Fig. 10.1
Macroscopic and microscopic images of the Ca model. Macroscopic images (a) and microscopic images (b, c) using Elastica van Gieson (EVG) and hematoxylin and eosin (HE) stains. Bars indicate 100 μm
As an experimental system, the Ca model is highly reproducible qualitatively and quantitatively [3]. Fine-tuning of the inflammation can be achieved by adjusting the time the CaCl2 solution is applied. On the other hand, some limitations of the Ca model are that it does not progress to rupture and develops no atherosclerosis or mural thrombi. The procedure requires careful training of the operator, especially for the exposure of the aorta and removal of the connective tissue surrounding the adventitia. The training of the operator can be lengthy, because it takes 4–6 weeks for the aortic aneurysm to develop. For optimal results, the operator should adjust the procedure based on the extent of inflammation and the dilation of the aorta. To shorten the training period, we suggest observing the aorta 1 week after the CaCl2 treatment. Adventitial inflammation of the entire circumference of the aorta ensures that the isolation and the CaCl2 treatment of the aorta were successful. At this time point, the aneurysm has not fully developed and the elastic lamellae, observed with Elastica van Gieson staining, should not be severely destructed. Once the circumferential inflammation is reproducibly obtained by the CaCl2 treatment, the aorta should be observed 6 weeks after the CaCl2 treatment to verify the aortic aneurysm develops as expected. Adjust the application time of the CaCl2 treatment without changing the surgical procedure to obtain the optimal results. By keeping the surgical technique and the CaCl2 treatment (concentration, volume, and application time) consistent, highly reproducible results can be obtained.
10.3 Tools
The tools and equipment that the authors use for making and analyzing the Ca model (Fig. 10.2) are listed below. The names of the manufacturers and the catalog numbers are provided for selected items as a reference for the readers. Similar products from other manufacturers may be used.
Fig. 10.2
Tools for the Ca model. (1) Physiological saline, (2) 1 mL syringe with 26G needle, (3) timer, (4) hair removal cream, (5) needle holder, (6) scissors, (7) forceps, (8) microsurgery forceps, (9) superfine cotton swabs, (10) cotton swabs, (11) microsurgical needles with 5-0 nylon suture thread, (12) large pieces of cotton, (13) small pieces of cotton, (14) 100 mm culture plate with rubber plate and scale
Cotton wool
Cotton swabs
Superfine cotton swabs
Scissors
Fine scissors (F.S.T. #14060-09)
Spring scissors (F.S.T. #15003-08)
Forceps (F.S.T. #11050-10)
Microsurgery forceps (F.S.T. #11253-20) × 2
Needle holder (Roboz #RS-7820)
Extra-thin pins (F.S.T. Minuten pins #26002-10)
Syringes (1 mL) with 26G needles
Infusion set
Microsurgical needles with 5-0 nylon suture thread
100 mm culture dish with black rubber plate and scale
Stereomicroscope for surgery with shadowless coaxial illumination (Carl Zeiss Stemi DV4 SPOT)
Microscope equipped with a calibrated digital camera and PC (Leica M165 C)
Anesthesia apparatus
Timer
Fiber optic illuminator or LED illuminator with flexible arms
Operating table
Heating plate
Hair removal cream
Anesthetics; sodium pentobarbital, isoflurane
70 % ethanol for disinfection
0.5 M CaCl2 solution
Physiological saline
Phosphate-buffered saline (PBS)
4 % paraformaldehyde (PFA) in PBS
10.4 Operation Procedure
In this instruction, the left and the right indicate those of the operator, not of the mouse. Fast the mice the day before the surgery. Dilute sodium pentobarbital (50 mg/mL) to 5 mg/mL with saline and administer it intraperitoneally at 0.08–0.1 mL/10 g body weight (40–50 mg/kg body weight) to the mouse for the induction of anesthesia. Maintain the anesthesia with 2 % isoflurane with appropriate adjustment of the concentration. Remove the abdominal hair with hair removal cream. Place the mouse on the operating table in the supine position. Sterilize the abdomen with 70 % ethanol. Make a skin incision of approximately 2.5 cm along the midline and cut the abdominal wall along the linea alba to open the peritoneal cavity (Fig. 10.3).
