Name: lumican extraction from amniotic membrane and determination of its storage temperature
Uploaded: 2022-10-14T14:30:00
Description: Watch this Scientific Journal Video about Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature at JoVE.com

Les auteurs n’ont pas d’intérêts financiers concurrents.

Toutes les procédures expérimentales ont été approuvées par le Comité d’examen institutionnel (projet n° CEI-2020/06/04). La MG a été obtenue auprès de la banque d’amnions de l’Instituto de Oftalmologia Conde de Valenciana (à partir de sujets humains déidentifiés), qui est préparée comme décrit par Chávez-García et al.17.
1. Préparation de l’extrait membranaire amniotique
<ol><li>Obtenez 100 mg d’AM de la banque d’amnion...

<ol>
	<li>Jirsova, K., Jones, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+in+ophthalmology:+properties,+preparation,+storage+and+indications+for+grafting-a+review.">Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review.</a> Cell and Tissue Banking. 18 (2), 193-204 (2017).</li><li>Witherel, C., Yu, T., Concannon, M., Dampier, W., Spiller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+human+cryopreserved+viable+amniotic+membrane+in+a+pro-inflammatory+environment+in+vitro.">Immunomodulatory effects of human cryopreserved viable amniotic membrane in a pro-inflammatory environment in vitro.</a> Cellular and Molecular Bioengineering. 10 (5), 451-462 (2017).</li><li>Ruiz-Ca&#241;ada, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+stimulates+cell+migration+by+modulating+transforming+growth+factor-&#946;+signalling.">Amniotic membrane stimulates cell migration by modulating transforming growth factor-&#946; signalling.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (3), 808-820 (2017).</li><li>Yeh, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+lumican+glycoprotein+purified+from+human+amniotic+membrane+promotes+corneal+epithelial+wound+healing.">Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing.</a> Investigative Opthalmology &amp; Visual Science. 46 (2), 479 (2005).</li><li>Navas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-Inflammatory+and+anti-fibrotic+effects+of+human+amniotic+membrane+mesenchymal+stem+cells+and+their+potential+in+corneal+repair.">Anti-Inflammatory and anti-fibrotic effects of human amniotic membrane mesenchymal stem cells and their potential in corneal repair.</a> Stem Cells Translational Medicine. 7 (12), 906-917 (2018).</li><li>Maga&#241;a-Guerrero, F., Dom&#237;nguez-L&#243;pez, A., Mart&#237;nez-Aboytes, P., Buentello-Volante, B., Garfias, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amniotic+membrane+mesenchymal+stem+cells+inhibit+neutrophil+extracellular+traps+through+TSG-6.">Human amniotic membrane mesenchymal stem cells inhibit neutrophil extracellular traps through TSG-6.</a> Scientific Reports. 7, 12426 (2017).</li><li>Garfias, Y., Zaga-Clavellina, V., Vadillo-Ortega, F., Osorio, M., Jimenez-Martinez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+is+an+immunosuppressor+of+peripheral+blood+mononuclear+cells.">Amniotic membrane is an immunosuppressor of peripheral blood mononuclear cells.</a> Immunological Investigations. 40 (2), 183-196 (2010).</li><li>Koob, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+properties+of+dehydrated+human+amnion/chorion+composite+graft:+implications+for+chronic+wound+healing.">Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing.</a> International Wound Journal. 10 (5), 493-500 (2013).</li><li>Miyagi, H., Thomasy, S., Russell, P., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+hepatocyte+growth+factor+in+corneal+wound+healing.">The role of hepatocyte growth factor in corneal wound healing.</a> Experimental Eye Research. 166, 49-55 (2018).</li><li>Chen, S., Mienaltowski, M., Birk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+corneal+stroma+extracellular+matrix+assembly.">Regulation of corneal stroma extracellular matrix assembly.</a> Experimental Eye Research. 133, 69-80 (2015).</li><li>Karamanou, K., Perrot, G., Maquart, F., Br&#233;zillon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumican+as+a+multivalent+effector+in+wound+healing.">Lumican as a multivalent effector in wound healing.</a> Advanced Drug Delivery Reviews. 129, 344-351 (2018).</li><li>Theocharis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-matrix+interactions:+focus+on+proteoglycan-proteinase+interplay+and+pharmacological+targeting+in+cancer.">Cell-matrix interactions: focus on proteoglycan-proteinase interplay and pharmacological targeting in cancer.</a> FEBS Journal. 281 (22), 5023-5042 (2014).</li><li>Saika, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lumican+in+the+corneal+epithelium+during+wound+healing.">Role of lumican in the corneal epithelium during wound healing.</a> Journal of Biological Chemistry. 275 (4), 2607-2612 (2000).</li><li>Dom&#237;nguez-L&#243;pez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+conditioned+medium+(AMCM)+reduces+inflammatory+response+on+human+limbal+myofibroblast,+and+the+potential+role+of+lumican.">Amniotic membrane conditioned medium (AMCM) reduces inflammatory response on human limbal myofibroblast, and the potential role of lumican.</a> Molecular Vision. 27, 370-383 (2021).</li><li>Vij, N., Roberts, L., Joyce, S., Chakravarti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumican+regulates+corneal+inflammatory+responses+by+modulating+Fas-Fas+Ligand+signaling.">Lumican regulates corneal inflammatory responses by modulating Fas-Fas Ligand signaling.</a> Investigative Opthalmology &amp; Visual Science. 46 (1), 88 (2005).</li><li>Mahbod, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+extract+preparation:+What+is+the+best+method.">Amniotic membrane extract preparation: What is the best method.</a> Journal of Ophthalmic and Vision Research. 9 (3), 314-319 (2014).</li><li>Ch&#225;vez-Garc&#237;a, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ophthalmic+indications+of+amniotic+membrane+transplantation+in+Mexico:+an+eight+years+Amniotic+Membrane+Bank+experience.">Ophthalmic indications of amniotic membrane transplantation in Mexico: an eight years Amniotic Membrane Bank experience.</a> Cell and Tissue Banking. 17 (2), 261-268 (2015).</li><li>Stenn, K. S., Link, R., Moellmann, G., Madri, J., Kuklinska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispase,+a+neutral+protease+from+Bacillus+polymyxa,+is+a+powerful+fibronectinase+and+type+IV+collagenase.">Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase.</a> Journal of Investigative Dermatology. 93 (2), 287-290 (1989).</li><li>Bhatnagar, B. S., Bogner, R. H., Pikal, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+stability+during+freezing:+separation+of+stresses+and+mechanisms+of+protein+stabilization.">Protein stability during freezing: separation of stresses and mechanisms of protein stabilization.</a> Pharmaceutical Development and Technology. 12 (5), 505-523 (2007).</li><li>McClain, A. K., McCarrel, T. M. <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+four+different+freezing+conditions+and+time+in+frozen+storage+on+the+concentration+of+commonly+measured+growth+factors+and+enzymes+in+equine+platelet-rich+plasma+over+six+months.">The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months.</a> BMC Veterinary Research. 15 (1), 292 (2019).</li><li>Tamhane, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+amniotic+membrane+transplantation+as+an+adjunct+to+medical+therapy+as+compared+with+medical+therapy+alone+in+acute+ocular+burns.">Evaluation of amniotic membrane transplantation as an adjunct to medical therapy as compared with medical therapy alone in acute ocular burns.</a> Ophthalmology. 112 (11), 1963-1969 (2005).</li><li>Shtein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+serum-based+eye+drops+for+treatment+of+ocular+surface+disease.">Autologous serum-based eye drops for treatment of ocular surface disease.</a> Ophthalmology. 127 (1), 128-133 (2020).</li><li>Shahriari, H., Tokhmehchi, F., Reza, M., Hashemi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+effect+of+amniotic+membrane+suspension+and+autologous+serum+on+alkaline+corneal+epithelial+wound+healing+in+the+rabbit+model.">Comparison of the effect of amniotic membrane suspension and autologous serum on alkaline corneal epithelial wound healing in the rabbit model.</a> Cornea. 27 (10), 1148-1150 (2008).</li><li>Schuerch, K., Baeriswyl, A., Frueh, B., Tappeiner, C. <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+amniotic+membrane+transplantation+for+the+treatment+of+corneal+ulcers.">Efficacy of amniotic membrane transplantation for the treatment of corneal ulcers.</a> Cornea. 39 (4), 479-483 (2019).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+transplantation+for+persistent+corneal+ulcers+and+perforations+in+acute+fungal+keratitis.">Amniotic membrane transplantation for persistent corneal ulcers and perforations in acute fungal keratitis.</a> Cornea. 25 (5), 564-572 (2006).</li><li>Guo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+effectiveness+between+amniotic+membrane+homogenate+and+transplanted+amniotic+membrane+in+healing+corneal+damage+in+a+rabbit+model.">A comparison of the effectiveness between amniotic membrane homogenate and transplanted amniotic membrane in healing corneal damage in a rabbit model.</a> Acta Ophthalmologica. 89 (4), 315-319 (2011).</li><li>Sabater, A., Perez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+use+for+management+of+corneal+limbal+stem+cell+deficiency.">Amniotic membrane use for management of corneal limbal stem cell deficiency.</a> Current Opinion in Ophthalmology. 28 (4), 363-369 (2017).</li><li>Ahmad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autolysis+of+bovine+skin,+its+endogenous+proteases,+protease+inhibitors+and+their+effects+on+quality+characteristics+of+extracted+gelatin.">Autolysis of bovine skin, its endogenous proteases, protease inhibitors and their effects on quality characteristics of extracted gelatin.</a> Food Chemistry. 265, 1-8 (2018).</li><li>Mullegama, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+extraction+from+human+biological+samples.">Nucleic acid extraction from human biological samples.</a> Methods in Molecular Biology. 1897, 359-383 (2019).</li><li>Skog, M., et al. <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+enzymatic+digestion+on+cultured+epithelial+autografts.">The effect of enzymatic digestion on cultured epithelial autografts.</a> Cell Transplantation. 28 (5), 638-644 (2019).</li></ol>

Dans cette étude, la présence de lumican a été analysée dans le TEA et sa corrélation directe avec sa stabilité dans différentes conditions de stockage. Fait intéressant, lorsque la concentration totale de protéines dans la TEA a été quantifiée, la concentration de protéines a augmenté après le stockage. Les preuves suggèrent trois mécanismes qui pourraient modifier la concentration de protéines dans l’entreposage congelé : la dénaturation à froid, la concentration congelée de solutés et le dép...

L’un des traitements les plus utilisés pour les affections cornéennes est la greffe de membrane amniotique; Cependant, au cours des dernières années, de nouvelles propositions ont émergé pour l’utilisation de divers composants du tissu amniotique comme traitements alternatifs et adjuvants. Parmi les composants les plus étudiés de la FA figurent ceux obtenus à partir de l’extrait d’AM (AME)1,2,3,4,5,6,7. La MA contient de multiples facteurs solubles tels que des protéines antiangiogéniques, des interleukines (IL), des inhibiteurs tissulaires des métalloprotéinases (TIMPs), des protéines anti-inflammatoires médiées par TSG-6 qui inhibent les pièges extracellulaires des neutrophiles, des facteurs de croissance: facteur de croissance épidermique (EGF), facteur de croissance transformant (TGF) (alpha et bêta), facteur de croissance kératinocytaire (KGF), facteur de croissance hépatocytes (HGF) et lumican, qui maintient la transparence cornéenne en régulant la fibrillogenèse du collagène1, 2,3,4,5,6,7,8,9.
Le lumican est un petit protéoglycane riche en leucine (SLRP), l’un des principaux composants extracellulaires de la collagénase interstitielle dans la matrice du stroma cornéen, responsable de l’organisation des fibres de collagène et du maintien de la transparence cornéenne 4,10,11. Les protéoglycanes sont des molécules de la matrice extracellulaire (MEC), qui sont les principales dans la réalisation de la signalisation cellulaire et le maintien de l’homéostasie intracellulaire12. Il a été rapporté que les protéines ECM conduisent les processus cellulaires de prolifération, de différenciation et de migration pendant la cicatrisation des plaies11.
Les preuves indiquent la participation possible du lumican dans le processus de réépithélialisation cornéenne. Saika et al., dans une étude, ont montré qu’après une lésion cornéenne, le lumican pouvait être détecté dans les kératocytes cornéens entre les 8 premières heures et jusqu’à 3 jours après la blessure. Présentant la plus forte concentration de lumican le deuxième et le troisième jour, ce protéoglycane est par la suite indétectable le septième jour13. Ces données suggèrent la participation du lumican dans l’activation du processus de réépithélialisation cornéenne. D’autre part, dans une autre étude, il a été rapporté que l’absence de lumican retarde la réépithélialisation; Fait intéressant, l’ajout de Lumican pourrait accélérer le processus de réépithélialisation 4,11,13. De même, une étude récente a rapporté que le lumican peut moduler les fonctions inflammatoires des fibroblastes du limbe cornéen14, ce qui suggère un rôle pour le lumican en tant que modulateur de la réponse inflammatoire, antifibrotique et ré-épithélialisante. De même, le lumican peut moduler la réponse cornéenne en interagissant avec des molécules de signalisation telles que Fas-FasL. De plus, l’absence de lumican dans un modèle Lum-/- souris knockout a démontré que l’absence de signalisation lumican empêche une réparation cornéenne adéquate15.
Principalement, cette méthode vise à démontrer un moyen réalisable et accessible d’extraire le lumican de la FA. Avec cette méthode avantageuse d’extraction du lumican, il est possible d’obtenir des concentrations similaires de protéines, ce qui diminue le temps de traitement et le rend plus pratique pour les chercheurs par rapport aux études précédentes16. De plus, ce lumican AME pourrait être utilisé comme adjuvant pour les processus de réparation et de réépithélialisation de la cornée.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 N H2SO4 stop solution</td>
			<td>R&#38;D Systems</td>
			<td>DY994</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm Petri dish</td>
			<td>Symlaboratorios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18 G Needle (1.2 mm x 40 mm)</td>
			<td>BD Becton Dickinson&#160;</td>
			<td>305211</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>Z606340</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL plastic syringe&#160;</td>
			<td>BD Becton Dickinson&#160;</td>
			<td>302562</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>30119401</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>SARSTEDT</td>
			<td>821581</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Amniotic membrane</td>
			<td>Instituto de Oftalmologia Conde de Valenciana Amnion Bank</td>
			<td></td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Balanced salt solution</td>
			<td>Bausch + Lomb</td>
			<td>BSS-403802</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRender&#160;</td>
			<td>BioRender</td>
			<td></td>
			<td>figures design&#160;</td>
		</tr>
		<tr>
			<td>Compact Rocker</td>
			<td>BioRad</td>
			<td>970822DD</td>
			<td>Mod. 5202SD-BIO</td>
		</tr>
		<tr>
			<td>complete, EDTA-free, Protease inhibitor 
			cocktail tablets</td>
			<td>Roche</td>
			<td>11 873 580 001</td>
			<td>Protease Inhibitor</td>
		</tr>
		<tr>
			<td>Daiggner vortex Genie 2</td>
			<td>A.Daigger &#38; Co. , INC</td>
			<td>22220A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plate spectrometer</td>
			<td>Thermo Labsystems&#160;</td>
			<td>35401106</td>
			<td>Multiscan</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism&#160;</td>
			<td>GraphPad Software, Inc</td>
			<td>version 9</td>
			<td>statistical analysis and graphic program&#160;</td>
		</tr>
		<tr>
			<td>Human lumican DuoSet ELISA kit</td>
			<td>R&#38;D Systems</td>
			<td>DY2846-05</td>
			<td>includes human Lumican capture antibody</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>Forma Scientific&#160;</td>
			<td>3326 S/N 36481-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light Microscope</td>
			<td>Olympus&#160;</td>
			<td>6A13921</td>
			<td>to confirm de-epithelialization&#160; Mod.CK2</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Forma Scientific&#160;</td>
			<td>14753-567</td>
			<td>Mod.1184</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipettor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen Tank</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Mod. Biocan 20</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>R&#38;D Systems</td>
			<td>DY006</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Modified Lowry Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23240</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealers</td>
			<td>R&#38;D Systems</td>
			<td>DY992</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent diluent</td>
			<td>R&#38;D Systems</td>
			<td>DY995</td>
			<td>1% BSA in PBS, pH 7.2-7.4, 0.2 &#956;m filtered</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>centurion scientific Ltd&#160;</td>
			<td>15877</td>
			<td>Mod. K2015R</td>
		</tr>
		<tr>
			<td>Rubber policeman cell scraper</td>
			<td>NEST</td>
			<td>710001</td>
			<td>for mechanical de-epithelialization</td>
		</tr>
		<tr>
			<td>Scalpel knife</td>
			<td>Braun</td>
			<td>BB521</td>
			<td>No. 10 or 21</td>
		</tr>
		<tr>
			<td>Streptavidin-HRP 40-fold concentrated&#160;</td>
			<td>R&#38;D Systems</td>
			<td>part 893975</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate tetramethylbenzidine (TMB) solution</td>
			<td>R&#38;D Systems</td>
			<td>DY999</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed tweezers</td>
			<td>Invent Germany</td>
			<td>6b</td>
			<td>inox&#160;</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>PISA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td>R&#38;D Systems</td>
			<td>WA126</td>
			<td>0.05% Tween 20 in PBS, pH 7.2-7.4</td>
		</tr>
	</tbody>
</table>

lumican extraction from amniotic membrane and determination of its storage temperature

Lumican est un petit protéoglycane riche en leucine dans la membrane amniotique humaine (AM) qui favorise l’épithélialisation cornéenne et l’organisation des fibres de collagène, en maintenant la transparence cornéenne. Dans le présent travail, une méthode d’extraction de protéines de la FA pour obtenir du lumican est proposée. De plus, la stabilité du lumican dans l’extrait AM (AME) stocké à différentes températures et périodes est évaluée. 100 mg de MA ont été décongelés et déépithélialisés mécaniquement. La MA désépithélialisée a été congelée et broyée jusqu’à l’obtention d’une poudre fine, qui a été solubilisée avec 2,5 mL de tampon saline avec des inhibiteurs de protéase et centrifugée pour l’extraction des protéines. Le surnageant a été recueilli et conservé à -20 °C, 4 °C et à température ambiante (RT) pendant 6, 12, 20 et 32 jours. Par la suite, le lumican a été quantifié dans chaque TEA. Cette technique permet un protocole accessible et acquis pour l’extraction du lumican à partir de la FA. La concentration de lumican a été affectée par le temps de stockage et les conditions de température. Le Lumican dans le TEA de 12 jours stocké à -20 °C et 4 °C était significativement plus élevé que les autres TEA. Cette extraction de lumican pourrait être utile pour développer des traitements et des solutions pharmaceutiques. D’autres études sont nécessaires pour déterminer les utilisations du lumican AME dans la réépithélialisation et le processus de cicatrisation.

Les résultats sont présentés sous forme de valeur moyenne ±écart-type (ET). Les tests t de Student et l’analyse de la variance (ANOVA) ont été effectués. Les valeurs de p < 0,05 ont été considérées comme statistiquement significatives. L’analyse statistique a été effectuée à l’aide d’un logiciel de statistiques (voir le tableau des matières).
La quantité totale de protéines dans le TEA a été influencée par le temps et les conditi...

Watch this Scientific Journal Video about Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature at JoVE.com

Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature

Le présent protocole décrit l’extraction du lumican de la membrane amniotique (AM) et leurs conditions de stockage sous forme d’extrait de MA (AME) à -20 °C, 4 °C et à température ambiante (RT) pendant 6, 12, 20 et 32 jours pour quantifier ses protéines et sa concentration en lumicane.

Extraction de lumican à partir d’une membrane amniotique et détermination de sa température de stockage

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen ...

Cette méthode vise à démontrer une technique simple d’extraction de protéines, utile pour l’obtention de protéines abondantes et d’autres facteurs. Cette technique pourrait être utilisée pour extraire d’autres facteurs et protéines présents dans la membrane amniotique et aussi, pour obtenir des protéines d’autres sources telles que les animaux et les tissus végétaux. Commencez par décongeler 100 milligrammes de la membrane amniotique obtenue à partir de la banque d’amnions.Une fois décongelé, ajoutez 10 millilitres de la solution saline équilibrée stérile à une boîte de Petri et lavez la membrane pendant deux minutes. Ensuite, retirez la solution saline équilibrée utilisée dans un bécher et répétez le processus jusqu’à ce qu’aucune trace de glycérol ne soit visible dans la boîte de Pétri. Ensuite, ajoutez 10 millilitres de dispase II à la membrane et incuber à 37 degrés Celsius et 5% de dioxyde de carbone pendant 30 minutes.À la fin de l’incubation, effectuer une désépithélialisation mécanique avec un grattoir de cellule de policier en caoutchouc. Ensuite, visualisez la membrane au microscope inversé pour confirmer la dé-épithélialisation. Ensuite, lavez la membrane amniotique désépithélialisée avec la solution saline équilibrée stérile comme démontré précédemment, et placez-la dans un tube microcentrifuge de deux millilitres.Ensuite, immergez le tube contenant la membrane dans de l’azote liquide pendant 40 minutes. Après la congélation avec de l’azote liquide, broyer manuellement la membrane pendant deux à trois minutes dans un mortier, pré-refroidi à moins 85 degrés Celsius, jusqu’à l’obtention d’une poudre fine. Ensuite, ajoutez 2,5 millilitres de solution inhibitrice de protéase et solubilisez la membrane finement réduite.Nettoyez les murs de mortier à l’aide d’un couteau à scalpel et transférez le mélange dans un tube de cinq millilitres. Tourbillonner le tube pendant 30 secondes. Ensuite, homogénéiser le mélange tissulaire en centrifugeant d’abord à 34 G pendant 20 minutes à quatre degrés Celsius, puis en le centrifugeant immédiatement à nouveau à 3, 360 G pendant 20 minutes à quatre degrés Celsius.À la fin de la centrifugation, recueillir le surnageant, qui est l’extrait de membrane amniotique, dans un nouveau tube. Ensuite, aliquote 0,7 millilitre dans un tube microcentrifuge différent de deux millilitres pour différents points de temps et températures. La quantité totale de protéines et la concentration de lumican dans l’extrait de membrane amniotique ont été affectées par le temps et les conditions de stockage.La concentration de protéines basales était similaire parmi tous les extraits de membrane amniotique. Cependant, une concentration significativement élevée de protéines a été notée dans l’extrait stocké pendant 32 jours à quatre degrés Celsius et 20 jours à moins 20 degrés Celsius. Le lumican dans l’extrait de membrane amniotique était significativement plus élevé dans l’échantillon stocké pendant une période de 12 jours par rapport aux échantillons stockés pendant 30, 20 et six jours à quatre et moins 20 degrés Celsius.Ces résultats suggèrent que la durée et la température d’entreposage appropriées pour atteindre la concentration la plus élevée de lumican sont de 12 jours à moins 20 degrés Celsius. Faites preuve du plus grand soin lors de la mise à la terre de la membrane amniotique gelée. La mise à la terre doit être douce car elle pourrait tomber du mortier.D’autres études sont nécessaires pour déterminer le rôle de l’extrait membranaire amniotique de lumican dans les cellules épithéliales cornéennes et pour déterminer la dose idéale de lumican pour la réépithélialisation cornéenne.

lumican-extraction-from-amniotic-membrane-determination-its-storage

Research

JoVE Journal

Medicine

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Technique de Subnormothermic<em&gt; Ex Vivo</em&gt; Foie de perfusion pour l&#39;entreposage, de l&#39;évaluation et de réparation des marginaux greffes du foie

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

Recherche

Médecine

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modélisation encéphalopathie de prématurité Utilisation prénatale hypoxie ischémie avec un lipopolysaccharide intra-amniotique chez le rat

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Using an Ingestible Telemetric Temperature Pill to Assess Gastrointestinal Temperature During Exercise

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of heat-related disorders. Therefore, accurate measurement of Tc during exercise is of great importance, especially in athletes who have to perform in challenging ambient conditions. In the current literature a number of methods have been described to measure the Tc (esophageal, external tympanic membrane, mouth or rectum). However, these methods are suboptimal to measure Tc during exercise since they are invasive, have a slow response or are influenced by environmental conditions. Studies described the use of an ingestible telemetric temperature pill as a reliable and valid method to assess gastrointestinal temperature (Tgi), which is a representative measurement of Tc. Therefore, the goal of this study was to provide a detailed description of the measurement of Tgi using an ingestible telemetric temperature pill. This study addresses important methodological factors that must be taken into account for an accurate measurement. It is recommended to read the instructions carefully in order to ensure that the ingestible telemetric temperature pill is a reliable method to assess Tgi at rest and during exercise.

This study describes an accurate, reliable and non-invasive technique to continuously measure gastrointestinal temperature during exercise. The ingestible telemetric temperature pill is suitable to measure gastrointestinal temperature in laboratory settings as well as in field based settings.

En utilisant une température Pill ingérable télémétrique pour évaluer Température gastro pendant l&#39;exercice

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of ...

Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often performed for neuroprotection after ischemic insult (e.g. hemorrhagic stroke, subarachnoid hemorrhage, cardiac arrest, or other hypoxic injury). Cooling from febrile states treats fever and reduces the negative effects of hyperthermia on injured neurons. Patients are warmed in the operating room to prevent inadvertent perioperative hypothermia, which is known to cause increased blood loss, wound infections, and myocardial injury, while also prolonging recovery time. There are many reported approaches for temperature management, including improvised methods that repurpose standard supplies (e.g., ice, chilled saline, fans, blankets) but more sophisticated technologies designed for temperature management are typically more successful in delivering an optimized protocol. Over the last decade, advanced technologies have developed around two heat transfer methods: surface devices (water blankets, forced-air warmers) or intravascular devices (sterile catheters requiring vascular placement). Recently, a novel device became available that is placed in the esophagus, analogous to a standard orogastric tube, that provides efficient heat transfer through the patient&#39;s core. The device connects to existing heat exchange units to allow automatic patient temperature management via a servo mechanism, using patient temperature from standard temperature sensors (rectal, Foley, or other core temperature sensors) as the input variable. This approach eliminates vascular placement complications (deep venous thrombosis, central line associated bloodstream infection), reduces obstruction to patient access, and causes less shivering when compared to surface approaches. Published data have also shown a high degree of accuracy and maintenance of target temperature using the esophageal approach to temperature management. Therefore, the purpose of this method is to provide a low-risk alternative method for controlling patient temperature in critical care settings.

This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management.

Transfert de chaleur oesophagienne pour le contrôle de la température du Patient et de la gestion de la température ciblée

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often ...

Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ion channels and electrogenic pumps that regulate Vm in disease conditions could potentially alter Vm, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record Vm in healthy and diseased states are essential. This method will allow modulating Vm using different pharmacological agents to restore Vm. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record Vm from cannulated resistance vessels such as the middle cerebral artery using the microelectrode impalement method. Middle cerebral arteries are allowed to gain myogenic tone in a myograph chamber, and the vessel wall is impaled using high resistance microelectrodes. The Vm signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the Vm of a vessel wall without damaging the cells and without changing the membrane resistance.

The primary goal of this article is to provide details of how to record membrane potential (Vm) from the middle cerebral artery using the microelectrode impalement method. The cannulated middle cerebral artery is equilibrated to gain myogenic tone, and the vessel wall is impaled using high resistance microelectrodes.

Méthode d'empalement de microélectrode pour enregistrer le potentiel de membrane d'une artère cérébrale moyenne cannulated

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the effects of nitrosated compounds. Measuring NO metabolites is essential to studying NO activity both at vascular levels and in other tissues, especially in the experimental settings where exogenous NO is administered. Ozone-based chemiluminescence assays allow precise measurements of NO and NO metabolites in both fluids (including plasma, tissue homogenates, cell cultures) and gas mixtures (e.g., exhaled breath). NO reacts with ozone to generate nitrogen dioxide in an excited state. The consequent light emission allows photodetection and the generation of an electric signal reflecting the NO content of the sample. Aliquots from the same sample can be used to measure specific NO metabolites, such as nitrate, nitrite, S-nitrosothiols, and iron-nitrosyl complexes. In addition, NO consumed by cell-free hemoglobin is also quantified with chemiluminescence analysis. An illustration of all these techniques is provided.

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

Tests basés sur la chimiluminescence pour la détection de l’oxyde nitrique et de ses dérivés à partir de l’autoxidation et des composés nitrosés

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the ...

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial infarction, arrhythmia, atherosclerosis, cardiogenic pulmonary edema, pulmonary embolism, and cerebral embolism. At the same time, it is urgent to break through the continuous heart blood sampling technique in live rats to evaluate the effectiveness of distinctive ethnic medicine therapy. In this study, a blood microdialysis probe was implanted in the right jugular vein of rats in a precise and noninvasive surgical procedure. Cardiac blood samples were then collected at a rate of 2.87 nL/min to 2.98 mL/min by connecting to an online microdialysis sample collection system. Even more momentously, the acquired blood samples can temporarily be stored in microdialysis containers at 4 &#176;C. The microdialysis-based online continuous blood collection program from rat heart has greatly guaranteed the quality of blood samples, advancing and invigorating the scientific rationality of the research on systemic cardiovascular diseases and evaluating ethnomedicine therapy from the perspective of hematology.

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

The present protocol describes a simple and efficient method for the real-time and dynamic collection of rat heart blood using the microdialysis technique.

Extraction dynamique continue de sang du cœur de rat par une technique de microdialyse non invasive

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial ...

Extraction économique de l’ADN de diatomées à l’aide d’un kit modifié

Extraction of Diatom DNA from Water Samples and Tissues

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

Pleins feux sur l’auteur : Test de diatomées pour l’examen médico-légal de la noyade 

This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

Extraction de l’ADN des diatomées à partir d’échantillons d’eau et de tissus

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...