Name: 쥐 췌장 창 수술
Uploaded: 2023-10-06T13:20:00
Description: Watch this Scientific Journal Video about Murine Pancreatic Window Surgery at JoVE.com

murine pancreatic window surgery

Implanted Stabilized Window를 사용한 쥐 췌장 영상

Benign and malignant pancreatic diseases are potentially life-threatening, with considerable gaps in the understanding of their pathophysiology. Pancreatitis-inflammation of the pancreas-is the third major cause of gastrointestinal disease-related hospital admissions and readmissions in the US and is associated with substantial morbidity, mortality, and socioeconomic burden1. Ranked as the third leading cause of cancer-related death2, pancreatic ductal adenocarcinoma (PDAC) accounts for most pancreatic malignancies3 and portends a poor 5-year survival rate of only 11%2. The leading cause of cancer-related mortality in PDAC is overwhelming metastatic burden. Unfortunately, most patients present with metastatic disease. Therefore, understanding the dynamics of metastasis in PDAC is a critical unmet need in the field of cancer research.
The mechanisms underpinning inflammation and the metastatic cascade of the pancreas are poorly understood. A major contributor to this gap in knowledge is the inability to observe pancreatic cellular dynamics in vivo. Direct observation of these cellular dynamics promises to unveil critical targets to leverage and improve the diagnosis and treatment of those with pancreatic disease.
Intravital imaging (IVI) is a microscopy technique that allows researchers to visualize and study biological processes in living animals in real time. IVI allows high-resolution, direct visualization of intracellular and microenvironmental dynamics in vivo and within the native environment of the biological process in question. Therefore, IVI allows in vivo observation of healthy and pathologic processes.
Contemporary whole-body imaging modalities such as MRI, PET, and CT offer excellent views of entire organs and can reveal pathologies, even before the onset of clinical symptoms4. They are unable, however, to attain single-cell resolution or reveal the earliest stages of disease-pancreatitis or malignancy.
Previous research has used single-cell resolution IVI to observe benign and malignant diseases of skin5,6, breast7, lung8, liver9, brain10, and pancreatic tumors11, leading to insights into mechanisms of disease progression12. However, the murine pancreas poses significant obstacles to achieving single-cell resolution using IVI, primarily due to its deep visceral location and high compliance. Moreover, it is a branched, diffusely distributed organ within the mesentery that connects to the spleen, small intestine, and stomach, making it challenging to access. The tissue is also highly sensitive to motion caused by adjacent peristalsis and respiration. Minimizing movement of the pancreas is essential for single-cell resolution microscopy, as motion artifacts of even a few microns can blur and distort images, making tracking the dynamics of individual cells impossible13.
To perform IVI, an abdominal imaging window (AIW) must be surgically implanted9,11. To implant the AIW surgically, a metal window frame is sutured into the abdominal wall. Afterward, the organ of interest is attached to the frame using cyanoacrylate adhesive. While this is sufficient for some rigid internal organs (e.g., liver, spleen, rigid tumors), attempts at imaging the healthy murine pancreas are compromised by suboptimal lateral and axial stability due to the tissue&#39;s compliant texture and complex architecture14. To address this limitation, Park et al.14 developed an imaging window specifically designed for the healthy pancreas. This Pancreas Imaging Window (PIW) minimizes the influence of intestinal movement and breathing by incorporating a horizontal metal shelf within the window frame, just below the coverslip, stabilizing the tissue and maintaining its contact with the cover glass. While the PIW offers increased lateral stability, we found that this window still demonstrates axial drift and additionally prevents the imaging of large solid tumors due to the narrow gap between the metal shelf and coverslip15.
To address these limitations, we developed the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP), an implantable imaging window capable of achieving stable long-term imaging of both the healthy and diseased pancreas (Figure 1)15. Here, we provide a comprehensive protocol for the surgical procedure used to implant the SWIP. Although the primary objective was to study the dynamic mechanisms involved in metastasis, this method can also be utilized to explore various aspects of pancreas biology and pathology.

저자 스포트라이트: 첨단 생체 내 이미징을 통한 췌장 질환 이해의 혁명 

쥐 췌장의 생체 내 이미징을 위한 안정화된 창

Employing high-resolution intravital imaging and an improved method of pancreas stabilization, we aim to enhance comprehension of the physiological dynamics of pancreatic adenocarcinoma, pancreatitis, and the healthy pancreas. This approach makes it possible to address gaps in the pathophysiological understanding, potentially advancing treatment strategies for these high-risk conditions. Single-cell resolution intravital imaging has illuminated the physiology and mechanisms of disease progression in various tissues.However, the pancreas presents unique challenges due to its deep placement, compliance, and susceptibility to motion artifacts, hindering effective access for observation for both benign and malignant conditions. Our SWIP technique enhances murine pancreas stabilization compared to prior abdominal imaging windows. The uniquely designed frame features etched lines for the use of micro-cartography, which enables consistent region relocalization across multiple imaging sessions.This facilitates the measurement of subcellular dynamics over days, providing valuable insights. The SWIP protocol enables stable, high-resolution, single-cell intravital imaging of the murine pancreas in normal and diseased states. It's especially beneficial for prolonged 3D and 4D imaging, offering insights into cellular interactions and disease mechanisms.Invaluable for unraveling pancreatic physiology and pathology, this technique visualizes single cells and their context in vivo.

Watch this Scientific Journal Video about Murine Pancreatic Window Surgery at JoVE.com

쥐 췌장 창 수술

먼저 마취된 쥐를 가열된 수술대 위에 오른쪽에 위치시켜 왼쪽 복부를 노출시킵니다. 마우스의 앞쪽 끝과 뒷다리를 종이 테이프로 고정하여 비장이 잘 보이도록 합니다. 다음으로 소독약을 넉넉하게 도포하여 수술 부위의 피부를 소독합니다.발가락 꼬집기를 투여하여 마우스가 완전히 마취되었는지 확인합니다. 집게를 사용하여 왼쪽 상복부의 피부를 들어 올리고 카스트로비에호 가위를 사용하여 피부와 근육층을 10mm 원형으로 절개합니다. 비장에 부착된 췌장을 찾아 십자수를 배치해야 하는 방향을 결정합니다.5/0 실크 봉합사를 사용하여 식별된 위치의 근육층에 첫 번째 바늘을 놓습니다. 3-5 노트로 끝을 고정하십시오. 절개 부위를 직접 꿰매십시오.그런 다음 약 5cm의 꼬리를 자르고 남겨 둡니다. 이번에는 원래 스티치에 수직으로 스티칭을 반복합니다. 그런 다음 췌장을 조심스럽게 들어 올려 십자수 위에 놓습니다.구멍에서 약 1mm 떨어진 곳에 지갑 끈 스티치를 놓고 피부와 근육층을 교차시킵니다. 원형 절개 가장자리가 창의 홈 안에 위치하도록 창틀을 배치합니다. 이제 5/0 실크를 단단히 묶어 이식 된 창을 고정하십시오.100마이크로리터의 액체 시아노아크릴레이트 접착제를 10mm 주사기에 넣습니다. 약 10초 동안 티슈에 섬세한 압축 공기를 가하여 건조시킵니다. 집게를 사용하여 창틀의 바깥쪽 가장자리를 잡고 조심스럽게 들어 올려 창틀 바닥에서 췌장이 분리되도록 합니다.췌장 조직을 피하면서 창의 홈을 따라 액체 시아노아크릴레이트 접착제의 얇은 층을 분배합니다. 그런 다음 진공 픽업을 사용하여 5mm 커버 슬립을 들어 올립니다. 커버 슬립을 광학 창틀 중앙에 부드럽게 놓고 접착제가 굳을 수 있도록 약 25초 동안 가벼운 압력을 유지합니다.집게를 사용하여 진공 픽업에서 커버 슬립을 분리합니다. 그런 다음 십자수 봉합사를 조여 췌장을 커버 슬립에 고정하고 봉합사 끝을 자릅니다. 마지막으로 마우스에서 테이프를 제거합니다.이소플루란 기화기를 끄고 마우스를 깨끗한 케이지로 옮깁니다. 췌장 조직의 증가된 측면 및 축 안정성은 침전 기간 후에 관찰되었습니다. SWIP를 사용한 이미징은 복부 및 췌장 이미징 창에 비해 낮은 수준의 드리프트를 보여주었습니다.쥐 췌장의 연속 영상은 세룰레인 처리 후에 수행되었습니다. 소엽 재배치는 이틀 연속 관찰되었습니다. SWIP는 장기 이미징에도 사용할 수 있어 액포 운동성을 시각화하고 정량화할 수 있습니다.예를 들어, 쥐 췌장에서 액포의 평균 속도는 PBS에 비해 세룰레인 처리 후 약 10% 증가했습니다. 세룰레인 처리는 또한 세포 내 구조의 평균 회전 빈도를 10% 증가시켰지만, 순 속도, 방향성 또는 처리 간 누적 거리에는 유의한 차이가 없었습니다. SWIP를 사용하면 종양 세포 이동을 시각화하고 캡처할 수 있습니다.종양 세포의 집단 클러스터 세포 이동은 짧은 기간 동안 관찰되었습니다. 종양 세포와 대식세포의 단세포 이동도 관찰되었습니다.

Research

JoVE Journal

Cancer Research

Murine Pancreatic Window Surgery

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...