Aortic Cross-Clamping to Provide Differential Fixation by Perfusion

Abstract

An intricate network of regulation between the brain and the pancreas modulates hormone secretion and organ function. Dysfunction of the brain-pancreas axis occurs in disease states such as diabetes and pancreatitis. Given the delicate nature of the mouse brain, procurement for tissue and cellular analysis is facilitated by fixation by perfusion with paraformaldehyde (PFA). The brain is hardened by PFA during the preservation process, but this hardening also occurs in the pancreas, as well as the remainder of the intra-abdominal organs. This hardening makes the pancreas friable and difficult to dissect without damaging and fragmenting the organ. Additionally, this fixation may preclude the ability to perform analytic techniques such as western blot and quantitative PCR (qPCR) simultaneously. Performing a simple cross-clamping of the thoracic aorta allows for differential perfusion of organs and maximal use of limited samples from a single animal. The brain can be perfused with PFA without compromising tissue collection of the pancreas and other intra-abdominal organs. This simple maneuver allows for greater tissue collection and analysis per mouse in studies evaluating the brain-pancreas or brain-gut axis. © 2021 Wiley Periodicals LLC. Basic Protocol: Differential fixation by perfusion using aortic cross-clamp.

Keywords: aortic cross-clamping; brain; brain-pancreas axis; cross-clamping; metabolism; mouse; pancreas.

Figure 1.

(A) Arterial anatomy overlying major organ systems of an adult mouse. The X indicates the area in which the aortic clamp is placed to separate the blood flow superiorly (head, neck, and forelimbs) from the blood supply for the abdominal contents and hind limbs. This allows fixative to flow to the head but not to the abdomen. The sternum and heart are retracted superiorly and to the mouse’s right to expose inferior thoracic cavity. (B) Magnified view of the boxed area in A. The inferior vena cava (IVC), esophagus (E), and the aorta (Ao) are exposed within this area after removing overlying fat and connective tissue. (C) Bulldog clamp placed on the aorta and occluding blood flow.

Figure 2.

(A) The exposed mouse skull with important sutures for dissection identified. (B) Cut along the sutures as indicated by the dotted line. (C) Remove the skull posterior to the coronal suture by retracting laterally. (D) Remove the skull anterior to the coronal suture by pulling toward the nose of the mouse. (E) Remove any remaining skull to expose the brain.