Name: a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation
Uploaded: 2017-07-26T15:00:00.000+00:00
Duration: 8 min 46 s
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 방에 보관했다. 복강 내 주사에 의해 전달 된 과량의 마취제를 사용하여 마우스를 희생시켰다. 1. 마우스 PNX 수술 <ol><li> 복막 내 (IP) 주사를 통해 sodium phenobarbital (120 mg / kg body weight)과 buprenorphine (0.1 mg / kg body weight)으로 마우스를 완전히 마취하십시오. 쥐가 더 이상 발가락에 눌려지지 않을 때 수술을하십시오. </li><li> 화학 요원으로 마우스의 왼쪽 흉부에있는 머리카락을 제거하십시오.흉내 치료 (~ 3 x 3 cm 2 영역). </li><li> 복부 측면이 조작자를 향하게하여 삽관 플랫폼에 각 마우스를 고정시킵니다 ( 그림 1A ). </li><li> 마우스 혀를 당기고 유도 카테터 18 ( 그림 1A )에 대한 노치가 들어있는 작은 동물 후두경으로 성대를 밝히십시오. </li><li> 호흡 중 성대의 움직임을 관찰하여 성대를 구분하십시오. 20G의 정맥 내 삽관 캐뉼라를 전방 각으로 기관에 부드럽게 삽입하십시오 19 . </li><li> 우측 가로 누운 위치에 마우스를 위치시키고 기계적 인공 호흡기에 연결 캐 뉼러 (예를 들어, 제어 된 압력은, 재료의 도표 참조). 마우스 가슴 ( 그림 1B )의 호흡 움직임을 관찰하여 기관에 캐뉼라의 삽입을 확인합니다. </li><li> 흡기 압력을 설정하십시오.f 인공 호흡기를 12cm H 2 O에 놓고 호흡 속도를 분당 120 회 호흡으로 설정합니다 ( 그림 1B ). </li><li> 베타 딘과 70 % 에탄올로 수술 부위의 피부를 오염 제거하십시오. </li><li> Noyes Spring Scissors (절삭 날 14 mm, 팁 직경 0.275 mm)로 피부와 근육을 절단하여 5 번째 늑간 간격의 공간에서 2 - 3 cm 외측 개흉술 절개를합니다 ( 그림 2B , C ). thoracotomy 절차에 사용되는 수술 악기는 사용하기 전에 소독됩니다. </li><li> 5 번째 늑간에서 1.5 cm 절개하여 왼쪽 폐를 노출시킵니다 ( 그림 2D , E ). 수술 중 고열 소작 기계를 사용하여 출혈을 중지하십시오. </li><li> 무뚝뚝한 팁 겸자 ( 그림 2F )와 가슴에서 왼쪽 폐엽의 3 분의 1을 들어 올린 다음 면봉을 사용하여 왼쪽 전체를 꺼내십시오폐 ( 그림 2G ). </li><li> 왼쪽 폐엽의 폐동맥과 기관지를 확인하십시오 ( 그림 2G ). </li><li> hilum에서 기관지와 혈관을 실크 수술 봉합사로 단단히 묶고 결찰 후 3 - 4 mm 지점에서 왼쪽 폐엽을 잘라냅니다 ( 그림 2H , I ). 참고 : 기흉을 일으킬 수있는 왼쪽 고관절의 봉합 매듭을 자르지 않도록주의하십시오 ( 즉 , 흉부의 공동에 공기 또는 가스가 발생할 수 있음) . </li><li> 1 봉합으로 가슴 벽을 닫은 다음 5 ~ 6 개의 중단 봉합을 사용하여 근육 층과 피부 층을 순차적으로 꿰맬 수 있습니다. 각 봉합사 사이에 3 - 4 mm 간격을 두십시오 ( 그림 2M , 2N ). 참고 : 외과 봉합 바늘을 심장에서 멀리 떨어지십시오. 부주의 한 심장 천자로 인해 즉시 사망 할 수 있습니다. </li><li> 포비돈 요오드로 수술 부위를 소독합니다. </li><li> 애프터외과 수술에서 마우스를 38 ° 열 패드에 놓고 마우스를 인공 호흡 운동이 시작될 때까지 인공 호흡기에 연결하십시오 ( 그림 2O ). </li></ol> 2. 보형물 삽입 <ol><li> PNX 절차의 1.1-1.13 단계를 수행하십시오 (즉, 마우스의 왼쪽 폐엽이 제거 될 때까지). </li><li> 무딘 집게 ( 그림 2J )를 사용하여 실리콘 보철의 중심을 고정하십시오 (길이 12 mm, 두께 3 mm, 너비 7 mm, 0.2 g, 타원체 모양). 실리콘 삽입물을 삽입하기 전에 소독하십시오. </li><li> 한 손으로 집게로 늑골을 잡고 흉강을 노출시킨 다음 다른 손으로 왼쪽 빈 가슴 흉강에 인공 삽입물을 삽입하십시오. 참고 : 삽입 각도는 보철의 정면과 흉부 표면 사이에서 약 45도입니다 ( 그림 2K ,L). 인공 삽입물 삽입시 매우 부드럽게하십시오. 과도한 힘은 흉막 파열로 이어질 것입니다. </li><li> 보철물이 왼쪽 빈 흉강을 차지하도록 무딘 집게로 보철물의 방향을 조정하십시오. </li><li> 마우스 PNX 절차의 1.14-1.16 단계를 수행하십시오. </li></ol> 3. ILV 측정 <ol><li> 일회용 혈청 피펫 (10 mL)에서 꺼낸 플런저, 바늘 어댑터가 달린 40 cm 길이의가요 관, 유속 조절 밸브 및 18 G 바늘로 구성된 맞춤 장치 ( &quot;팽창 튜브&quot;)를 준비하십시오. 조립 후 테이프로 보드에 피펫을 고정시킵니다 ( 그림 3A ). 피펫 상단과 실험대 사이의 거리는 최소 30cm 이상이어야합니다. </li><li> 55 ° C의 수 욕조에서 500 mL의 예비 가열 된 1x 인산 완충 생리 식염수 (PBS)에 20 g의 PFA를 용해시켜 신선한 4 % 파라 포름 알데히드 (PFA) 고정 용액을 수동으로 흔들어서 준비하십시오용액이 깨끗해질 때까지 매 10 분마다. 실온으로 냉각시킨 후, 용액을 0.45 ㎛ 필터로 여과한다. 주의 : PFA를 취급 할 때는 적절한 개인 보호 장비 (PPE)를 착용하십시오. </li><li> 과량의 마취제 (0.8 % 페노바르비탈 나트륨, 1,000 U / mL 헤파린)로 마우스를 희생시킵니다. </li><li> 폴리스티렌 해부 플레이트에 각 마우스를 고정하고 70 % 알콜로 뿌리십시오. </li><li> 조심스럽게 마우스 가슴을 열고 가위를 사용하여 흉골을 잘라내어 폐엽을 철저히 노출시킵니다. </li><li> 기관을 가위로 가위로 과도한 조직을 제거하십시오. 기관과 식도를 분리하십시오. </li><li> 복부 대동맥을 잘라 심장의 오른쪽 심실에 25 게이지 바늘을 삽입합니다. 이 삽입 전에 바늘을 20 mL 주사기에 연결하십시오. 폐가 흰색으로 변할 때까지 천천히 1x PBS를 심장에 밀어 넣어 혈액 세포를 제거하십시오. 일반적으로 폐 혈관을 치우기 위해 5 - 10 mL PBS가 필요합니다. </li><li> 채우기e 4 % 신선한 PFA로 인플레이션 튜브를 주문 제작하고 인플레이션 튜브에서 모든 거품을 제거하십시오. </li><li> 팽창 튜브의 18 게이지 바늘을 기관에 삽입하고 유체 누출을 피하기 위해 혈관 클립으로 기관을 클립하십시오. </li><li> 25cm의 일정한 압력 경폐 / H 2 O 2 (20)에 4 % PFA와 폐 팽창. 완전히 확장 된 폐를 달성하기 위해 2 시간 동안 실온에서 폐를 품어. 이 &quot;예비 고정&quot;단계는 폐 형태를 보존하는 데 중요합니다. <ol><li> 팽창 튜브를 모니터링하여 초기 4 % PFA 볼륨의 값을 기록하고 최종 볼륨을 기록하십시오. 내부 폐 부피는 초기 4 % PFA 체적에서 최종 4 % PFA 체적을 뺀 값과 같습니다. </li></ol></li><li> 기관지를 가리고 가위로 가볍게 주변의 결합 조직에서 폐를 해부합니다 (폐를 손상시키지 않음). 폐를 손상시키지 않도록 매우 조심하십시오. </li><li> 인큐4 ℃에서 12 시간 동안 4 % PFA로 채워진 50-mL 원추형 튜브에서 폐를 흔들어 쉐이커 (50 rpm)에서 부드럽게 흔들어줍니다. 조직 처리 및 염색으로 진행하십시오 (4 절 참조). </li></ol> 4. 조직 Embedding, 단면 및 Hematoxylin &amp; Eosin (H &amp; E) 염색 <ol><li> 고정 후 Noyes Spring Scissors를 사용하여 폐에서 심장 및 과도한 결합 조직을 잘라냅니다. 폐 엽을 기관과 연결하는 기관지를 잘라내어 각 폐엽을 부드럽게 분리하십시오. </li><li> 궤도 진탕 기 (50 rpm)에서 50 mL 1x PBS (30 분 / 세척)로 폐 엽을 3 ~ 4 회 씻어냅니다. </li><li> 최종 세척 후, 조직이 50 ML 원추형 튜브 (약 12 H)의 바닥에 싱크 때까지 4 ° C에서 30 % 자당 솔루션 (1x PBS)에 그들을 immersing하여 폐엽을 cryoprotect. </li><li> 조직을 임베딩하고 cryosectioning하기 전에 포셉과 튜브에서 폐엽 샘플을 제거하고, 액세스를 유지(sory lobes)을 이용하여 부엽 시료의 표면에서 남아있는 자당 용액을 추출한 다음 최적 절단 온도 (OCT) 화합물이 들어있는 페트리 접시에 시료를 약 30 분 동안 담그십시오. </li><li> Cryomolds를 사용하여 액체 질소로 OCT 임베디드 액세서리 엽 샘플을 고정합니다. 로브의 가장 큰 표면적을 몰드 바닥에 평행하게 위치시킵니다. </li><li> 조직학 분석을위한 cryosectioning 동안 각 샘플에 대한 총 세 μm의 두께 섹션을 준비합니다. 처음 1 mm 조직을 버리고 10 μm 두께의 부분을 하나씩 수집하고 0.5 mm 조직을 버리고 다른 부분을 수집 한 다음 0.5 mm 조직을 버리고 세 번째 (최종) 부분을 수집합니다. </li><li> H &amp; E 염색을하기 전에 1 시간 동안 공기 건조하십시오. </li><li> H &amp; E 얼룩 수행 <ol><li> 수돗물을 3 ~ 4 차례 변경 한 다음 신선한 hematoxylin에서 2 분 동안 얼룩을 지우십시오; 흐르는 수돗물 아래의 섹션을 헹구십시오; 과량의 헤 마톡 실린을 제거하기 위해 1 % HCl-70 % 에탄올 용액에 2 번 섹션을 담그십시오. </li><li> 3 분 동안 신선한 에오 신의 섹션 얼룩; 95 % 에탄올에서 2 번 연속으로 30 번 세척하고 100 % 에탄올로 2 번 30 번 세척하여 절편을 탈수시킵니다. 30 초 동안 크실렌의 단면을 깨끗이하고, 신선한 크실렌에서 1 회 제거 단계를 반복한다; 유리 coverslips를 사용하여 마운트 매체와 슬라이드를 탑재하십시오. </li></ol></li></ol> 5. MLI의 정량화 <ol><li> 명 시야 현미경을 사용하여 H &amp; E로 염색 한 액세서리 로브 섹션 (20X 배율)의 디지털 이미지를 수집하십시오. </li><li> MLI를 정량화하려면 3 개의 섹션으로 구성된 적절한 영역 (동맥 및 정맥, 주요기도 및 폐포 덕트 없음)에서 총 15 개의 중첩되지 않는 뷰 (1,000 μm x 1,000 μm)를 무작위로 선택하십시오. </li><li> 10 개의 고르게 분포 된 수직선과 정의 된 len의 균등하게 분포 된 10 개의 수평선이있는 격자 배치눈금자 도구를 사용하여 선택한 영역에서 gth (1,000 μm); 따라서 각 라인은 100 μm 떨어져 있습니다 ( 그림 4B ). </li><li> 하나의 절편 값을 인접한 두 개의 폐포 상피 사이의 직선 길이로 정의하십시오. 1,000 μm 길이 선을 따라 모든 절편 값을 측정하십시오. </li><li> 각 그리드에 대해 가로 10 개, 세로 1,000 개, 세로 10 개 사이의 모든 절편 값을 계량화하십시오. 참고 : MLI는 각각의 액세서리 로브에 대해 준비된 3 개의 섹션 중에서 분석 된 총 15 개의 그리드로부터 절편 길이의 평균값입니다. </li></ol> 6. ISA의 계산 <ol><li> 등식 1을 사용하여 ISA를 계산하십시오 ( 소개 참조). ILV의 측정에 대해서는 3 장을 참조하고 MLI의 정량화에 대해서는 5 장을 참조하십시오. </li></ol>

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저자는 공개 할 것이 없습니다.

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

a-standardized-method-for-measuring-internal-lung-surface-area-via

Research

JoVE Journal

Developmental Biology

13.1K 조회수.  Peking University.  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. 

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

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

마우스 폐 삽관 법의 간단한 방법

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

연구

의학

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

마우스 폐 내피 표면 층의 생체 측정

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

폐에 암 전이를 조사하기위한 영구적 인 창

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

암 연구학

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

담배 연기에 노출 된 마우스의 폐 폐기종 및 작은기도 리모델링 자동 측정

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

보상 성 폐 성장의 마우스 전폐 절제술 모델

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

마우스 폐의 압력 볼륨 곡선의 측정

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

마우스 폐 암 연구에 유용한 점안 및 고정 방법

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

복 포도 당 내성 검사, 폐 기능 측정 및 폐의 고정 비만 및 장애인된 대사 폐 결과에 미치는 영향 연구

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

면역학 및 감염병학

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Orthotopic 폐 암 세포 Engraftment의 효율성 증가 사전 조건 Bleomycin와 쥐의 기도

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

<meta charset="utf8"></head><body>생쥐 폐 이식에서 세포 역학의 실시간 모니터링

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>저자 스포트라이트: 생체 내 이미징을 통한 폐 이식 면역학의 발전

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

이식된 마우스 폐의 생체 내 2광자 현미경

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...