hilar clamping and arterial blood gas evaluation of reperfused lungs in mice for ischemia research

Mouse Model for Studying Lung Ischemia-Reperfusion Injury

IRI during organ transplantation is a major risk factor for primary graft dysfunction and later episodes of graft rejection1,2. During transplantation, warm ischemia time is defined as the period of time from donor aortic cross-clamp to initiation of cold perfusion and from organ removal from ice to organ implantation. Cold storage time is defined as the period of time from the start of cold perfusion to the removal of the organ from ice3. Warm ischemia is more deleterious for later organ function than cold ischemia4,5,6, and its underlying mechanisms warrant further study in pre-clinical models. Additionally, organ transplantation from donation after cardiac death (DCD) is associated with longer warm ischemic times than traditional donation after brain death (DBD)7. While the use of DCD donors can expand the donor pool and increase lung utilization, further pre-clinical studies to evaluate the effects of warm ischemia on post-transplant lung function are needed. Below, we describe a model of warm IRI in mice via the left pulmonary hilar clamp.
Several animal models of lung hilar clamping have been developed and adapted over the last several years and may include the use of an atraumatic microvascular clamp8,9,10,11,12,13, a Rumel tourniquet14,15, or suture ligation16 as the hilar clamp. The crux of the hilar clamp is that it must be reversible and cause minimal or no damage to the hilar structures so that reperfusion can be achieved. Here, we describe our hilar clamp technique in mice that involves reversible suture ligation of the left pulmonary hilum with a slipknot. This method occludes the pulmonary arterial inflow, venous outflow, and airflow in and out of the mainstem bronchus. The main benefit of a slipknot over a vascular clamp, clip, or tourniquet is that the chest can be closed during prolonged periods of ischemia, thereby minimizing insensible fluid and heat loss in the mouse. We provide a protocol for obtaining reliable arterial blood gas (ABG) measurements and for measuring cell extravasation after hilar clamping.
This hilar clamp technique holds an important place in the wider study of lung transplantation. Compared to small animal models of orthotopic lung transplantation, the hilar clamp technique can isolate the effects of IRI without the addition of surgical anastomotic trauma or allogenicity17. Additionally, the hilar clamp technique can be more easily and quickly mastered than the mouse lung transplant. In fact, using hilar clamp techniques, several important mechanisms in the pathogenesis of IRI have been identified in the past decade, such as TLR4, NADPH oxidase, and adenosine A2A receptor14,18,19,20. In the following protocol, we present a reliable, teachable, and reproducible method of hilar clamping as a tool for studying lung IRI.

Author Spotlight: Insights into the Effect of Ischemia Reperfusion on Lung Transplantation

Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury

We are interested in studying the effects of ischemia-reperfusion injury on outcomes following lung transplantation with the goal of understanding underlying cellular and molecular mechanisms. Neutrophils that enter the lung contribute to ischemia-reperfusion injury, or IRI. We've recently discovered that B cells proceed neutrophils in entering the lung, and they play a role in the recruitment of neutrophils following IRI.Left lung transplantation in the mouse is technically challenging, and requires full-time, dedicated training over several months to achieve consistent results. Additionally, it introduces allergenicity and surgical anastomotic trauma. Thus, we need a more accessible technique that can isolate the study of ischemia-reperfusion injury.Using our hilar clamp model, we have found that B-cells enter the lung early after ischemia-reperfusion injury, and they recruit classical monocytes in a B-cell receptor into TLR4 dependent fashion. This ultimately results in increased neutrophil extravasation into the lung. Our protocol involves reversible suture ligation of the left pulmonary hilum with a slip knot.The pliable and unobtrusive nature of the suture allows for chest closure, which is useful for studying longer periods of warm ischemia, as chest closure minimizes heat and insensible fluid losses in the mouse.

Hilar Clamping and Arterial Blood Gas Evaluation of Reperfused Lungs in Mice for Ischemia Research

hilar-clamping-arterial-blood-gas-evaluation-reperfused-lungs-mice

Research

JoVE Journal

Medicine

178 ビュー.  Washington University, St. Louis. まず、麻酔して挿管したマウスを右の側褥位に置きます。両方の前足を左上隅に、後足を左下隅に持っていきます。切開前に左胸部を70%アルコールで消毒します。第4肋間腔の皮膚を切開して開いた後、ハンドヘルド焼灼ペンを使用して、皮下層と筋肉層内の目に見える血管を凝固させます。はさみで、広背筋と前鋸筋を皮膚切開の長さに沿って分割します。一対の...

ビデオ: 虚血研究のためのマウスにおける再灌流肺のヒラークランプと動脈血ガス評価

Ischemia reperfusion injury (IRI) during lung transplantation is a major risk factor for post-transplant complications, including primary graft dysfunction, acute and chronic rejection, and mortality. Efforts to study the underpinnings of IRI led to the development of a reliable and reproducible mouse model of left lung hilar clamping. This model involves a surgical procedure performed in an anesthetized and intubated mouse. A left thoracotomy is performed, followed by careful lung mobilization and dissection of the left pulmonary hilum. The hilar clamp involves reversible suture ligation of the pulmonary hilum with a slipknot, which stops the arterial inflow, venous outflow, and airflow through the left mainstem bronchus. Reperfusion is initiated by careful removal of the suture. Our laboratory uses 30 min of ischemia and 1 h of reperfusion for the experimental model in the current investigations. However, these time periods can be modified depending on the specific experimental question. Immediately prior to sacrifice, arterial blood gas can be obtained from the left ventricle after a 4 min period of right hilar clamping to ensure that the PaO2 values obtained are attributed to the injured left lung alone. We also describe a method to measure cell extravasation with flow cytometry, which involves intravenous injection of a fluorochrome-labeled antibody specific for the cell(s) to be studied prior to sacrifice. The left lung can then be harvested for flow cytometry, frozen or fixed, paraffin-embedded immunohistochemistry, and quantitative polymerase chain reaction. This hilar clamp technique allows for&#160;detailed study of the cellular and molecular mechanisms underlying IRI. Representative results reveal decreased left lung oxygenation and histologic evidence of lung injury following hilar clamping. This technique can be readily learned and reproduced by personnel with and without microsurgical experience, leading to reliable and consistent results and serving as a widely adoptable model for studying lung IRI.

The protocol outlines a feasible, reliable, and reproducible method of left pulmonary hilar clamping that can be used to study lung ischemia-reperfusion injury in mouse models.