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

ويود المؤلفون أن يعترفوا بالمعهد الوطني للعلوم البيولوجية، بيجين، على المساعدة. وأيد هذا العمل من قبل مؤسسة العلوم الطبيعية لبلدية بكين (رقم Z17110200040000).

جميع الإجراءات المستخدمة في هذا البروتوكول نفذت وفقا للتوصيات الواردة في المبادئ التوجيهية لرعاية واستخدام الحيوانات المختبرية للمعهد الوطني للعلوم البيولوجية، بكين. تم إيواء الفئران الذكور من نوع سد-1 البالغ عمرها 8 أسابيع في منشأة خالية من مسببات الأمراض (سف) حتى ...

<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>

في هذا البروتوكول، ونحن نقدم أوصاف مفصلة عن قياس المعلمات الرئوية بعد الماوس اليسار الرئة ينكس وزرع الاصطناعية. ويعتبر عيسى الآن مقياسا رئيسيا لتقييم وظيفة الجهاز التنفسي في العديد من الأمراض الرئوية وفي تجديد السنخية الناجم عن الإصابة. ومع ذلك، على الرغم من أن مجت?...

الوظيفة الأساسية للرئة هي تبادل الأكسجين وثاني أكسيد الكربون بين الأوعية الدموية والغلاف الجوي. أمراض الرئة مثل خلل التنسج القصبي الرئوي (بد)، مرض الانسداد الرئوي المزمن (كوبد)، والتهابات الجهاز التنفسي الحادة، ويؤدي إلى انخفاض عيسى 2 . وقد طور الباحثون الذين يدرسون أمراض الرئة العديد من الطرق الكمية لتقييم التغيرات المورفولوجية في الرئتين، بما في ذلك ملي، إلف، وعدد من وحدات تبادل الغاز، عيسى، والالتزام أنسجة الرئة 2 ، 3 . دراسات رائدة من قبل ويبل وآخرون. 4 و دوغويد إت آل. 5 معا أن المعیار الدولي للتدقیق یمکن استخدامھ کمقیاس مباشر لقدرة تبادل الغاز الرئوي في الرئتین البشریة ویمکن استخدامھ کمعیار لتحدید شدة انتفاخ الرئة. وقد استخدم عدد من الدراسات التي نشرت في السنوات الخمس الماضية المعلمات المورفولوجية الرئة (على سبيل المثال، </eم&gt; عيسى و ملي) لتقييم التغيرات المورفولوجية والوظيفية في الرئتين من الفئران خلال التنمية 6 وخلال الانتعاش من إصابة ينكس 1 ، 7 . ويحسب عيسى باستخدام المعادلة 1 8 و 9 : <img alt="معادلة" src="/files/ftp_upload/56114/56114eq1.jpg" /> ، حيث إلف هو حجم الرئة الداخلية و ملي هو وسيط المعلمة التي تمثل المجال الجوي الطرفية الرئوية حجم 10 . ينكس، إزالة جراحية واحدة أو أكثر من فصوص الرئة، تم الإبلاغ على نطاق واسع للحث على تجديد السنخية في العديد من الأنواع، بما في ذلك البشر 11 ، الفئران 1 ، الكلاب 12 ، الفئران 13 ، والأرانب 14 ، 15 . مسمارذ من الرئتين الفئران في أربعة عشر يوما بعد بنكس أظهرت أن كلا من التوسع في الحويصلات الهوائية الموجودة مسبقا وتشكيل دي نوفو من الحويصلات الهوائية تسهم في استعادة عيسى، إلف، وعدد من الحويصلات الهوائية في أنسجة الرئة المتبقية 1 . لقد أظهرنا نحن وآخرون أن إدخال مواد مثل الإسفنج أو الشمع أو بدلة على شكل مخصص في التجويف الصدري الفارغ بعد بنكس ( أي زرع الأطراف الاصطناعية) يضعف تجديد السنخية. ومن الثابت الآن أن القوة الميكانيكية تعمل باعتبارها واحدة من أهم العوامل لبدء تجديد السنخية 1 ، 16 ، 17 . وقد سلطت هذه الدراسات الضوء على فعالية استخدام قيم عيسى من بنكس المعالجة والرئة الاصطناعية مزروع كمعيار لتقييم كميا تجديد السنخية. ومن المعروف أن التحيز المراقب تؤثر تأثيرا كبيرا فازهري لمعلمات المورفولوجية الرئة (على سبيل المثال، MIL وILV). ويمكن استخدام بروتوكولات موحدة لتفادي هذا التحيز في تحديد كل من إلف و ملي، وهما البارامترات المستخدمة في حساب عيسى. هنا، ونحن نقدم بروتوكولات مفصلة للغاية، موحدة لقياس هذه المعلمات الرئة. الأهم من ذلك، القدرة على تحديد بدقة عيسى وعد بتحسين موثوقية واستنساخ الدراسات من وظائف الرئة في نماذج تجديد السنخية الناجم عن إصابة وينبغي أن تسهل الاكتشافات الميكانيكية في أمراض رئوية متعددة.

<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">Vessel clips</td>
			<td>Fine Science Tools</td>
			<td>18374-44</td>
			<td></td>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">Surgical suture</td>
			<td>Jinhuan Medical Product Co., LTD.</td>
			<td>F602</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mouse intubation platform</td>
			<td>Penn-Century, Inc</td>
			<td>Model MIP</td>
			<td></td>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">TOPO Small Animal Ventilator</td>
			<td>Kent Scientific</td>
			<td>RSP1006-05L</td>
			<td></td>
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			<td height="21" style="height:21px;">Thermal pad</td>
			<td>Stuart equipment</td>
			<td>SBH130D</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital sodium salt</td>
			<td>Sigma</td>
			<td>P3761</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin Solution</td>
			<td>Sigma</td>
			<td>GHS132</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Eosin Y solution, alcoholic</td>
			<td>Sigma</td>
			<td>HT110116</td>
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		<tr height="21">
			<td height="21" style="height:21px;">10 ml Pipette</td>
			<td>Thermo Scientific</td>
			<td>170356</td>
			<td></td>
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		<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>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cryosection machine</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Disposable Base Molds</td>
			<td>Fisher HealthCare</td>
			<td>22-363-553</td>
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		<tr height="21">
			<td height="21" style="height:21px;">18 gauge needle</td>
			<td>Becton Dickinson</td>
			<td>305199</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Povidone iodine</td>
			<td>Fisher Scientific</td>
			<td>19-027132</td>
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			<td height="21" style="height:21px;">70% ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP82011</td>
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			<td height="21" style="height:21px;">Infusion sets for single use</td>
			<td>Weigao</td>
			<td>SFDA 2012 3661704</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Gibco</td>
			<td align="right">10010023</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tapes</td>
			<td>3M Scotch</td>
			<td style="width:136px;">8915</td>
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			<td height="21" style="height:21px;">Cotton pad</td>
			<td>Vinda</td>
			<td>Dr.P</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Silicone prosthesis</td>
			<td>Custom made</td>
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		<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>
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			<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.

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

منطقة سطح الرئة الداخلية (عيسى) هو معيار حاسم لتقييم مورفولوجيا الرئة وعلم وظائف الأعضاء في أمراض الرئة والتجدد السنخي الناجم عن الاصابة. نحن تصف هنا طريقة موحدة التي يمكن أن تقلل من التحيز قياس عيسى في كل من استئصال رئوي الرئوي ونماذج الفئران زرع الاصطناعية.

طريقة موحدة لقياس المساحة الداخلية الرئة السطحية عن طريق استئصال رئوي الماوس وزرع الاصطناعية

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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