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

저자는 북경 생물 과학 연구소 (Beijing Institute of Biological Sciences)에 도움을 청한다. 이 연구는 베이징 시립 자연 과학 재단 (No. Z17110200040000)의 지원을 받았다.

이 프로토콜에 사용 된 모든 절차는 국립 생물 과학 연구소의 실험실 동물의 관리 및 사용에 관한 지침의 권장 사항에 따라 수행되었습니다. 8 주된 CD-1 수컷 마우스를 실험이 수행 될 때까지 특정 병원균이없는 (SPF) 시설에 수용했다. 수술은 완전히 마취 된 마우스를 사용하여 수행되었습니다 ( 즉 , 발가락 핀치 반응없이). 수술 후 쥐는 충분한 음식과 담수가있는 따뜻하고 습기가 많은 방?...

<ol>
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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. 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이 프로토콜에서는 마우스 왼쪽 폐 PNX 및 보철물 삽입 후 폐 매개 변수 측정에 대한 자세한 설명을 제공합니다. ISA는 현재 많은 폐 질환 및 부상 유발 폐포 재생에서 호흡 기능 평가의 주요 척도로 간주됩니다. 그러나 폐 연구 공동체가 ISA의 유용성에 관해서는 유용한 척도로 동의하고 있지만 현재까지는 ISA를 계산하는 데 사용 된 두 가지 매개 변수 인 ILV 및 MLI 측정의 표준화에 대해서는 거의 고?...

폐의 기본 기능은 혈관과 대기 사이의 산소와 이산화탄소의 교환입니다. 기관지 폐 이형성증 (BPD), 만성 폐색 성 폐 질환 (COPD) 및 급성 호흡기 감염과 같은 폐 질환은 ISA 2 를 감소시킵니다. 폐 질환을 연구하는 연구자들은 MLI, ILV, 기체 교환 단위 수, ISA 및 폐 조직 적합성 2 , 3을 포함하여 폐의 형태 학적 변화를 평가하기위한 몇 가지 정량적 방법을 개발했습니다. Weibel 등의 선구자 연구 4 및 Duguid et al. 5는 함께 ISA가 인간의 폐에서 폐 가스 교환 용량의 직접 척도로 사용될 수 있음을 입증했으며 기종의 심각성을 판단하는 기준으로 사용할 수있다. 지난 5 년 동안 발표 된 여러 연구에서 폐 형태 학적 매개 변수 ( 예 : </em&gt; ISA 및 MLI)가 개발 중에 6 및 부상 PNX 1 (7)로부터 복구시 쥐의 폐에 형태 학적 및 기능적 변화를 평가한다. ISA는 식 1 8 , 9를 사용하여 계산됩니다. <img alt="방정식" src="/files/ftp_upload/56114/56114eq1.jpg" /> , ILV 내부 폐 용적이며 MLI는 말초 폐 공역 10 크기를 나타내는 매개 변수이다. 하나 이상의 폐엽의 외과 적 제거 인 PNX는 인간 11 , 마우스 1 , 개 12 , 쥐 13 및 토끼 14 , 15를 비롯한 많은 종에서 폐포 재생을 유도하는 것으로 널리보고되었습니다. 스터드PNX 후 14 일째 생쥐의 폐는 기존 폐포의 팽창과 폐포의 새로운 형성 모두 남아있는 폐 조직에서 ISA, ILV 및 폐포의 수를 회복시키는 데 기여한다고보고했다 1 . 우리와 다른 사람들은 스폰지, 왁스 또는 주문형 보철물과 같은 재료를 PNX ( 즉 , 보철물 삽입) 후 빈 흉부 공동에 삽입하면 폐포 재생을 손상시키는 것으로 나타났습니다. 기계적 힘은 폐포 재생을 시작하는 가장 중요한 요인 중 하나로 작용한다고 확고하게 확립되었습니다 1 , 16 , 17 . 이러한 연구는 PNX 치료 및 인공 삽입물 폐에서 ISA 값을 사용하여 폐포 재생을 정량적으로 평가하는 기준으로 사용하는 것이 효과적이라는 것을 강조했습니다. 옵서버 바이어스는 측정 된 VA에 유의 한 영향을 미치는 것으로 알려져 있습니다.폐 형태 학적 매개 변수 ( 예 : MIL 및 ILV)에 대한 정보. 표준화 된 프로토콜은 ISA 계산에 사용 된 두 가지 매개 변수 인 ILV와 MLI를 결정할 때이 편향을 없애기 위해 사용할 수 있습니다. 여기서는 이러한 폐 매개 변수를 측정하기위한 매우 상세한 표준화 된 프로토콜을 제공합니다. 중요하게도, ISA를 정확히 정량화하는 능력은 손상 유발 폐포 재생 모델에서 폐 기능 연구의 신뢰성과 재현성을 향상시키고 다중 폐 질환에서 기계적 발견을 촉진해야한다고 약속한다.

<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> 우리는 여기서 PNX 치료군과 인공 삽입물 (인공 삽입물)을 사용한 실험을 수행했습니다. 이러한 그룹은 이전에 발표 된 연구 그룹의 연구에서 사용한 그룹과 동일합니다 14 . 마우스 PNX 및 보철물 삽입 절차는 그림 2에 나와 있습니다. 8 주 된 CD-1 수컷 마우스가 수술과 정량화에 사용...

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

내부 폐 표면적 (ISA)은 폐 질환 및 부상 유발 폐포 재생에서 폐 형태 및 생리학을 평가하는 데 중요한 기준입니다. 폐 절제술 및 인공 삽입물 마우스 모델에서 ISA의 측정 편향을 최소화 할 수있는 표준화 된 방법을 설명합니다.

마우스 폐 전 절제술 및 보철물 삽입을 통한 내시경 표면 측정을위한 표준화 된 방법

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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