Surgical preparation of rats and mice for intravital microscopic imaging of abdominal organs

Abstract

Intravital microscopy is a powerful research tool that can provide insight into cellular and subcellular events that take place in organs in the body. However, meaningful results can only be obtained from animals whose physiology is preserved during the process of microscopy. Here I discuss the importance of preserving the overall state of health of the animal, methods of anesthesia, surgical techniques for intravital microscopy of various abdominal organs, methods to maintain and monitor the physiology of the animal during microscopy and associated peri- and post-operative recovery considerations.

Keywords: Analgesia; Anesthesia; Animal surgery; Aseptic technique; Imaging window; Intravital microscopy.

Figure 1

Flow diagram for selection of anesthetic agent in rats. Isoflurane is generally the agent of choice because it is easy to titrate to effect for cases of long or short duration, or in rats that are physiologically unstable. In addition, emergence and recovery from Isoflurane are rapid in comparison to injectable agents. Inactin is ideal in non-survival cases in male rats that are physiologically stable because it has a long duration of action that eliminates the need for redosing. Iso= Isoflurane 1–2% vapor with a flow of Oxygen at 1l/min. Ket= Ketamine cocktail 60–100 mg/kg Intraperitoneal injection (typically with Xylazine 5–10 mg/kg +/- Acepromazine 2.5 mg/kg). Pento= Sodium Pentobarbital 30–60 mg/kg Intraperitoneal injection. In survival cases Isoflurane is combined with Buprenorphine HCl subcutaneously at a dose of 0.01–0.05 mg/kg for analgesic purposes.

Figure 2

Flow diagram for selection of anesthetic agent in mice. Isoflurane is generally the agent of choice because it is easy to titrate to effect for cases of long or short duration, and emergence and recovery from Isoflurane are rapid in comparison to injectable agents. Iso= Isoflurane 1–2% vapor with a flow of Oxygen at 1l/min. Ket= Ketamine cocktail 90–100 mg/kg intraperitoneal injection (xylazine 2.5–5.0 mg/kg +/- acepromazine 1.0–2.5 mg/kg). In survival cases Isoflurane is combined with Buprenorphine HCl subcutaneously at a dose of 0.05–2.0 mg/kg for analgesic purposes.

Figure 3

Externalization of the kidney, spleen and pancreas. A left flank vertical incision provides access to the kidney (A), spleen (B) and pancreas (C). The incision in the figure has been retracted medially and laterally to depict the anatomical relationships of the three organs. The incision length should be no longer than 1.0 cm to prevent retraction of the externalized organ back into the abdominal cavity. Once the organ of interest is externalized, the animal is placed left side down on the microscope stage and organ rests in the glass bottom dish.

Figure 4

Externalization of the liver for imaging purposes. A horizontal incision just below the level of the xyphoid process allows for delivery of the liver and placement in a customized glass bottom imaging dish once the animal is placed face down on the microscope stage.