Introduction
Recent studies have demonstrated that exposure of the developing brain to anesthetics is associated with neurobehavioral abnormalities including cognitive impairments. The result from less than 1 h of general anesthesia in early infancy provides no evidence of neurocognitive deficits at age 2 or 5 years compared with awake-regional anesthesia (Davidson et al.
2016; McCann et al.
2019). However, some clinical studies of longer or repeated exposures have linked childhood anesthesia to subsequent language impairment, cognitive abnormalities, and learning disabilities (Warner et al.
2018; Wilder et al.
2009). Therefore, there is significant concern that anesthesia exposure in early infancy may have deleterious effects on the developing brain. Persistent memory and learning impairments were observed in postnatal day 7 (P7) rats exposed to a combination of anesthetics for 6 h because of extensive neuronal apoptosis and neurodegeneration (Jevtovic-Todorovic et al.
2003). Furthermore, P7 rats exposed to 1 minimum alveolar concentration (MAC) of isoflurane for 4 h showed reduced proliferation of the neural progenitor cells and defective spatial reference memory (Stratmann et al.
2009b). The exposure of P7 rats to sevoflurane also caused short-term or long-term memory deficits. The exposure of P6 mice to 3% sevoflurane for 6 h caused learning defects and abnormal social behaviors resembling autism spectrum disorders (Satomoto et al.
2009).
The exposure to 3.4% isoflurane for 6 h inhibited proliferation of primary cultured NSCs (Chen et al.
2020) and decreased mRNA levels of
Ki67 and
Sox2 in the rat neural progenitor cells (NPCs) (Sall et al.
2009). Isoflurane suppressed self-renewal of primary NSCs in a dose- and time-dependent manner (Hou et al.
2015). Sevoflurane also inhibited the self-renewability of mouse embryonic stem cells via the GABAAR-ERK signaling pathway (Wang et al.
2016).
Neurogenesis persists throughout the life of adult mammals in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus (Zhao et al.
2008). NSCs can differentiate into several committed neural cell types, including neurons, astrocytes, and oligodendrocytes (Kempermann et al.
2004). The expression of doublecortin (
DCX) gene is temporally regulated during neurogenesis, and is typically expressed during the first 2 weeks after the birth of the neurons. The expression of neuronal nuclei (
NeuN) gene begins during the first few days of neuronal cell development and persists in the mature neurons. In the granule cells, expression of the calcium-binding protein, calretinin, is observed between the 2nd and 4th weeks of neurogenesis (Zhao et al.
2008). It has been shown that isoflurane or sevoflurane can induce inhibition of neurogenesis in the hippocampus of rodent animals (Jia et al.
2020; Li et al.
2021a).
Microglia is the main regulator of neuroinflammation and the only immune cell type that permanently resides in the central nervous system (CNS) alongside neurons (Tambuyzer et al.
2009; Tay et al.
2017). Microglial cells play a dual role in neurogenesis based on the nature and duration of brain inflammation (Ekdahl et al.
2009; Whitney et al.
2009). Activated microglial cells inhibit neurogenesis via pro-inflammatory cytokines, such as TNF-α and IL-6 (Cacci et al.
2008; Monje et al.
2003; Ryan and Nolan
2016). Foreign antigens or changes in the brain homeostasis activate microglial cells, which subsequently promote neuroinflammation and suppress hippocampal neurogenesis (Walton et al.
2006). Furthermore, anesthetics such as isoflurane or sevoflurane promote secretion of inflammatory cytokines from the microglia (Zhang et al.
2013). Both in vivo and in vitro studies have demonstrated that activated microglial cells inhibit neurogenesis via neuroinflammation (Monje et al.
2003). However, little is known about the interaction between microglia and NSCs and anesthetics-induced neurodevelopmental toxicity or cognitive dysfunction.
VEGFR2 (VEGF receptor 2) is the main receptor that regulates VEGF-A-mediated trophic effects in the CNS (Licht et al.
2011). VEGFR2 signaling promotes proliferation, migration, and differentiation of the NSCs (Jin et al.
2002; Sun et al.
2006). VEGFR2 is significantly expressed in the NPCs (Ogunshola et al.
2002). Furthermore, learning and memory can be regulated via VEGFR-2 (Deyama et al.
2020). In a diabetic foot ulcer rat model, Nrf2 overexpression can increase VEGFR2 phosphorylation, promotes proliferation and angiopoiesis in endothelial progenitor cells by reducing levels of inflammatory cytokines such as IL-6 and TNF-α (Li et al.
2018). Besides, in an ischemic hind limb model, ischemic wound healing may be associated with enhanced levels of phosphorylated VEGF receptors through reduction of inflammatory response (Li et al.
2021b). Thus, we postulated that VEGFR2 phosphorylation might be inhibited by neuroimflammation.
We therefore set out to determine whether isoflurane and sevoflurane can induce neurodevelopmental toxicity and cognitive dysfunction through the interaction between microglia and NSCs and whether its effects are associated with the changes of VEGFR2 phosphorylation.
Material and Methods
Animals
All animal experimental protocols were approved by the Animal Care and Use Committee of Wenzhou Medical University (Wenzhou, Zhejiang, China), and all procedures were performed following the National Institutes of Health (NIH, Bethesda, MD, USA) guidelines of animal care. The P7 rats were housed under a 12:12 h light–dark cycle at 22–24 ℃ ambient temperature with their parents. They were randomly assigned to the neonatal rats control (CON), neonatal rats anesthetized with isoflurane (ISO), and sevoflurane (SEV) groups.
Anesthesia in Rats
The P7 rats were anesthetized at 1 minimum alveolar concentration (MAC) as determined by tail clamping. The 1 MAC of isoflurane or sevoflurane was determined to be 1.1% or 2.0% concentrations, respectively. The ISO or SEV group was flushed continuously with isoflurane or sevoflurane and 30% oxygen for 4 h. The CON group received 30% oxygen for 4 h at the identical condition. During anesthesia, neonatal rats breathed spontaneously. The temperature of the chamber floor was kept at 37 ℃ and was covered with soda lime. The concentration of isoflurane was continuously monitored using a gas analyzer (ARYM-0054 Vamos, Dräger, Germany). When the rats were exposed to anesthetics in chambers, respiratory rate and invasive arterial blood pressure were monitored and blood was sampled for blood gas analysis. Anesthesia was terminated by discontinuing isoflurane or sevoflurane. Then the rats were kept in a chamber containing 30% oxygen until the return of righting reflex. Subsequently, they were returned to their home cages with their parents.
BrdU Administration
The rats were administered bromodeoxyuridine (BrdU; Sigma, St. Louis, MO; 20 mg/ml) at 100 mg/kg/d for 4 d (intraperitoneally; dissolved in PBS) after isoflurane or sevoflurane exposure.
BrdU has been a principal marker for mitotic cells in studies of the neurogenesis (Gratzner
1982). This method labels freshly divided neural stem/progenitor cells and benefits from its long-term retention in divided cells and its passage to their daughter cells (Schmuck et al.
2014). This feature can be used to trace the cell lineage and cell survival.
Hemodynamic Monitoring and Blood Gas Analysis
Blood pressure and blood gases were measured in a separate cohort (n = 8/group) as previously described to confirm whether such an anesthesia regimen affects cardiorespiratory function. Briefly, arterial blood was sampled in the ISO or SEV rats via a 24-gauge arterial puncture needle (IntroCan®-W, Braun Medical Inc., Bethlehem, PA, USA) through the abdominal aorta using a dissecting microscope (PS100, Nikon, Tokyo, Japan). The mean arterial blood pressure (MAP) was measured by an anesthesia monitor (M3046, Philips Medical System, Boeblingen, Germany). The blood sample (0.2-0.03 ml) was immediately analyzed to determine pH, arterial oxygen, and carbon dioxide using the blood gas analyzer (GEM Premier 3000, Bedford, MA, USA).
Open-field Test
The open-field test (OFT) was done at 2 weeks after the isoflurane or sevoflurane exposure. The animals were brought to the experimental room 5–20 min before testing to allow habituation. Each animal was placed in a corner square of the open field and faced the corner. Subsequently, each animal was observed for 5 min each time for three times. After 5 min, the animal was removed. Then the movement distance, time spent, and number of entries to the central region of the animal were recorded.
Morris Water Maze (MWM)
The MWM was done at 2 weeks (P21) and 6 weeks (P49) after the isoflurane or sevoflurane exposure. Before the trials, all rats were placed in the water of the swimming arena with a 6 cm diameter platform submerged 0.5–1 cm above the surface of the water on day 0. Each rat was allowed to swim for 120 s to locate the platform. The rats that had vision problems or did not swim were removed from the arena and excluded from further experiments. Subsequently, spatial acquisition trials were conducted, where the platform was submerged 0.5–1 cm below the surface of the water. The animals underwent four trials each day in the pool at four different starting positions facing the tank wall. A time limit of 2-min per trial allowed for rats to find the platform within a 30-min inter-trial interval. The animals not finding the platform within the time allotted were guided onto the platform for 15 s. The swim speed, distance (path length), and time (escape latency) in finding the platform were calculated from the recorded videos using the MWM software (SLY-WMS Morris water maze, Shuolinyuan, Beijing, China). On day 6, a probe trial was performed, during which the platform was removed, and animals were placed in a novel start position 180° from the original platform position to swim freely for 30 s. The percentage of time spent in the target quadrant and the time on platform–site crossovers were recorded.
T Maze
The T maze was done at 2 weeks and 6 weeks after the isoflurane or sevoflurane exposure. Before trials, each animal was maintained at 90%-95% of its free-feeding body weight. Then the animals were placed into a T maze for 3 min each day for successive four days. For the forced trials, the reward food (milk) was placed in the food well at one goal arm, and the other goal arm door was blocked. After opening the central partition at the start arm, each animal, which was placed in the start area, ran for the reward food. When the animal consumed all the reward food, the animal was returned to the start arm and the start arm door was then closed. Then, for rewarded alternation trials, each animal was stood in the start area facing away from the goal arms for 15 s. After 15 s, the central partition and the doors of the goal arms were removed. The animal was allowed to choose between the two open goal arms. The time was allowed to consume the reward if correct. If the animal chose incorrectly the animal was removed after a time period equivalent to the time normally used to consume the reward to ensure that it had definitely discovered that the sample well was empty. As with rewarded alternation, each trial took no more than 2 min. The two trials (forced trials and rewarded alternation trials) were alternated, and the reward food at one goal arm was random, and the numbers of reward food were equal for two goal arms. The numbers of correct alternations were recorded for each animal.
Western Blot
Eight animals from each group were deeply anesthetized with 5% chloral hydrate and transcardially perfused with normal saline 5 days after anesthetics exposure. The brains were quickly removed. The hippocampus tissue was homogenized to a mixture composed of RIPA lysis buffer, phosphatase, and protease inhibitors, and incubated for 30 min on ice. The lysate was then sonicated and centrifuged at 12,000 rpm for 30 min at 4 ℃. The protein samples were quantitated using bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Eugene, OR, USA) and the concentrations were measured using a spectrophotometer (Multiskan MK3, Thermo scientific). Subsequently, the samples were admixed with 5 × sample buffer, equalized using double-distilled H2O, and heated for 5 min at 100 ℃. An equal amount of protein from each sample was separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel and then electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories Inc., Hercules, CA, USA). The blots were blocked with 10% skim milk in Tris-buffered saline and Tween 20 (0.1%) (TBST) for 2 h at room temperature (RT) and incubated at 4 ℃ overnight with mouse Nestin antibody (1:500, MAB353, Millipore), rabbit Sox2 antibody (1:1000, ab97959, Abcam), rabbit cd11b antibody (1:500, DF6476, Affinity), rabbit IL-6 antibody (1:500, DF6087, Affinity), rabbit IL-1β (1:500, AF5103, Affinity), rabbit TNF-α (1:500, AF7014, Affinity), rabbit VEGFR2 (1:500, AF6281, Affinity), rabbit p-VEGFR2 (1:500, AF8022, Affinity), or rabbit β-actin antibody (1:1000, AP0060, Bioworld Technology). After incubation with the primary antibody, the blots were incubated for 2 h at RT with secondary antibodies [goat anti-rabbit antibodies (1:5000, 111–035-003), and goat anti-mouse antibodies (115–035-003), Jackson]. Between steps, the blots were washed with TBST. The blots were visualized using ECL western blot detection system (ImageQuant LAS 4000 Mini). The bands were analyzed by Quantity One software version 4.6.2 (Bio-Rad Laboratories Inc.).
Immunohistochemistry
Six animals from each group were deeply anesthetized with 5% chloral hydrate and transcardially perfused with normal saline with simultaneous exsanguination from the right atrium, and then with 4% paraformaldehyde in 0.1 M phosphate buffer at a pH of 7.4, 5 days after anesthesia exposure (P12) and 24 h after the MWM (P28 and P56). The brains were removed and kept in 4% paraformaldehyde at 4 ℃ overnight. Subsequently, the brains were consecutively dipped into 70%, 85%, 95%, and 100% ethanol. Serial coronal 5 µm sections were cut in paraffin blocks using a microtome (Leica R2016), and at least three slides from each animal were used for staining. Briefly, sections were deparaffinized with xylene, rehydrated with a series of graded ethanol, and washed in distilled water and then in PBS. The antigen retrieval was performed incubating in 10 mM sodium citrate buffer at a pH of 6.0 for 20 min in a microwave oven at 100 ℃. The sections were blocked in 5% BSA in PBS for 30 min and were then incubated with primary antibodies (mouse anti-Nestin, 1:100, Millipore; rabbit anti-Sox2, 1:100, Millipore; rabbit anti-GFAP, 1:500, Dako; sheep anti-BrdU, 1:500, Abcam; rabbit anti-DCX, 1:400, Abcam; rabbit anti-NeuN, 1:200, Abcam; rabbit anti-IBA1, 1:400, Wako) overnight at 4 ℃. Subsequently, the sections were washed at RT, incubated in fluorophore-conjugated secondary antibodies (donkey anti-sheep, 1:500, ab150177, Abcam; donkey anti-mouse, 1:500, ab150112, Abcam; donkey anti-rabbit, 1:500, ab150068, Abcam; donkey anti-rabbit, 1:500, ab150073, Abcam) for 50 min, and then washed. The tissue sections were then incubated in DAPI (1:1000, Sigma) and then washed with PBS. Finally, the sections were mounted, viewed and quantitated using a microscope (Nikon E100) captured with 200 × magnification. The NSC proliferation (BrdU/Nestin, BrdU/Sox2), numbers (GFAP/Sox2) and differentiation (BrdU/DCX, BrdU/NeuN) of the NSC, microglia activation (IBA1
+), and the number of neuronal nuclei in the DG (DAPI) were examined. Five random sections per brain were immunostained, and photomicrographs were captured at × 200 magnification. The average of positive cell numbers of five random sections represented one brain. The total number of cells in the hippocampus was assessed by counting the neuronal nuclei stained with DAPI. All counting by investigators was blinded. All quantifications were determined by calculating the percentage of IBA1
+ or double-labeled positive cells to the total cells of cross-sectional hippocampus area in five random sections per rat using Image-Pro Plus software version 6.0. A ratio was calculated and results presented as levels of expression (positive/total cells). The methods were analyzed as previously described (Shen et al.
2013).
Statistical Analysis
All the data were expressed as mean ± SEM except those data derived from the probe trials of MWM that were expressed as median and interquartile range. The data of spatial acquisition trials were analyzed by a two-way ANOVA with repeated measures (isoflurane or sevoflurane exposure as one factor between subjects and day as a repeated measure factor) followed with the LSD post hoc test comparison. MANOVA was used to test the main effects for a group at each time point. The data of the probe trial were analyzed using a one-way ANOVA (isoflurane or sevoflurane as one variable). The level of protein expression and the levels of markers in each group were analyzed with one-way ANOVA followed by the LSD post hoc test. A p-value less than 0.05 was considered statistically significant. The SPSS software (SPSS for Windows, version 24.0, SPSS, Chicago, IL, USA) was used to analyze the data.
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