuelle d&#39;une méthodologie de collecte et de mesure biochimiques médiateurs inflammatoires et nociceptifs au site de la plaie chirurgicale suite à une césarienne. Ce bio-essai humain a été utilisé pour déterminer les corrélations entre la plaie et les concentrations de cytokines sériques et médicaments médiées par des changements dans la plaie cytokines, des chimiokines et neuropetides.

Collecte et de mesure des médiateurs biochimiques exsudat de la plaie Dans Plaies chirurgicales

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

Research

JoVE Journal

Medicine

11.9K vues.  Stanford University School of Medicine. Cet article fournit une description détaillée et visuelle d'une méthodologie de collecte et de mesure biochimiques médiateurs inflammatoires et nociceptifs au site de la plaie chirurgicale suite à une césarienne. Ce bio-essai humain a été utilisé pour déterminer les corrélations entre la plaie et les concentrations de cytokines sériques et médicaments médiées par des changements dans la plaie cytokines, des chimiokines et neuropetides.

Vidéo: 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).

Mesure biochimique de l&#39;hypoxie néonatale

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

Recherche

Médecine

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.

Collecte de salive et de mesure du cortisol salivaire et l&#39;alpha-amylase dans Frail communautaires résidant personnes âgées par les aidants familiaux

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 mesure de la fréquence cardiaque chez la souris par M mode non invasive échocardiographie

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 ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

Démonstration de la peau plaies Modèle Rat ischémique

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

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.

À l’aide de In Vitro imagerie de cellules vivantes pour explorer les agents chimiothérapeutiques livré par nanoparticules à base de lipides

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.

Le prélèvement d’échantillons de cheveux pour cheveux analyse de Cortisol dans les Afro-américains

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.

Protocole pour créer des blessures chroniques chez les souris diabétiques

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.

Abrasion épithéliale cornéenne avec oculaire Burr comme modèle pour la cornée, la cicatrisation

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.

Ciblage gris Rami Communicantes dans une sympathectomie lombaire chimique sélective

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

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily practice because of the limited awareness and insufficient experience of medical and nursing personnel. There are case reports suggesting that maggot therapy can be applied and supported by lay caregivers, provided they are supervised and informed by physicians/nurses. The foregoing observation suggests that the method should be considered for implementation by a wider group of caregivers if accepted and meticulously supervised by trained and experienced medical staff. The concerns related to the therapeutic use of maggots in certain regions seem understandable, but are not supported by scientific facts. It should be noted that many therapeutic agents (including brood) used in medicine are of natural origin, and are associated with low production costs and high possibilities of implementation in the course of therapy. By analyzing the literature and using our own clinical and research experience, we have come to conclusions related to using larvae therapy, as a quick and safe method providing cleaning and revitalization in the process of treating wounds of various etiologies, especially pressure ulcers. In the current study, medical-grade Lucilia sericata maggots were applied to remove necrotic tissue from deep pressure sores. The treatment is mostly accepted by both caregivers and patients. In most cases, it is conducted by trained and experienced medical personnel in home and outpatient settings. Over the course of the conducted analyzes involving the collected specimens, there were no statistically significant relationships (p&gt; 0.05) confirmed between the wound surface successfully cleared by brood and variables, such as time from wound formation, location, surface size, and the depth of damage to the tissue structure. The lack of statistical dependence may result from the small size of the studied group. Based on the current findings, we have formulated the following conclusions: Maggot Debridement Therapy (MDT) is a fast and effective method enabling the preparation of the wound bed. The use of MDT in outpatient settings is safe and acceptable for patients and their caregivers.

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Maggots of Lucilia sericata were used in the debridement of deep pressure sores in outpatient settings for the treatment of pressure sores involving the complete skin thickness. The technique presented in the article does not pose risks for the patient.

Application des larves de Lucilia sericata dans le débridement des plaies sous pression en ambulatoire

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily ...