Name: a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation
Uploaded: 2017-07-26T15:00:00
Description: Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

Les auteurs souhaitent remercier l&#39;Institut national des sciences biologiques de Beijing pour l&#39;assistance. Ce travail a été soutenu par la Fondation municipale des sciences naturelles de Beijing (n ° Z17110200040000).

Toutes les procédures utilisées dans ce protocole ont été effectuées conformément aux recommandations des Lignes directrices pour la prise en charge et l&#39;utilisation des animaux de laboratoire de l&#39;Institut national des sciences biologiques de Pékin. Les souris mâles CD-1 de 8 semaines ont été logées dans une installation spécifique de pathogène (SPF) jusqu&#39;à ce que les expériences aient été réalisées. Les chirurgies ont été effectuées en utilisant des souris complètement anesthésiée...

<ol>
	<li>Liu, Z., 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=MAPK-Mediated+YAP+Activation+Controls+Mechanical-Tension-Induced+Pulmonary+Alveolar+Regeneration.">MAPK-Mediated YAP Activation Controls Mechanical-Tension-Induced Pulmonary Alveolar Regeneration.</a> Cell Rep. 16 (7), 1810-1819 (2016).</li><li>Thurlbeck, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+surface+area+and+other+measurements+in+emphysema.">Internal surface area and other measurements in emphysema.</a> Thorax. 22 (6), 483-496 (1967).</li><li>Knudsen, L., Weibel, E. R., Gundersen, H. J. G., Weinstein, F. V., Ochs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+air+space+size+characteristics+by+intercept+(chord)+measurement:+an+accurate+and+efficient+stereological+approach.">Assessment of air space size characteristics by intercept (chord) measurement: an accurate and efficient stereological approach.</a> J Appl Physiol. 108 (2), 412-421 (2010).</li><li>Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Morphometry of the Human Lung. , (1963).</li><li>Duguid, J. B., Young, A., Cauna, D., Lambert, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+internal+surface+area+of+the+lung+in+emphysema.">The internal surface area of the lung in emphysema.</a> J Pathol Bacteriol. 88, 405-421 (1964).</li><li>Branchfield, K., 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=Pulmonary+neuroendocrine+cells+function+as+airway+sensors+to+control+lung+immune+response.">Pulmonary neuroendocrine cells function as airway sensors to control lung immune response.</a> Science. 351 (6274), 707-710 (2016).</li><li>Ding, B. -. 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=Endothelial-derived+angiocrine+signals+induce+and+sustain+regenerative+lung+alveolarization.">Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization.</a> Cell. 147 (3), 539-553 (2011).</li><li>Dunnill, 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=Quantitative+methods+in+the+study+of+pulmonary+pathology.">Quantitative methods in the study of pulmonary pathology.</a> Thorax. 17 (4), 320-328 (1962).</li><li>Weibel, E. R., Gomez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+human+lung.+Use+of+quantitative+methods+establishes+fundamental+relations+between+size+and+number+of+lung+structures.">Architecture of the human lung. Use of quantitative methods establishes fundamental relations between size and number of lung structures.</a> Science. 137 (3530), 577-585 (1962).</li><li>Thurlbeck, W. 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+internal+surface+area+of+nonemphysematous+lungs.">The internal surface area of nonemphysematous lungs.</a> Am Rev Respir Dis. 95 (5), 765-773 (1967).</li><li>Butler, J. P., 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=Evidence+for+adult+lung+growth+in+humans.">Evidence for adult lung growth in humans.</a> N Engl J Med. 367 (16), 244-247 (2012).</li><li>Hsia, C. C. W., Herazo, L. F., Fryder-Doffey, F., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensatory+lung+growth+occurs+in+adult+dogs+after+right+pneumonectomy.">Compensatory lung growth occurs in adult dogs after right pneumonectomy.</a> J Clin Invest. 94 (1), 405-412 (1994).</li><li>Thurlbeck, S. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumonectomy+in+Rats+at+Various+Ages.">Pneumonectomy in Rats at Various Ages.</a> Am Rev Respir Dis. 120 (5), 1125-1136 (1979).</li><li>Cagle, P. T., Langston, C., Thurlbeck, W. 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+Age+on+Postpneumonectomy+Growth+in+Rabbits.">The Effect of Age on Postpneumonectomy Growth in Rabbits.</a> Pediatr Pulmonol. 5 (2), 92-95 (1988).</li><li>Langston, 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=Alveolar+multiplication+in+the+contralateral+lung+after+unilateral+pneumonectomy+in+the+rabbit.">Alveolar multiplication in the contralateral lung after unilateral pneumonectomy in the rabbit.</a> Am Rev Respir Dis. 115 (1), 7-13 (1977).</li><li>Cohn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+Affecting+The+Postnatal+Growth+of+The+Lung.">Factors Affecting The Postnatal Growth of The Lung.</a> Anatomical Record. 75 (2), 195-205 (1939).</li><li>Hsia, C. C., Wu, E. Y., Wagner, E., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+mediastinal+shift+after+pneumonectomy+impairs+regenerative+alveolar+tissue+growth.">Preventing mediastinal shift after pneumonectomy impairs regenerative alveolar tissue growth.</a> Am J Physiol Lung Cell Mol Physiol. 281 (5), L1279-L1287 (2001).</li><li>Das, S., MacDonald, K., Chang, H. -. Y. S., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+mouse+lung+intubation.">A simple method of mouse lung intubation.</a> J Vis Exp. (73), e50318 (2013).</li><li>Liu, S., Cimprich, J., Varisco, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+pneumonectomy+model+of+compensatory+lung+growth.">Mouse pneumonectomy model of compensatory lung growth.</a> J Vis Exp. (94), (2014).</li><li>Silva, M. F. R., Zin, W. A., Saldiva, P. H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airspace+configuration+at+different+transpulmonary+pressures+in+normal+and+paraquat-induced+lung+injury+in+rats.">Airspace configuration at different transpulmonary pressures in normal and paraquat-induced lung injury in rats.</a> Am J Respir Crit Care Med. 158 (4), 1230-1234 (1998).</li><li>Yilmaz, 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=Noninvasive+quantification+of+heterogeneous+lung+growth+following+extensive+lung+resection+by+high-resolution+computed+tomography.">Noninvasive quantification of heterogeneous lung growth following extensive lung resection by high-resolution computed tomography.</a> J Appl Physiol. 107 (5), 1569-1578 (2009).</li><li>Voswinckel, 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=Characterisation+of+post-pneumonectomy+lung+growth+in+adult+mice.">Characterisation of post-pneumonectomy lung growth in adult mice.</a> Eur Respir J. 24 (4), 524-532 (2004).</li><li>Ravikumar, P., 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=Regional+Lung+Growth+and+Repair+Regional+lung+growth+following+pneumonectomy+assessed+by+computed+tomography.">Regional Lung Growth and Repair Regional lung growth following pneumonectomy assessed by computed tomography.</a> J Appl Physiol. 97, 1567-1574 (2004).</li><li>Gibney, B. 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=Detection+of+murine+post-pneumonectomy+lung+regeneration+by+18FDG+PET+imaging.">Detection of murine post-pneumonectomy lung regeneration by 18FDG PET imaging.</a> EJNMMI Res. 2 (1), (2012).</li><li>Mu&#241;oz-Barrutia, A., Ceresa, M., Artaechevarria, X., Montuenga, L. M., Ortiz-De-Solorzano, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+lung+damage+in+an+elastase-induced+mouse+model+of+emphysema.">Quantification of lung damage in an elastase-induced mouse model of emphysema.</a> Int J Biomed Imaging. 2012, (2012).</li></ol>

Dans ce protocole, nous fournissons des descriptions détaillées sur la mesure des paramètres pulmonaires après le PNX du poumon gauche et l&#39;implantation de la prothèse. L&#39;ISA est maintenant considérée comme une mesure clé pour l&#39;évaluation de la fonction respiratoire dans de nombreuses maladies pulmonaires et dans la régénération alvéolaire induite par une blessure. Cependant, bien que la communauté de recherche pulmonaire soit d&#39;accord sur l&#39;utilité de l&#39;ISA comme mesure utile, à...

La fonction fondamentale du poumon est l&#39;échange d&#39;oxygène et de dioxyde de carbone entre les vaisseaux sanguins et l&#39;atmosphère. Les maladies pulmonaires telles que la dysplasie bronchopulmonaire (BPD), la maladie pulmonaire obstructive chronique (MPOC) et les infections respiratoires aiguës entraînent une diminution de l&#39;ISA 2 . Les chercheurs qui étudient la maladie pulmonaire ont développé plusieurs méthodes quantitatives pour évaluer les changements morphologiques dans les poumons, y compris MLI, ILV, nombre d&#39;unités d&#39;échange de gaz, ISA et compliance tissulaire pulmonaire 2 , 3 . Études pionnières par Weibel et al. 4 et Duguid et al. 5 établissent ensemble que l&#39;ISA peut être utilisé comme mesure directe de la capacité d&#39;échange de gaz pulmonaire dans les poumons humains et peut être utilisé comme critère pour déterminer la gravité de l&#39;emphysème. Un certain nombre d&#39;études publiées au cours des cinq dernières années ont utilisé des paramètres morphologiques pulmonaires ( p. Ex. </eM&gt; ISA et MLI) pour évaluer les changements morphologiques et fonctionnels dans les poumons de la souris pendant le développement 6 et pendant la récupération de la lésion PNX 1 , 7 . ISA est calculé en utilisant l&#39; équation 1 8 , 9 : <img alt="Équation" src="/files/ftp_upload/56114/56114eq1.jpg" /> , Où ILV est le volume pulmonaire interne et MLI est un paramètre intermédiaire qui représente l&#39;espace périphérique pulmonaire taille 10 . PNX, l&#39;ablation chirurgicale d&#39;un ou plusieurs lobes de poumon, a été largement rapportés pour induire la régénération alvéolaire chez de nombreuses espèces, y compris les humains, les souris 11 1, 12 les chiens, les rats et les lapins 13, 14, 15. Un goujonY des poumons de souris à quatorze jours après la PNX a montré que l&#39;expansion des alvéoles préexistants et la formation de novo des alvéoles contribuent à la restauration de l&#39;ISA, de l&#39;ILV et du nombre d&#39;alvéoles dans les tissus pulmonaires restants 1 . Nous et d&#39;autres ont montré que l&#39;insertion de matériaux tels que l&#39;éponge, la cire ou une prothèse sur mesure dans la cavité thoracique vide suivant la PNX ( c&#39;est -à- dire l&#39; implantation de la prothèse) entrave la régénération alvéolaire. Il est maintenant établi que la force mécanique fonctionne comme l&#39;un des facteurs les plus importants pour l&#39;initiation de la régénération alvéolaire 1 , 16 , 17 . De telles études ont mis en évidence l&#39;efficacité de l&#39;utilisation des valeurs ISA des poumons traités par la PNX et de la prothèse comme critère pour évaluer quantitativement la régénération alvéolaire. Le biais d&#39;observateur est connu pour influencer de manière significative les valeurs mesuréesLues pour les paramètres morphologiques pulmonaires ( p . Ex. , MIL et ILV). Des protocoles standardisés peuvent être utilisés pour éviter ce biais en déterminant à la fois ILV et MLI, qui sont les deux paramètres utilisés dans le calcul de l&#39;ISA. Ici, nous fournissons des protocoles hautement détaillés et standardisés pour mesurer ces paramètres pulmonaires. Fait important, la capacité de quantifier avec précision l&#39;ISA promet d&#39;améliorer la fiabilité et la reproductibilité des études de la fonction pulmonaire dans les modèles de régénération alvéolaire induite par les blessures et devrait faciliter les découvertes mécanistes dans de multiples maladies pulmonaires.

<table width="881">
	<tbody>
		<tr height="21">
			<td height="21" style="width:411px;height:21px;">Low cost cautery kit</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:136px;">18010-00</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Noyes scissors</td>
			<td>Fine Science Tools</td>
			<td>15012-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Castroviejo Micro Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vessel clips</td>
			<td>Fine Science Tools</td>
			<td>18374-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">I. V. Cannula-20 gauge</td>
			<td>Jinhuan Medical Product Co., LTD.</td>
			<td>29P0601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical suture</td>
			<td>Jinhuan Medical Product Co., LTD.</td>
			<td>F602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse intubation platform</td>
			<td>Penn-Century, Inc</td>
			<td>Model MIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Laryngoscope</td>
			<td>Penn-Century, Inc</td>
			<td>Model LS-2-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TOPO Small Animal Ventilator</td>
			<td>Kent Scientific</td>
			<td>RSP1006-05L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal pad</td>
			<td>Stuart equipment</td>
			<td>SBH130D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital sodium salt</td>
			<td>Sigma</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin Solution</td>
			<td>Sigma</td>
			<td>GHS132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin Y solution, alcoholic</td>
			<td>Sigma</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml Pipette</td>
			<td>Thermo Scientific</td>
			<td>170356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cryosection machine</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Base Molds</td>
			<td>Fisher HealthCare</td>
			<td>22-363-553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 gauge needle</td>
			<td>Becton Dickinson</td>
			<td>305199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone iodine</td>
			<td>Fisher Scientific</td>
			<td>19-027132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP82011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Infusion sets for single use</td>
			<td>Weigao</td>
			<td>SFDA 2012 3661704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Gibco</td>
			<td align="right">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tapes</td>
			<td>3M Scotch</td>
			<td style="width:136px;">8915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton pad</td>
			<td>Vinda</td>
			<td>Dr.P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone prosthesis</td>
			<td>Custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brightfield microscope</td>
			<td>Olympus</td>
			<td>VS120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ruler tool</td>
			<td>Adobe Photoshop</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

<html> Nous avons effectué ici une expérience avec un groupe traité par PNX et un groupe d&#39;implantation de prothèse (implanté en prothèse). Ces groupements sont identiques aux groupements utilisés dans une étude publiée précédemment de notre groupe de recherche 14 . Le PNX de la souris et les procédures d&#39;implantation de la prothèse sont présentés à la figure ...

Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

La zone de surface pulmonaire interne (ISA) est un critère critique pour évaluer la morphologie pulmonaire et la physiologie dans les maladies pulmonaires et la régénération alvéolaire induite par les blessures. Nous décrivons ici une méthode standardisée qui peut minimiser le biais de mesure pour ISA dans les modèles de la pneumonectomie pulmonaire et de la souris implantation de la prothèse.

Une méthode standardisée pour mesurer la surface interne du poumon par la pneumonie et la prothèse

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

The overall goal of this procedure is to accurately measure the internal surface area of a mouse lung after pneumonectomy and prosthesis implantation. This method helps to analyze the morphological and the physiological functions of the lung in mechanical force-induced adult lung regeneration. The main advantage of this technique is to accurately quantify internal lung surface area, which promise to increase the reliability and the reproducibility of lung function status.Prepare the mouse for surgery on an intubation platform with its ventral side accessible. Then pull out the tongue and inspect the vocal chords with a small animal laryngoscope that is compatible with guiding catheters. Then gently insert a 20 gauge intravenous intubation canula into the trachea at an interior angle.Next, put the mouse in a right lateral recumbent position and connect the canula to a mechanical ventilator. Observe the chest to judge the insertion of the canula into the trachea. Set the inspiratory pressure to 12 centimeters of water and set the respiratory rate to 120 breaths per minute.Now, clean the exposed skin with at least three alternating scrubs of Betadine and 70%ethanol. Then begin the surgery with a two to three centimeter posterolateral thoracotomy incision into the fifth intercostal space. Penetrate the musculature using Noyes spring scissors.Next, make a 1.5 centimeter incision to expose the left lung. As needed throughout the surgery, use a high temperature cauterizer to stop bleeding. Then, lift 1/3 of the left lung lobe from the chest with blunt tip forceps, and use a cotton swab to pull out the entire left lung.Now, identify the pulmonary artery and bronchi of the left lung lobe, and tightly ligate the bronchi and vessels at the hilum with a soaked surgical suture. Next, cut out the left lung lobe three to four millimeters from the ligation, being careful not to cut off the suture, which could result in pneumothorax. Next, close the chest wall with one suture.Then, stitch the muscle layer and the skin layer sequentially, using five to six interrupted sutures with three to four millimeter gaps. Be sure to keep the surgical suture needle away from the heart. Cardiac puncture is lethal.Lastly, disinfect the surgical area with povidone-iodine. Then, place the mouse on a 38 degrees Celsius thermal pad. Keep using the ventilator until spontaneous breathing returns.Perform the previously described operation, but just after removing the lung, prepare to insert the prosthesis. Using blunted forceps, grip the center of the ellipsoid silicone prothesis. Next, while holding the rib with forceps to expose the thoracic cavity, very gently insert the prothesis.Enter at a 45 degree angle between the frontal plane of the prosthesis and the thoracic surface. Using blunt forceps, ensure that the prosthesis is fully within the left empty thoracic cavity. Now, close the chest and monitor the mouse as previously described.For this procedure, assemble a custom inflation tube that consists of a plunger removed from a 10 milliliter serological pipette, a 40 centimeter long flexible tube with a needle adapter, a flow rate control valve, and an 18 gauge needle. Once assembled, secure the pipette to a sideboard with tape. The distance between the top of the pipette and the bench must be at least 30 centimeters.After euthanizing the mouse by anesthetic injection, secure it to a sterilized board and carefully open the chest to cut out the sternum and expose the lung lobes. Then, expose the trachea. Next, fill the inflation tube with freshly made 4%PFA and remove all the bubbles from the tube.Then, insert the needle into the trachea. To avoid leakage, clip the trachea with vessel clips. Now, inflate the lung with fixative at a constant transpulmonary pressure of 25 centimeters of water.Incubate the lungs at room temperature for two hours for full expansion. After the inflation, take note of the volume displacement of fixative. Now, ligate the trachea and gently dissect out the lungs intact from the surrounding connective tissues.Use scissors and be very gentle. Next, incubate the lungs in a 50 milliliter conical tube filled with 4%PFA for 12 hours at four degrees Celsius with gentle shaking on a shaker. Then proceed with processing and staining as described in the text protocol.To quantify the MLI, use images of the agent used in sections collected under bright-field at 20x magnification. From three different lung sections, randomly select a total of 15 non-overlapping views, each about one square millimeter, and then vent them. Only use areas without arteries and veins, major airways, or alveolar ducts.For the analysis, draw a 10 by 10 grid over each selected area using a ruler tool. The grid lines are thus about 100 microns apart. Then, to find an intercept along the line as the length between two adjacent alveolar epithelia.For example, the double-headed arrows illustrate the lengths. Count all these lengths along each grid line, and the MLI is the average value of the intercept lengths. Using the described protocols, PNX-treated mice were compared with prosthetic implanted mice.The mice used were eight-week-old CD-1 males, and their left lung lobes were resected. The prosthesis mimics the size and shape of the left lung lobe and was inserted into the chest after the left lung lobe was removed. 14 days after the surgery, a custom made inflation tube was used to determine the internal volume of the remaining right lung.The average volume of the right lung in the five PNX-treated mice was approximately 1.4 milliliters, which was significantly greater, 1.05 milliliters for the five prosthesis-implanted mice. The MLI values in the remaining right lungs were significantly greater as well. Using these parameters, the internal lung surface area was calculated and was found to be significantly smaller in the prosthesis-implanted mice.After watching this video, you should have a good understanding of how to calculate the internal lung surface area for the mechanical force-induced alveolar regeneration mouse model.

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