Animals
Rabbits (Japanese white, 6 weeks old, 2.5–3.0 kg, male) were purchased from KITAYAMA LABES CO.,LTD. (Nagano, Japan) and used with a 1-week acclimation period in individual cages. For induction of anesthesia, 5 mg/kg (0.25 mL/kg) of xylazine was administered intramuscularly, and 35 mg/kg (0.7 mL/kg) of ketamine was administered intramuscularly approximately 5 minutes later. Anesthesia was maintained by ear vein infusion (50 mL/h) of Ringer’s lactate solution with 4% v/v ketamine. Rabbits were euthanized at the end of the experiment by blood liberation by cutting the abdominal aorta while maintaining anesthetic depth.
Materials
For topical hemostatic agents, gelatin sponge (Gelfoam®, Pfizer Inc., New York, NY) and oxidized cellulose (SURGICEL® Nu-Knit Absorbable Hemostat, Ethicon Inc., New Brunswick, New Jersey), which are commonly used in neurosurgery, were selected. These two topical hemostatic agents were used alone or in combination with fibrin glue (BOLHEAL®, KM Biologics Co., Ltd., Kumamoto, Japan) and the results were compared in a total of four groups (Table 1). Fibrin glue consists of two fluids, a fibrinogen solution and a thrombin solution, and is a biological adhesive that gels quickly when mixed.
Procedures
As a rabbit venous bleeding model, rabbits were operated on under anesthesia and, after exposure of the posterior vena cava, bleeding was induced by direct puncture of only the upper wall of the posterior vena cava with a 19G needle. Procedure was performed with neurosurgical pads (BemsheetsTM, Kawamoto Corporation, Osaka, Japan) and an irrigation/suction device (Kamiyama Irrigation Suction Version I, Ohwa Tsusho Co., Ltd., Tokyo, Japan) using saline. After confirming the bleeding point while controlling the bleeding, the sample was securely applied to the bleeding point and compressed and fixed for 10 seconds. Achievement of hemostasis was assessed macroscopically and signs of slight bleeding were considered a failure. If no bleeding was observed after the 10 second hemostatic procedure, a 30 second wash water pressure load was added using the irrigation/suction device, and if no bleeding was observed after application of hydraulic pressure, hemostasis was judged to be achieved. If hemostasis was not achieved, the applied hemostatic sample was completely removed, a new hemostatic sample in the same group was used, and hemostasis was attempted again in the same manner. Hemostasis was attempted up to three times per bleeding point, and the number of attempts required to achieve hemostasis was counted. If hemostasis was not achieved after three hemostatic attempts, it was counted as “4”. Deviations in hemostasis procedures were excluded from the evaluation.
Groups 1 and 2 used gelatin sponges and Groups 3 and 4 used oxidized cellulose as samples. Groups 1 and 3 used 3 mm square trimmed hemostatic agents as intact samples. In groups 2 and 4, hemostatic agents trimmed to 3 mm square were soaked in 10 μL of fibrinogen solution immediately prior to use, applied to the bleeding point, and adhered to the bleeding point by dripping an appropriate amount of thrombin solution onto the sample and gelling. Additional movie files show this in more detail [see Additional file 1, 2].
In the posterior vena cava, four bleeding points were created and one test for each of the four groups was performed at each bleeding point to eliminate individual variability. The bleeding points were created at least 2 cm apart to avoid the effects of each test. In addition, the bleeding point and the group used for hemostasis were switched from one animal to another, and the number of applications to the distal and central sides of the blood vessels in each group was allocated equally. In determining the number of samples, it was necessary to conduct one set of four tests to ensure uniform allocation. To ensure reproducibility, this set was repeated three times. Sixteen rabbits were used. Sixteen tests were conducted for each group. Surgical procedures were performed using a laparoscopic endoscope (LTF-S-190-10, Olympus Corporation, Tokyo, Japan) with an enlarged field of view. This procedure was performed by a single expert.
Statistical analysis
Median calculations and graphs were performed using EZR (Saitama Medical Center, Jichi Medical University Site, Saitama, Japan). EZR is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria, version 2.13.0) [12].