Animal preparation
Eight adult male Sprague-Dawley rats were divided in 2 groups: in the Group 1 (n=4),
rats underwent a surgical procedure of small bowel ligation followed by 8 hrs macroscopical monitoring; in the Group 2 (n=4),
high resolution micro-US scannings (Visualsonics Vevo 2100) were performed before and after small bowel ligation.
In both cases,
injured bowel was processed for histological analysis.
The rats were maintained on a 12/12 h light/dark cycle and allowed free access to food and water.
They were anesthetized with Ketamine hydrochloride 100 mg/kg im (CU ChemieUetikon GmbH,
Lahr,
Germany) and Domitor 0,25 mg/kg im (Medethomidine hydrochloride,
Pfizer,
New York,
United States) injections.
Dolorex 0,1 mg/kg sc (Butarphanol,
Intervet,
Boxmeer,
The Netherlands) was used immediately before the intervention to ensure intra-operative analgesia.
Further injections of these drugs were provided throughout the intervention to maintain a sufficient state of anesthesia.
Each rat was allowed to breathe spontaneously.
Body temperature was monitored with a rectal probe and maintained at 37.0 ± 0.5 °C with a heating blanket regulated by a homeothermic blanket control unit (Harvard Apparatus Limited,
Edenbridge Kent,
United Kingdom).
After drug injection,
eight rats were prepared for surgery through abdomen trichotomy.
These areas were then washed with povidone iodine and alcohol.
Macroscopic Analysis
After SBO,
the anesthetized rats of group I were observed for 8 h.
The bowel was exposed on gauze moistened with saline to prevent excessive evaporative loss.
Photographs of the exposed bowel and mesentery were taken at predetermined time points.
At the end of the observation period,
all rats were euthanized with an intrapulmonary injection of Tanax 0.5 ml and the large bowel was excised for histological analysis.
Ultrasonography
US examinations were performed with a VisualSonicsVevo 2100 unit (VisualSonics Inc,
Toronto,
Canada) specifically customized for animal research.
Each rat was positioned on a dedicated table used to secure the animal and support the manipulation during imaging,
ensure the comfort during the imaging session and monitor the ECG and heart rate of the animals.
Body temperature was monitored by a rectal probe and maintained at 37.0 ± 0.5 °C with a heating blanket regulated by a homeothermic blanket control unit of which the US unit is equipped.
An appropriate amount of heated sonographic gel (Aquasonic 100; Parker Laboratories,
Inc,
Fairfield,
NJ) was applied on the abdomen.
The micro-US examination was performed in B-mode The MS550D (22–55 MHz) transducer was fixed on an appropriate arm to ensure a stationary position on the desired image plane.
Group II animals were examined at predetermined time-points.
Ultrasound images were acquired with the following setting parameters: 40 MHz,
gain: 22dB,
frame rate: 28,
depth: 15 mm,
width: 14.08 mm,
line density: high.
The examination was integrated with LAZR photoacustic imaging system to characterize the change of hemoglobin signal before and after occlusion.
At the end of the observation period,
all rats were euthanized with an intrapulmonary injection of 0.5 ml of Tanax (Embutramide+MebezoniumIodide+Tetracaine,
Intervet/Shering-Plough Animal Healt,
Boxmeer,
The Netherlands).
Histological analysis
For light microscopy,
the large bowel of both groups of rats,
from the cecum to the rectum,
including the mesentery,
was excised and stored in 10% buffered formalin acetate for at least 2 days.
The samples were divided into three segments: the cecum and proximal colon (first segment),
splenic flexure (second segment),
distal colon and rectum (third segment).
Three-mm sections were obtained at 10-mm intervals from each segment and embedded in paraffin.
Transverse sections 3 micron thick were cut and stained with hematoxylin-eosine.
The sections were mounted on chromealume-gelatine-coated slides,
dehydrated and coverslipped.
Slides were imaged with a Zeiss Axio Skope microscope equipped with a high-resolution digital camera (ORCA-HR C4742-95-12HR,
10 MP; Hamamatsu Photonics K.K.,
Hamamatsu City,
Japan)