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

Yazarlar, yardım için Pekin Ulusal Biyolojik Bilimler Enstitüsü&#39;nü kabul etmek istiyorlar. Bu çalışma Pekin Belediye Tabiat Bilimleri Vakfı (No. Z17110200040000) tarafından desteklenmiştir.

Bu protokolde kullanılan tüm prosedürler, Pekin Ulusal Biyolojik Bilimler Enstitüsünün Laboratuar Hayvanlarının Bakımı ve Kullanımı için Yönergelerdeki önerilere uygun olarak gerçekleştirildi. 8 haftalık CD-1 erkek fareler, deneyler gerçekleştirilene kadar spesifik bir patojen içermeyen (SPF) tesiste barındırıldı. Ameliyatlar tamamen anestezi uygulanmış fareler kullanılarak gerçekleştirildi (diğer bir deyişle çimdikleme tepkileri olmadan). Ameliyattan sonra fareler yeterli mikta...

<ol>
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Bu protokolde, fare sol akciğer PNX ve protez yerleştirildikten sonra pulmoner parametrelerin ölçümü hakkında ayrıntılı açıklamalar sağlarız. ISA, şimdi birçok pulmoner hastalıkta solunum fonksiyonlarının değerlendirilmesi ve yaralanmanın yol açtığı alveol rejenerasyonunda önemli bir metrik olarak düşünülmektedir. Bununla birlikte, pulmoner araştırma topluluğu ISA&#39;nın yararlı bir metriği olarak yararlı olduğu konusunda anlaşmaya rağmen, günümüze kadar ISA hesaplamak için k...

Akciğerin temel işlevi, kan damarları ile atmosfer arasındaki oksijen ve karbon dioksit değişimidir. Bronkopulmoner displazi (BPD), kronik obstrüktif akciğer hastalığı (COPD) ve akut solunum yolu enfeksiyonları gibi akciğer hastalıkları, ISA 2 azalmasına neden olur. Akciğer hastalığını araştıran araştırmacılar, MLI, ILV, gaz değişim birimi sayısı, ISA ve akciğer dokusu uyumluluğu 2 , 3 dahil olmak üzere akciğerlerdeki morfolojik değişiklikleri değerlendirmek için çeşitli niceliksel yöntemler geliştirmiştir. Weibel ve ark.nın öncü çalışmaları 4 ve Duguid ve ark. 5 , ISA&#39;nın insan akciğerlerindeki akciğer gaz değişim kapasitesinin doğrudan bir ölçümü olarak kullanılabileceğini ve amfizemin ciddiyetini belirlemek için bir kriter olarak kullanılabileceğini birlikte ortaya koymuştur. Son beş yılda yayınlanan bir dizi çalışma, akciğer morfolojik parametrelerini ( örn. </eM&gt; ISA ve MLI) 6 gelişme sırasında ve yaralanma PNX 1 , 7 kurtarma sırasında farelerin akciğerlerinde morfolojik ve fonksiyonel değişiklikleri değerlendirmek için. ISA, Denklem 8 , 9 kullanılarak hesaplanır: <img alt="Denklem" src="/files/ftp_upload/56114/56114eq1.jpg" /> , Burada ILV, iç akciğer hacmi ve MLI, pulmoner periferik hava sahası büyüklüğünü 10 gösteren aracı bir parametredir. PNX, bir veya daha fazla akciğer lobunun cerrahi olarak çıkarılması, insanlar 11 , fareler 1 , köpekler 12 , sıçanlar 13 ve tavşanlar 14 , 15 dahil olmak üzere birçok türe ait alveol rejenerasyonunu uyardığı yaygın olarak bildirilmiştir. SaplamaOn dört gün sonra fareler, akciğer y sonrası PNX önceden var olan alveoller genişlemesi ve alveollere de novo oluşumu hem ISA, ilv restorasyonu ve kalan akciğer dokularında 1 alveol sayısını katkı göstermiştir. Biz ve diğerleri, PNX&#39;yi takiben boş göğüs boşluğuna sünger, balmumu veya özel şekil verilmiş bir protez gibi materyallerin sokulduğunu ( örn. , Protez implantasyonu) alveol rejenerasyonunu engellediğini gösterdik. Artık mekanik kuvvetin alveol rejenerasyonunu başlatmak için en önemli faktörlerden biri olarak işlev gördüğü tam olarak bilinmektedir 1 , 16 , 17 . Bu tür çalışmalar, alveol rejenerasyonunu kantitatif olarak değerlendirmek için bir kriter olarak PNX ile tedavi edilen ve Protez implante edilen akciğerlerden alınan ISA değerlerinin etkinliğini vurgulamıştır. Gözlemci önyargılarının ölçülen vayı önemli derecede etkilediği bilinmektedirAkciğer morfolojik parametreleri ( örn. , MIL ve ILV). ISA hesaplamasında kullanılan iki parametre olan hem ILV hem de MLI&#39;yi belirlemede bu önyargıdan kurtulmak için standart protokoller kullanılabilir. Burada, bu akciğer parametrelerini ölçmek için oldukça ayrıntılı ve standartlaştırılmış protokoller sunuyoruz. Daha da önemlisi, ISA&#39;yı doğru bir şekilde ölçebilme özelliği, yaralanmaya bağlı alveolar rejenerasyon modellerinde akciğer fonksiyonlarının güvenilirliğini ve tekrarlanabilirliğini artırmayı ve çoklu pulmoner hastalıklarda mekanik keşifleri kolaylaştırmayı taahhüt eder.

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			<td height="21" style="width:411px;height:21px;">Low cost cautery kit</td>
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			<td>Fine Science Tools</td>
			<td>15012-12</td>
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			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-12</td>
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			<td height="21" style="height:21px;">Castroviejo Micro Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
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			<td height="21" style="height:21px;">Vessel clips</td>
			<td>Fine Science Tools</td>
			<td>18374-44</td>
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			<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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			<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>
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			<td height="21" style="height:21px;">Small Animal Laryngoscope</td>
			<td>Penn-Century, Inc</td>
			<td>Model LS-2-M</td>
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			<td>Kent Scientific</td>
			<td>RSP1006-05L</td>
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			<td>Stuart equipment</td>
			<td>SBH130D</td>
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			<td height="21" style="height:21px;">Pentobarbital sodium salt</td>
			<td>Sigma</td>
			<td>P3761</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>
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			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
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			<td>Leica</td>
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			<td>Fisher HealthCare</td>
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			<td>Becton Dickinson</td>
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			<td height="21" style="height:21px;">Povidone iodine</td>
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			<td height="21" style="height:21px;">Brightfield microscope</td>
			<td>Olympus</td>
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</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> Burada, PNX ile işleme tabi tutulan bir grup ve bir protez implantasyonu (Protez yerleştirilmiş) grubu ile bir deney gerçekleştirdik. Bu gruplamalar, araştırma grubumuzdan daha önce yayınlanmış bir çalışmada kullanılan gruplamalarla aynıdır 14 . Fare PNX ve protez yerleştirme prosedürleri Şekil 2&#39;de gösterilmektedir. Ameliyatlar ve kantifikasy...

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

Akciğer hastalıklarında ve yaralanmaya bağlı alveol rejenerasyonunda akciğer morfolojisi ve fizyolojisini değerlendirmek için iç akciğer yüzey alanı (ISA) kritik bir kriterdir. Burada hem akciğer pnömonektomisinde hem de protez implantasyon fare modellerinde ISA için önyargı önlemeyi en aza indirecek standart bir yöntem açıklanmaktadır.

Fare Pnömonektomi ve Protez İmplantasyonu ile İç Akciğer Yüzey Alanının Ölçümü için Standart Bir Yöntem

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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JoVE Journal

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