Major reagent
Perillaldehyde essential oil (PAEEO) was obtained by Sigma-Aldrich (St. Louis, MO). Primary antibodies including NR2B, TRPM2, β-actin were purchased from Santa Cruz Company. Mouse neuron growth factor (mNGF) was purchased from the First Affiliated Hospital of Xinxiang Medical University (Xinxiang, China) and prepared in saline consisting of 1% Tween 80.
Animals
Male Wistar rats weighing 260–270 g were supplied by the Experimental Animal Center of Henan Province (license number: SYXK-Yu-005-0012). All rats were housed individually in cages and given free access to food and water. The living temperature and humidity range was 25 ± 1 °C and 55% ± 5% with a 12 h light/dark cycle. The study was maintained in accordance with the guidelines outlined by the National Institutes of Health Guide for the Care and Use of Laboratory. The experiment protocol was reviewed and approved by the Animal Care and use Committee of the Xinxiang Medical University.
Permanent common carotid artery occlusion
VD rats were established by bilateral common carotid arteries occlusion (2-vessel occlusion [2VO]). Rats were weighed and intraperitoneally injected with 30 mg/kg pentobarbital sodium for anesthesia. The bilateral common arteries were exposed and separated from the vagus nerve, then ligated with 5–0 silk thread. The sham operation group was operated in the same way, but it was not arterial ligated. During the whole operation, the operation should be as mild as possible to reduce the pain of animals.
Primary culture of hippocampal neurons
After pregnant rats were anesthetized, the abdomen of pregnant rats were disinfected with 75% alcohol, and then the fetal rats were carefully taken out. The fetal rats were immersed in 0.9% normal saline and 75% alcohol successively at 4 °C for 1 min each. The fetal rats were decapitated, and the heads were immediately immersed in artificial cerebrospinal fluid at 0 °C for 10 s. The surgical scissors kept parallel to the decapitation surface of rats. The olfactory bulb end of the transverse plane of the rat skull was cut horizontally, and then the longitudinal cutting was carried out along the coronal plane of both sides of the rat skull. After cutting the rat skull, the head of the olfactory bulb end was clamped firmly with surgical tweezers, and the whole skull was lifted up to remove the rat brain Hemispheres were immediately immersed in artificial cerebrospinal fluid at 0 °C for 2 min. Under the stereomicroscope, the hippocampal tissue was quickly separated with ophthalmic scissors and fine tweezers, and quickly transferred to the culture dish of 5 mL balanced salt solution on the ice bag.
The 0.5 mm × 0.5 mm square hippocampal tissue blocks were cut with ophthalmic scissors, and 0.25% trypsin was added into each hippocampal tissue block until the final concentration of trypsin was 0.125%. Put the culture dish into the cell incubator for incubation, take out the culture dish every 5 min and gently shake it to make sure that the hippocampal tissue is evenly distributed in the culture dish. After 15 min, 1 mL fetal bovine serum was added to the culture dish and mixed with pipette. Trypsin digestion was stopped and the supernatant was sucked away. 3 mL of culture medium was added to the culture dish, and the cells were repeatedly blown with the gun head 10 times. In the process of operation, the suspension should be slowly sucked and blown at a uniform speed. The cell suspension was seeded in a poly lysine coated cell culture plate or dish for culture.
Experimental scheme in vivo
The essential oil was first dissolved in DMSO at the concentration of 10 g/mL, and then suspended in saline containing 1% Tween80 to prepare for three concentrations of low, medium, and high doses of 50 mg/kg, 100 mg/kg, and 150 mg/kg for rats daily drinking after 2VO surgical operation. Mouse neuron growth factor (mNGF) was administered by a single intraperitoneal injection. All rats were randomly divided into eight groups of six animals. Group 1: Saline group (Sham); Group 2: Vascular dementia group (Model); Group 3: Perillaldehyde group (Sham + PAE-150 mg/kg); Group 4: Vascular dementia + Perillaldehyde (50 mg/kg) group (PAE-50 mg/kg); Group 5: Vascular dementia + Perillaldehyde (100 mg/kg) group (PAE-100 mg/kg); Group 6: Vascular dementia + Perillaldehyde (150 mg/kg) group (PAE-150 mg/kg). Group 7: Vascular dementia + mNGF (20 mg/kg) group (mNGF). Group 8: Vascular dementia + Perillaldehyde (150 mg/kg) + mNGF (20 mg/kg) group (PAE-mNGF). Before sacrifice, the learning and memory behavior of rats were assessed. The hippocampus of rats was isolated for morphological analysis.
Morris water maze (MWM) test
The Morris water maze test (MWM) was used for memory and learning behavior assessment [18]. On the 1st to 4th day of the experiment, rats were put into the water maze with their heads facing the pool wall in the order of East, West, South and North. Each experiment was limited to 60 s. If the rats found the platform within 60 s after entering the water, they were allowed to stay on the platform for 10 s; if the rats could not find the platform within 60 s after entering the water, the experimenters placed the rats on the platform and stayed for 10 s. Each rat was trained 4 times a day with an interval of 20 min. On the 5th day of the experiment, the platform was removed, and the rats were put into any water entry point in the water maze. The swimming track of rats within 1 min was recorded by a video recording system (Columbus Instruments, USA). Swim distance in quadrant (m), Swim distance out quadrant (m), Swim distance (m), Swim distance in quadrant/Swim distance, Swim time in quadrant (s), Swim time out quadrant (s), Swim time in quadrant/Swim time, Swim speed in quadrant (mm/s), Swim speed out quadrant (mm/s), Average swim speed (mm/s), Number of escape were analyzed by image pro plus medical image processing system.
HE staining
The rats of each group were anesthetized with chloral hydrate (350 mg/kg, ip), and then perfused with PBS and 4% paraformaldehyde. The whole brain was removed and then post-fixed in 4% paraformaldehyde. After regular perfusion and fixation, the brain segments containing hippocampus were obtained and paraffin embedded. The frontal slices were cut continuously with a thickness of 5 μm using a microtome (Leica RM2135, Nussloch, Germany). The sections were stained with hematoxylin and eosin and collected pictures with digital pathological section Scanner (3DHISTECH, Pannoramic SCAN, EU). HE staining was carried out to detect the global morphology of tissues as described previously [19].
Golgi staining
The hippocampal tissue was cut into 80 μm thick slices by cryosectioning machine at − 20 to − 22 °C. According to the instructions, the sections were stained with Golgi staining kit (Hito Golgi-Cox Optimstain kit, Hitobiotec, USA). After the sections were sealed with neutral gum, they were dried at room temperature. The number of dendritic spines and morphology of neurons were observed. Under the microscope, 5 neurons were located in each group, and 3 segments of dendrites with grade 2–3 branches were selected respectively. The measurement range of 100 μm length was marked, and the number of dendritic spines was counted.
Electron microscope quantitative analysis
Each group of specimens took 30 cell fields, each field took a more appropriate range, unity square large multiple was 1000 times for ultrastructural observation and photography. Using SIS electron microscope image analysis software to test, under the interactive operation mode, the mitochondria and nuclei of each picture are respectively traced by the cursor, and the primary parameters such as perimeter, area, roundness, long axis, short axis, as well as the area of each picture are tested. According to the principle of miss's, the formula VX/VR = ax/AR (i.e. the ratio of cross-sectional area of two structures on two-dimensional section image, etc.) is derived. According to its volume ratio in three-dimensional space, the relevant secondary parameters volume density (Vv), shape factor (PE), average area (s) and average perimeter (L) are calculated. The thickness of presynaptic membrane and the width of postsynaptic space were measured.
TUNEL assay
TUNEL is a common method for detecting DNA fragmentation that results from apoptosis signaling cascades. One Step TUNEL Apoptosis Assay Kit was purchased from Beyotime Biotechnology and operated according to the instructions. All the images were collected in the digital pathological section scanner (3D HISTECH, Pannoramic SCAN, EU).
Western Blotting
Western blot was used to detect the expression of TRPM2 and NR2B in hippocampus tissues. After anesthesia, the hippocampal tissues were taken out and homogenized on in RIPA buffer with 1 mM PMSF. The proteins were quantified by BCA Protein Assay kit (Thermo Scientific), separated by SDS-PAGE, transferred to PVDF membrane and detected by specific antibody. Band intensity was measured by densitometry (model GS-700, Imaging Densitometer; Bio-Rad). The ratio of β-actin to NR2B and TRPM2 was used to represent the expression of NR2B and TRPM2 in hippocampus respectively.
Immunohistochemistry and confocal laser scanning
For immunohistochemistry staining, the hippocampal tissue slices were frozen and sectioned with a cryostat microtome (8 µm). The sections were incubated for 10 min for antigen recovery, and then blocked with 5% BSA for 30 min. Brain sections were incubated with a primary antibody overnight at 4 °C to detect the expression of TRPM2 and NR2B respectively. Then incubated with secondary antibodies for 1 h at room temperature and detected with DAB. For immunofluorescence staining, brain sections and cells were blocked with 5% BSA and then incubated with primary antibodies overnight at 4 °C to detect the expression of neurons (Neu N), the colocalization between TRPM2 and NR2B respectively. Subsequently, the sections and cells were washed three times with PBST and incubated with secondary fluorescent antibody in the dark for 2 h at room temperature and tested after mounting with DAPI. All the images were collected in the digital pathological section scanner (3D HISTECH, Pannoramic SCAN, EU).
Fluorescence Ca2+ concentration detection
The Fluo3-AM calcium ion fluorescent probe (Ringer’s) was used to detect Ca2+ concentration. The signal sampling rate was adjusted to 1 Hz with a 488 nm argon laser. Most cells were delineated as the region of interest (ROI). The time series imaging of ZEN2009 software was used to observe spontaneous calcium shock imaging in the resting state of the neurons under LSM 510 system laser confocal microscope. Denoted by F/F0 Spontaneous calcium oscillations of neurons. F represents the relative fluorescence signal value of neuron cells, and F0 represents the average fluorescence signal value recorded within 2 min. F, Fmax and Fmin collected by LSM 510 system laser confocal microscope, enter the following formula:
$$\left[ {{\text{Ca}}^{2 + } } \right]_{{\text{i}}} = \left[ {\left( {{\text{F}} - {\text{F}}_{\min } } \right)/\left( {{\text{F}}_{\max } - {\text{F}}} \right)} \right] \times {\text{K}}_{{\text{d}}} \times {\text{S}}{.}$$
Among them, Kd is 224 mM; Fmin is the F value measured after the buffer lysed cells complexed with free calcium ions. The F value of the buffer solution contained 0.05% TritonX-100, 6.6 mM EGAT and 40 mM Tris–HCl buffer without Ca2+; the F value measured after the buffer solution with calcium ion concentration of 0.05% TritonX-100 is 2 mM. S is the ratio of the minimum fluorescence intensity to the maximum fluorescence intensity measured at 380 nm excitation wavelength.
MTT staining
Hippocampal tissue of rats was isolated and cultured for primary neurons. Cells in the logarithmic phase of the cell cycle were collected and cultured on cell plate. Thus, the 20 μL MTS solution was added to each well and continued to incubate for 1–2 h. Then the cell plate was put into the dtx880 multi-functional microplate reader, at the wavelength range of 490 nm to measure the light absorption value of each well. Finally, the cell growth curve with time and absorption value as abscissa and ordinate respectively was drawn to calculate the cell activity.
ELISA analysis
Animals were sacrificed and brains were quickly harvested. Then, the hippocampus was extracted by using a dissecting microscope and weighted and homogenized in liquid nitrogen. The content of nitric oxide (NO), malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were detected by ELISA according to the manufacturer’s instructions (FineTest, China).
Statistical analysis
Data are presented as the mean ± SEM. Raw data was statistically analyzed with Graph Pad Prism 5.0. MWM test escape latencies and swimming speeds were analyzed using two-way analysis of variance (ANOVA) with repeated measures. The other data was analyzed using one-way ANOVA. Fisher’s least-significant difference post hoc test was used to test the differences between two groups. The value of P < 0.05 was considered statistically significant.
