All animal experiments were approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University. Pregnant Sprague-Dawley rats were housed at 24 °C on a 12 h light: 12 h dark cycle with access to food and water ad libitum. Every time after injection, chambers with computer-controlled heater plates maintained a constant temperature of 36.5 ± 1 ℃ were used to ensure rat comfort and safety.
Experiment 1: To evaluate the effect of propofol on the Akt-p27 signaling pathway during the BGS, and the change in learning ability during adulthood, 7-day-old male rats (11–15 g) were divided into two groups (propofol-treated and control groups). In the propofol-treated group, The 7-day-old rats in the propofol-treated group received a single intraperitoneal (ip) injection of propofol (75 mg/kg) diluted in a fat emulsion. The rats in the control group received an equal volume of fat emulsion in a single injection (Figure 1A).
Figure 1. Effect of neonatal anesthesia with propofol on p-Akt/p27 signaling and neurocognitive function in adult rats. (A) Experimental protocol. Western blotting was performed 24 h after each treatment. Sixty-day-old rats underwent Morris water maze (MWM) testing. (B, C) The expression of p-Akt, total-Akt (B), and p27 (C) in the hippocampal DG was determined by western blotting; n = 6. (D–H) Spatial learning and memory function in adult rats was determined by the Morris water maze (MWM) test; n = 8. (D) Representative swimming paths of space exploration. (E) The escape latency of reaching the submerged platform in the training period. (F) The escape latency in the probe period. (G) The crossing over number to the previous platform within 60 s. (H) The time spent in the target quadrant. Data are presented as the mean ± SD. ip, intraperitoneal; PND, postnatal day; *P < 0.05, **P < 0.01
Experiment 2: To determine whether enhancement of Akt phosphorylation influenced the effects of propofol on neurogenesis in the DG and neurocognitive dysfunction in adulthood, male rat pups were randomly assigned to the following four groups: control group; propofol-treated group; SC79 (an Akt activator) group; and SC79 + propofol group. Rats in the SC79 and SC79 + propofol groups were pre-injected with SC79 solution into the hippocampus. Rats in the control and propofol groups received an equal volume of the solvent. Then, the rats in the propofol and SC79 + propofol groups received a single intraperitoneal injection of propofol (75 mg/kg), while the rats in the other groups were given an equal volume of fat emulsion. After injecting propofol and the fat emulsion, all experimental rats were treated with an intraperitoneal injection of 5-bromo-2-deoxyuridine [BrdU], 100 mg/kg; Sigma, St. Louis, MO, USA; (Figure 2A).
Figure 2. Effect of SC79 pre-treatment on the expression of p-Akt and p27 in the DG of rats exposed to propofol at PND-7. (A) Experimental protocol. (B, C) The expression of p-Akt, total-Akt (B), and p27 (C) in the DG was determined by western blots. Data are presented as the mean ± SD (n = 6). ACSF, artificial cerebrospinal fluid; h.i., hippocampal dentate gyrus injection; ip, intraperitoneal; PND, postnatal day; DG, hippocampal dentate gyrus; *P < 0.05, **P < 0.01.
SC79 was injected into the hippocampus of rats using a stereotaxic apparatus. Chloral hydrate (50 mg/kg, ip) was used for anesthesia. The head of the rat was positioned in a stereotaxic frame and a midline sagittal incision on the scalp was made with a scalpel. The layer of connective tissue of newborn rats over the skull was thin and clear, which facilitated easy location of the bregma; coordinates (2.3 mm posterior to the bregma, 2.2 mm lateral to the sagittal suture on the left and right sides, and 2.5 mm beneath the surface of the skull) were adjusted digitally. A puncture needle was used to drill a burr hole on both sides over the hippocampus. A microsyringe was filled with SC79 (20 μL). SC79 (0.045 μg/μL) dissolved in artificial cerebrospinal fluid (ACSF) with 5% DMSO was administered bilaterally (10 µL each side) at a rate of 2 µL/min. The microsyringe was then kept in place for 3−5 min to prevent solution overflow. After removing the microsyringe, we closed the skin incision using a suturing needle and surgical thread. The control animals underwent the same surgical procedure and received the same volume of ACSF with 5% DMSO, but without SC79.
Twenty-four hours after injecting BrdU and establishing deep anesthesia with chloral hydrate (50 mg/kg), the animals underwent cardiac perfusion with 4% paraformaldehyde after 0.9% normal saline. The brains were removed carefully, postfixed in 4% paraformaldehyde overnight, and submerged in 30% sucrose. After exposing the hippocampus, the brain was cut in coronal sections on a microtome at a thickness of 30 μm. The sections were incubated in 2N HCl at 37 °C for 30 min to expose the BrdU antigen, then thrice-washed in PBS (5 min/wash). The sections were treated with 10% goat serum in PBS with 0.3% Triton-X for 2 h at room temperature to block non-specific epitopes followed by incubation with the primary antibodies in PBS with 0.3% Triton-X at 4 °C overnight (Table 1). After 3 washes with PBS, the sections were incubated in the appropriate secondary fluorescent antibodies for 2 h at 37 °C. A skilled researcher blinded to the study observed the sections using image stacks on a laser scanning confocal microscope (Fluoview 1000; Olympus, Japan). Image-Pro Plus software was used to calculate the number of double-positive cells in the hippocampal DG.
Antibody name Specificity Host species Dilution Company Catalog number Nestin Neural stem cells Rabbit 1:100 Santa Cruz sc-23927 Nestin Neural stem cells Mouse 1:200 CST 33475 β-tubulin III Newborn neurons Rabbit 1:200 CST 5568 GFAP Astrocytes Rabbit 1:200 CST 3670 BrdU Newly generated cells Mouse 1:100 Santa Cruz sc-32323 p-Akt Rabbit 1:1,000 CST #4060 p27 Rabbit 1:1,000 CST #3686
Table 1. Primary antibodies
Twenty-four hours after injection of propofol, the rats were decapitated, and the hippocampal tissues were separated and removed. The tissues were homogenized together with lysis buffer and protease inhibitors, and clarified by centrifugation at 14,000 rpm for 15 min at 4 °C. The liquid supernatant represented the protein sample. Equal amounts of solubilized protein (20 μg) were subjected to sodium dodecyl sulfate 10% polyacrylamide gel, and the target proteins were transferred to nitrocellulose membranes. The blots were blocked with 5% skim milk powder diluted with PBST for 2 h at room temperature, then incubated overnight with the primary antibodies against p-Akt or p27 (Table 1). After thrice-washing with washing buffer (5 min/wash), the membranes were incubated with the appropriate secondary antibodies for 2 h at room temperature. The immunoreactive bands were detected with a chemiluminescence substrate. Image J software was used to calculate the grayscale value of the bands.
We used the Morris water maze (MWM) to test hippocampal-dependent spatial memory ability. All the control and propofol-treated animals were tested 2 months after treatment on day 7 after birth. A circular, black pool with a 150-cm diameter and a 50-cm depth was divided into four quadrants. The pool was filled with water at room temperature (23 ± 2) °C until the platform (10 cm diameter), which was placed inside the target quadrant, was submerged 2 cm below the surface. The platform location was kept in the same position throughout all trials. Rats were trained four times/d at 15-min intervals for 4 consecutive days during the memory acquisition trials (60 s/trial). The rats were allotted 60 s to find the platform during each trial from a random starting position; the trials were held in a dark and quiet laboratory. If the platform was located, the rat was allowed to rest on the platform for 15 s. If the rat did not locate the platform, the rat was gently guided to the platform, where the rat remained for 15 s; the escape latency was recorded as 60 s. The mean escape latency from four trials was recorded as the daily outcome. A probing trial was performed on the 5th day with the platform removed; specifically, the starting direction was opposite to the target quadrant. Each rat was allotted 60 s to probe the pool. The time required for the rat to arrive in the labeled quadrant of the previous platform, the number of crossovers to the labeled quadrant, and the time the rat spent in the target quadrant containing the previous platform within 60 s were recorded. A computerized video system was used to track the path of the rats in each trial. After every trial, rats were dried off and normothermia was assured before returning the rat to the chamber.
The novel object recognition test (NORT) and the object location test (OLT) are two well-established behavioral tests that are commonly used to evaluate cognitive performance, including memory and spatial cognitive ability. Both of the tests are based on the intrinsic preference of animals to spend more time exploring a novel object or location than a previously encountered object or location. The tests were conducted according to the method of a previous study . The results were recorded as the time spent exploring the new location or object (TN) and the old location or object (TO). The DI, which was used to evaluate the cognitive performance, was calculated as follows: (TN-TO)/(TN+TO).
SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA) was used to analyze statistics and GraphPad Prism 5 was used to create the graphs. Differences between two groups were analyzed using the Student's t-test and differences in escape latency in the MWM test among more than two groups were analyzed using two-way ANOVA. The other data were analyzed by one-way ANOVA with least significant difference (LSD) post-hoc analysis. The numerical data are presented as means ± SD. Statistical significance was considered at a P-value < 0.05.
Neonatal Exposure to Propofol Interferes with the Proliferation and Differentiation of Hippocampal Neural Stem Cells and the Neurocognitive Function of Rats in Adulthood via the Akt/p27 Signaling Pathway
- Received Date: 2021-12-04
- Accepted Date: 2022-02-11
|Citation:||MIAO Hui Hui, LIU Wen Bo, JIAO Xin Hao, SHAO Ke Jie, YUAN Ying Xuan, SHA Sha, ZHANG Qi Qi, YAN Jing, SUN Yin Ying, ZHOU Cheng Hua, WU Yu Qing. Neonatal Exposure to Propofol Interferes with the Proliferation and Differentiation of Hippocampal Neural Stem Cells and the Neurocognitive Function of Rats in Adulthood via the Akt/p27 Signaling Pathway[J]. Biomedical and Environmental Sciences, 2022, 35(4): 283-295. doi: 10.3967/bes2022.040|