Mechanical Stabilization of Mouse Carotid Artery for In Vivo Intravital Microscopy Imaging of Atherogenesis

Fig. 1

Surgical preparation and stabilization of the mouse carotid artery. Numbers indicate the different elements and tissues involved in the procedure. (a) Mice are immobilized in decubitus position, the carotid artery area is exposed, and skin around the surgical wound is fixed with sutures (white arrows). (b) The salivary gland (1) is fixed above the preparation with sutures (white arrows). (c) Hyoid muscles surrounding the carotid artery are separated and fixed with sutures (white arrows). Detail of hyoid muscles: omohyoid muscle (2 ), mastoid part of sternocephalic muscle (3 ), sternothyroid muscle (4). (d) Positioning of the metal support and glass coverslip for stabilization of the artery. The right vagus nerve (9) is carefully separated from the carotid artery (10 ). A flat metal piece with a beveled edge (6) is placed under the vessel, while the other side of the piece is fixed to a lateral holder (8 ). An image-grade round coverslip cut in half (5) is then placed above the artery and flexibly fixed to the bottom support using water-insoluble modeling clay (7 ). (e) General and detailed views of the stabilizer and of the platform. (f) Schematic representations of the stabilizer and tissues. (g) Schematic showing the positioning of the stabilizer. (h and i) General view of the mouse on the platform without (h) and with (i) the stabilizer positioned on carotid artery. (j) Image of the mouse prepared for observation under the microscope

3 Methods

3.1 Mouse Preparation

Inject intraperitoneally the anesthetic mixture (ketamine + medetomidine) and wait approximately 15 min until the mouse losses reflex response to toe or tail pinching (see Note 5 ).

Shave the neck and clean with 70 % ethanol to prevent contamination at the surgical site.

Immobilize mouse in decubitus position on the custom plastic platform as shown in Fig. 1 using water-resistant tape. Immobilize the head of the mouse by attaching its teeth to the platform using sutures and tape (Fig. 1a and see Note 6 ).

3.2 Surgery for Carotid Artery Exposure

Apply warm saline to the tissues throughout the procedure.

Using sharp/blunt serrated-edge scissors, make an ≈1-cm-long straight incision on the right part of the neck region, parallel to the trachea, from just below the chin to the top of the sternum just above the rib cage (see Note 7 ).

Using dull forceps, separate and blunt-dissect the skin on both sides of the incision and dissect the underlying tissue away from the skin circumferentially around the entire incision wound. Using sutures hung with water-resistant tape, maintain the skin away from incision wound (Fig. 1a and see Note 8 ).

Using the forceps, bluntly dissect the connective tissue away from the mouse’s right jugular (see Note 9 ).

Using the forceps, separate the fascia overlying the glandular tissue to expose the underlying glands, and gently dissect glandular tissue apart via blunt dissection to expose the muscular layer. Using sutures and tape, maintain the salivary gland away from the wound (Fig. 1b).

Using sutures and tape, maintain surrounding muscles (omohyoid muscle, mastoid part of the sternocephalic muscle, and the sternothyroid muscle) away from the carotid artery (Fig. 1c, f).

Expose the right carotid artery and carefully dissect it from the surrounding tissues (Fig. 1c, f and see Note 10 ).

Carefully separate the right vagus nerve from the carotid artery and surrounding tissues using dull forceps (Fig. 1c, f and see Note 11 ).

3.3 Carotid Artery Mechanical Stabilization for Intravital Imaging

Prepare the stabilizer (Fig. 1d–j). Fix the front end of the surgical blade to the plastic stick using water-resistant tape. Cut the image-grade round coverslip in half, and put it on the top of the back end of the surgical blade. Fix both elements flexibly using modeling clay (see Note 12 ). Using forceps, gently separate the coverslip from the surgical blade to obtain a separation of approximately 0.5–1 mm (Fig. 1f).

Place the beveled edge of the flat metal piece under the carotid artery, with the coverslip on its top (see Note 13 ).

Attach flexibly the stabilizer to the platform using modeling clay, as shown in Fig. 1f.

Using the back end of forceps, press gently on the coverslip and on the modeling clay to visually restrain motion in z-plane without interrupting blood flow (see Note 14 , Fig. 1d–f).

Place the stabilized region of the artery under the water-dipping objective of a multichannel epifluorescence microscope or a two-photon microscope (Fig. 1j and see Note 15 ).

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