Name: noninvasive intratracheal lipopolysaccharide instillation in mice
Uploaded: 2023-03-31T13:40:00
Description: Watch this Scientific Journal Video about Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice at JoVE.com

Dette arbejde blev støttet af National Natural Science Foundation of China (nr.: 81903902), China Postdoctoral Science Foundation (nr.: 2019M663457), Sichuan Science and Technology Program (nr.: 2020YJ0172) og Xinglin Scholar Research Premotion Project fra Chengdu University of TCM (nr.: QJRC2022053).

Dyreforsøgsprotokollen blev gennemgået og godkendt af forvaltningskomitéen for Chengdu University of Traditional Chinese Medicine (Record No. 2021-11). C57/BL-hanmus (20-25 g, 6-8 uger gamle) blev anvendt til nærværende undersøgelse. Musene blev holdt i et dyrekammer og var frie til at drikke og spise under forsøget.
1. Forberedelse
<ol><li>Sørg for, at intubationsplatformen består af en base, en stigerør, en papirclips, to elastikker og nogle strenge. Tag en snor,...

<ol>
	<li>Xia, Y., 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=Protective+effect+of+human+amnion+epithelial+cells+through+endotracheal+instillation+against+lipopolysaccharide-induced+acute+lung+injury+in+mice.">Protective effect of human amnion epithelial cells through endotracheal instillation against lipopolysaccharide-induced acute lung injury in mice.</a> Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 33 (1), 7-11 (2017).</li><li>Butt, Y., Kurdowska, A., Allen, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+lung+injury:+A+clinical+and+molecular+review.">Acute lung injury: A clinical and molecular review.</a> Archives of Pathology and Lab Medicine. 140 (4), 345-350 (2016).</li><li>Ware, L. B., Matthay, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome.">Acute respiratory distress syndrome.</a> Lancet. 398 (10300), 622-637 (2021).</li><li>An, N., Yang, T., Zhang, X. X., Xu, M. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bergamottin+alleviates+LPS-induced+acute+lung+injury+by+inducing+SIRT1+and+suppressing+NF-&#954;B.">Bergamottin alleviates LPS-induced acute lung injury by inducing SIRT1 and suppressing NF-&#954;B.</a> Innate Immunity. 27 (7-8), 543-552 (2021).</li><li>Liu, 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=Comparative+study+of+trans-oral+and+trans-tracheal+intratracheal+instillations+in+a+murine+model+of+acute+lung+injury.">Comparative study of trans-oral and trans-tracheal intratracheal instillations in a murine model of acute lung injury.</a> The Anatomical Record. 295 (9), 1513-1519 (2012).</li><li>Virag, J. A., Lust, R. 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(52), e2581 (2011).</li><li>Li, J., 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=Panaxydol+attenuates+ferroptosis+against+LPS-induced+acute+lung+injury+in+mice+by+Keap1-Nrf2/HO-1+pathway.">Panaxydol attenuates ferroptosis against LPS-induced acute lung injury in mice by Keap1-Nrf2/HO-1 pathway.</a> Journal of Translational Medicine. 19 (1), 96 (2021).</li><li>Zhou, Y., 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+epoxide+hydrolase+inhibitor+attenuates+lipopolysaccharide-induced+acute+lung+injury+and+improves+survival+in+mice.">Soluble epoxide hydrolase inhibitor attenuates lipopolysaccharide-induced acute lung injury and improves survival in mice.</a> Shock. 47 (5), 638-645 (2017).</li><li>Nosaka, N., 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=Optimal+tube+length+of+orotracheal+intubation+for+mice.">Optimal tube length of orotracheal intubation for mice.</a> Laboratory Animals. 53 (1), 79-83 (2019).</li><li>Cicero, L., Fazzotta, S., Palumbo, V. D., Cassata, G., Lo Monte, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia+protocols+in+laboratory+animals+used+for+scientific+purposes.">Anaesthesia protocols in laboratory animals used for scientific purposes.</a> Acta Bio-Medica: Atenei Parmensis. 89 (3), 337-342 (2018).</li><li>Ehrentraut, H., Weisheit, C. K., Frede, S., Hilbert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+acute+lung+injury+in+mice+by+direct+intratracheal+lipopolysaccharide+instillation.">Inducing acute lung injury in mice by direct intratracheal lipopolysaccharide instillation.</a> Journal of Visualized Experiments. (149), e59999 (2019).</li><li>Yang, 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=STAT6+inhibits+ferroptosis+and+alleviates+acute+lung+injury+by+regulating+P53/SLC7A11+pathway.">STAT6 inhibits ferroptosis and alleviates acute lung injury by regulating P53/SLC7A11 pathway.</a> Cell Death &amp; Disease. 13 (6), 530 (2022).</li><li>Aramaki, O., 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=Induction+of+operational+tolerance+and+generation+of+regulatory+cells+after+intratracheal+delivery+of+alloantigen+combined+with+nondepleting+anti-CD4+monoclonal+antibody.">Induction of operational tolerance and generation of regulatory cells after intratracheal delivery of alloantigen combined with nondepleting anti-CD4 monoclonal antibody.</a> Transplantation. 76 (9), 1305-1314 (2003).</li><li>Lan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+mammalian+target+of+rapamycin+(mTOR)+in+a+murine+model+of+lipopolysaccharide+(LPS)+-induced+acute+lung+injury+(ALI).">Activation of mammalian target of rapamycin (mTOR) in a murine model of lipopolysaccharide (LPS) -induced acute lung injury (ALI).</a> Peking Union Medical College. , (2010).</li><li>Lv, 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=Tenuigenin+ameliorates+acute+lung+injury+by+inhibiting+NF-&#954;B+and+MAPK+signalling+pathways.">Tenuigenin ameliorates acute lung injury by inhibiting NF-&#954;B and MAPK signalling pathways.</a> Respiratory Physiology &amp; Neurobiology. 216, 43-51 (2015).</li><li>Yang, H., Lv, H., Li, H., Ci, X., Peng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oridonin+protects+LPS-induced+acute+lung+injury+by+modulating+Nrf2-mediated+oxidative+stress+and+Nrf2-independent+NLRP3+and+NF-&#954;B+pathways.">Oridonin protects LPS-induced acute lung injury by modulating Nrf2-mediated oxidative stress and Nrf2-independent NLRP3 and NF-&#954;B pathways.</a> Cell Communication and Signaling. 17 (1), 62 (2019).</li><li>Im, G. 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=Improvement+of+orthotopic+lung+cancer+mouse+model+via+thoracotomy+and+orotracheal+intubation+enabling+in+vivo+imaging+studies.">Improvement of orthotopic lung cancer mouse model via thoracotomy and orotracheal intubation enabling in vivo imaging studies.</a> Laboratory Animals. 48 (2), 124-131 (2014).</li><li>Hamacher, J., 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=Microscopic+wire+guide-based+orotracheal+mouse+intubation:+description,+evaluation+and+comparison+with+transillumination.">Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination.</a> Laboratory Animals. 42 (2), 222-230 (2008).</li><li>Nelson, A. M., Nolan, K. E., Davis, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+orotracheal+intubation+in+mice.">Repeated orotracheal intubation in mice.</a> Journal of Visualized Experiments. (157), e60844 (2020).</li><li>Zhang, 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=Microvesicles+packaging+IL-1&#946;+and+TNF-&#945;+enhance+lung+inflammatory+response+to+mechanical+ventilation+in+part+by+induction+of+cofilin+signaling.">Microvesicles packaging IL-1&#946; and TNF-&#945; enhance lung inflammatory response to mechanical ventilation in part by induction of cofilin signaling.</a> International Immunopharmacology. 63, 74-83 (2018).</li><li>Feng, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+different+intratracheal+instillation+in+acute+lung+injury+model+of+mice.">Comparative study of different intratracheal instillation in acute lung injury model of mice.</a> Jilin University. , (2010).</li><li>Chen, J. 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=Comparison+of+acute+lung+injury+mice+model+established+by+intranasal+and+intratracheal+instillation+of+lipopolysaccharide.">Comparison of acute lung injury mice model established by intranasal and intratracheal instillation of lipopolysaccharide.</a> Pharmacology and Clinics of Chinese Materia Medica. 38 (02), 222-227 (2022).</li><li>Luckow, B., Lehmann, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+bronchoalveolar+lavage+in+mice+by+orotracheal+intubation+avoiding+tracheotomy.">A simplified method for bronchoalveolar lavage in mice by orotracheal intubation avoiding tracheotomy.</a> BioTechniques. 71 (4), 534-537 (2021).</li><li>Cai, Y., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+intratracheal+intubation+to+study+the+pathology+and+physiology+of+mouse+lung.">Noninvasive intratracheal intubation to study the pathology and physiology of mouse lung.</a> Journal of Visualized Experiments. (81), e50601 (2013).</li></ol>

I første omgang kiggede vi inde i mundhulen for at finde placeringen af luftrøret19. Under denne proces opdagede vi imidlertid, at luftrøret hos C57/BL-mus er smalt, hvilket gør det vanskeligt at finde den rigtige placering ved denne metode uden hjælp fra udstyr såsom et endoskop20. Ved yderligere udforskning fandt vi, at lyset fra intubatorlampen kunne trænge ind i overfladen af kroppen, så operatøren kunne bestemme kanylens position21....

Akut lungeskade (ALI) er et almindeligt klinisk syndrom. Under en række patogene faktorer fører forstyrrelsen af den fysiologiske barriere i lungeepitelcellerne og vaskulære endotelceller til øget alveolær permeabilitet og derved forårsager nedsat lungeoverensstemmelse, lungeødem og alvorlig hypoxæmi1. Akut respiratorisk distress syndrom (ARDS) er den mest alvorlige form for ALI. Ukontrolleret inflammation og oxidativ stressskade anses for at være hovedårsagerne til ALI og den mere alvorlige ARDS2. Når alveolære epitelceller er direkte skadet på grund af traumer, aktiveres den inflammatoriske responskæde af alveolære makrofager, hvilket fører til betændelse i lungen3. Globalt er der mere end 3 millioner patienter med akut ARDS om året, og de tegner sig for ca. 10% af indlæggelserne på intensivafdelinger; Derudover er dødeligheden i alvorlige tilfælde så høj som 46%4,5,6. Der er derfor behov for at etablere en passende dyremodel for ALI til undersøgelse af dets patogenese. Musen er det mest almindeligt anvendte forsøgsdyr i studiet af ALI, da dets luftveje kan simulere de menneskelige luftveje godt til ALI-undersøgelser. Desuden manifesterer ALI sig som massiv inflammatorisk celleinfiltration, øget pulmonal vaskulær permeabilitet og lungeødem. Ændringerne i inflammatoriske cytokiner i serum og forholdet mellem tør og våd vægt i lungerne afspejler graden af ALI7.
På nuværende tidspunkt inkluderer de vigtigste metoder til modellering af LPS-induceret ALI hos mus intranasal og kirurgisk trakeal intubation 8,9. Her foreslår vi en ny metode til at levere LPS i luftrøret via ikke-invasiv orofaryngeal intubation. Denne metode bruger en oplyst intubator til at finde musens luftrør og leverer derefter LPS ind i luftrøret og lungen. Denne metode leverer LPS til lungerne mere præcist end den intranasale leveringsmetode. Sammenlignet med kirurgisk trakeal intubation kræver denne metode ikke kirurgi, undgår at forårsage sår og reducerer smerter hos mus10. Derfor kan denne metode bruges til at etablere en mere overbevisende musemodel af ALI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lipopolysaccharide</td>
			<td>MERK</td>
			<td>L4130</td>
			<td>LPS</td>
		</tr>
		<tr>
			<td>Microliter Syringes</td>
			<td>SHANGHAI GAOGE INDUSTRY AND TRADE CO., LTD</td>
			<td>10028505008124</td>
			<td>To deliver LPS</td>
		</tr>
		<tr>
			<td>Mouse cannula</td>
			<td>RWD Life Science</td>
			<td>803-03008-00</td>
			<td>Mouse cannula</td>
		</tr>
		<tr>
			<td>Mouse intubation kit</td>
			<td>RWD Life Science</td>
			<td>903-03027-00</td>
			<td>Including a base, a riser, a intubator, a surgical forceps and some strings</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Biosharp life science</td>
			<td>BS-XG-03</td>
			<td>To verify the success of intubation</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Beijing Chemical Co., China</td>
			<td>20220918</td>
			<td>To anesthetize mice</td>
		</tr>
	</tbody>
</table>

noninvasive intratracheal lipopolysaccharide instillation in mice

Den akutte lungeskade (ALI) musemodel induceret af lipopolysaccharid (LPS) eller endotoksin er stadig blandt de mest almindeligt anvendte modeller i dyreforsøg med akut lungeskade eller akut inflammation. De nuværende mest almindeligt anvendte metoder i musemodeller for akut lungeskade er en intraperitoneal injektion af LPS og trakeostomi til trakeal infusion af LPS. Den førstnævnte metode mangler imidlertid lungemålretning og beskadiger andre organer, og sidstnævnte metode inducerer operativt traume, infektionsrisiko og en lav overlevelsesrate. Her anbefaler vi en ikke-invasiv orofaryngeal endotracheal intubationsmetode til LPS-instillation hos mus. I denne metode indføres LPS ikke-invasivt i luftrøret gennem orofaryngeal hulrum, der skal indføres i lungen ved hjælp af et apparat til endotracheal intubation. Denne metode sikrer ikke kun lungemålretning, men undgår også skader og risikoen for død hos dyrene. Vi forventer, at denne tilgang vil blive udbredt inden for akut lungeskade.

Den foreslåede metode til LPS-instillation hos mus blev verificeret ved at evaluere ekspressionen af det inflammatoriske cytokin TNF-α og lungetør-vådvægtforholdet 12 timer efter LPS-instillation. Der var fire grupper i eksperimentet: blank kontrol (uden behandling), kirurgisk intubation16, intranasal17,18 og ikke-invasiv orofaryngeal intubation (n = 6). Sammenlignet med blindprøvegruppen var serum-TN...

Watch this Scientific Journal Video about Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice at JoVE.com

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

Her foreslår vi en protokol for intratracheal lipopolysaccharid (LPS) levering via ikke-invasiv orofaryngeal endotracheal intubation. Denne metode minimerer traumet ved den kirurgiske procedure for dyret og leverer nøjagtigt LPS til luftrøret og derefter til lungerne.

Noninvasiv intratrakeal lipopolysaccharidinstillation hos mus

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of ...

Vores protokol foreslår en ny ikke-invasiv leveringsmetode af LPS fra luftrøret til lungen, mere præcist. Folk, der prøver denne metode for første gang, bør lægge stor vægt på at finde luftrørets position. For at forberede intubationsplatformen skal du føre strengen gennem de to huller og binde dens ender til de små fremspring øverst på stigrøret.Fastgør derefter to elastikker til hver ende af papirclipsen og tape dem fast på stigrørets bagside. Til sidst skal du fastgøre stigrøret til bunden i en 90 graders vinkel. Saml kanylen på en kanylepen, og tænd pennens lys.Desinficer derefter små kirurgiske tang og Pasteur-pipette med 70%alkohol. For at forberede testforbindelsesopløsningen tilsættes tre milligram lipopolysaccharid eller LPS i en milliliter pH 7,2 PBS. Derefter opløses 10 mg pentobarbitalnatrium i en milliliter normalt saltvand og filtreres opløsningen ved hjælp af et 0,45 mikrometer sprøjtefilter.Placer den bedøvede mus på intubationsplatformen. Fastgør derefter de øverste fortænder med snoren og de to forfødder med elastikkerne. Brug pincet til at trække tungen ud og holde den.Skub kanylen langsomt langs munden til mandibular epiglottis. Find luftrøret med kanylepennens lys, og indsæt langsomt kanylen i den. Træk derefter intubationspennen ud og lad kanylen være indeni.Indsæt Pasteur-pipetten i kanyleleddet, og tryk på hovedet. Efter vellykket endotracheal intubation ved hjælp af en mikrosprøjte med fladt hoved indsprøjtes den tilberedte LPS-opløsning ved tre milligram pr. Kg dosis gennem kanylen. Når du er færdig, skal du tage mikrosprøjten og kanylen ud.Fjern musen fra intubationsplatformen og læg den i et separat bur for at komme sig. Overhold musens åndedrætstilstand, indtil den genopretter og genvinder bevidstheden. 12 timer efter LPS trakeal instillation blev ekspressionen af et inflammatorisk cytokin, TNF-alfa, og lungetør-vådvægtforholdet evalueret og sammenlignet med andre instillationsmetoder.Serum-TNF-alfa-niveauerne i den ikke-invasive orofaryngeale intubationsgruppe var signifikant øget sammenlignet med blindprøvegruppen. Forholdet mellem tør og våd lungevægt blev også øget og nåede samme niveau som i den kirurgiske trakeale intubationsgruppe. Det er vigtigt ikke at ridse luftrøret i processen med at finde placeringen af luftrøret.Teknikken kan også bruges til at levere andre lægemidler, der skal nå lungen på en ikke-invasiv måde.

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