PMID: 25063071 DOI: 10.1016/S1995-7645(14)60066-3
2. Materials and methods
2.1. Animals
A total of 80 healthy 7-day-old SD rats, male or female,
weighing 12-18 g were selected. All animals were provided
by XX University Experimental Animal Center, and were
kept in a constant temperature 25 ℃, constant humidity
40% -50% environment, and had freely drank autoclaved
water.
2.2. Main reagents and instruments
Optical microscope was purchased from Japanese Nikon
company,German Leica Microtome was purchased from
Dalian Dajian Medical Devices Co., Ltd., Micro pipette and
homogenizer were purchased from the German Eppendorf
Company, -80 ℃ refrigerator were purchased from China
Haier Company. TUNEL assay kit was purchased from Roche
Company, IL-4, IL-1毬 and IgE radioimmunoassay kit were
purchased from Wuhan Boster Biological Engineering Co.,
Ltd., Ketamine (100 mg/10 mL) were purchased from Jiangsu
Hengrui Limited Company, propofol injection (200 mg/20 mL)
were purchased from Sichuan Shule Pharmaceutical
Corporation. Experimental animal cages, precision electronic
balance, 0.9% saline solution, hematoxylin, eosin staining
solution were provided by the laboratory.
2.3. Experimental methods
2.3.1. Experimental animal model and grouping methods
A total of 80 young rats were randomly divided into
four groups (the control group, experimental group A,
experimental group B, experimental group C) (n=20). All
young rats received the adaptive breeding for 1 week in
animal room. The animals in the control group received 0.9%
saline l mL by intraperitoneal injection every 2 h, continuous
for 3 times. The animals in experiment group A received 80 mg/kg
ketamine l mL by intraperitoneal injection every 2 h,
continuous for 3 times. The animals in experiment group B
received 80 mg/kg propofol 1 mL by intraperitoneal injection
every 2 h, continuous for 3 times. The animals in experiment
group C received 80 mg/kg ketamine and propofol 1 mL by
intraperitoneal injection every 2 h, continuous for 3 times.
The injection volume was 1 mL, and if it was less than l mL it
was supplemented by saline. Half of rats in each group were
randomly sacrificed after 15 min of anesthesia, the other half
underwent Morris water maze test 3 weeks later. All died
or abandoned animals in midway were supplemented by
modeling again.
2.3.2. Immune parameters detection
Using heparinization disposable 5 mL sterile syringe, 2 mL
blood was obtained by percutaneous puncture at the point
of maximal impulse and then it was injected into sterile EP
tube. After 30 min at 4 ℃, it was centrifuged at 3 000 r/min at
low temperature for 10 min. Serum was separated and stored
at -80 ℃ for the test. Serum IL-2, IL-4 and IL-10 levels
were detected by ELISA.
2.3.3. Brain tissue specimen collection, preparation and
indicators test
After blood collection, half of the young rats were randomly
perfusion needle was inserted to the ascending aorta from
the left ventricle, and fixed. The right auricle was cut. It
was washed at 4 ℃ saline by perfusion needle until the
effluent of the right atrium was clear. Then it was fixed
by 4% paraformaldehyde phosphate buffer. Hippocampal
was isolated from the brain tissue when the body tissues
and organs were hard, they were paraffin-embedded
and cut. Neuronal apoptosis detection was performed by
terminal deoxynucleotidyl transferase-mediated nick end
labeling (TUNEL) method. TUNEL-positive cells showed
brown particles in the nucleus. Six horizons were randomly
selected and average optical density was measured.
Positive intensity and the apoptotic index were calculated.
The formula was as follow: apoptotic index (AI) = MOD ×
Area% × 100, MOD represents the average gray level; area%
represents the percentage of the total positive nucleus area
in the total nucleus area. The other half young rats cerebral
was obtained quickly by sterile opening cranium, and brain
tissue was mixed with ice normal saline by homogenizer.
10% brain homogenate was prepared at 4 ℃, and centrifuged
at 3 000 r/min for 15 min. The supernatant was stored at
-80 ℃ for test. Whole brain IL-1毬 levels were detected by
ELISA.
2.3.4. Morris water maze test
Behavior of rats was observed by Morris water maze[3].
Round tank has four quadrants. A black platform was fixed
at the fourth quadrant, located 1 cm underwater. The rats
were put into the water of a randomly select quadrant,
swim tracks of the rats were recorded with a camera. How
long rats find the platform is the latency. After this test, the
platform was removed and the rats were put into water from
the same water-entering point, the times of crossing the
former platform were measured.
2.4. Statistical analysis
Data were expressed as mean依SD values and analyzed with
SPSS 13.0 software. After the variance test, the difference
between two groups was compared with single factor analysis
of variance. P<0.05 was considered as statistical significant
difference.